Adult neurogenesis is necessary for functional regeneration of a forebrain neural circuit.

In adult songbirds, new neurons are born in large numbers in the proliferative ventricular zone in the telencephalon and migrate to the adjacent song control region HVC (acronym used as proper name) [A. Reiner et al., J. Comp. Neurol. 473, 377-414 (2004)]. Many of these new neurons send long axonal projections to the robust nucleus of the arcopallium (RA). The HVC-RA circuit is essential for producing stereotyped learned song. The function of adult neurogenesis in this circuit has not been clear. A previous study suggested that it is important for the production of well-structured songs [R. E. Cohen, M. Macedo-Lima, K. E. Miller, E. A. Brenowitz, J. Neurosci. 36, 8947-8956 (2016)]. We tested this hypothesis by infusing the neuroblast migration inhibitor cyclopamine into HVC of male Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii) to block seasonal regeneration of the HVC-RA circuit. Decreasing the number of new neurons in HVC prevented both the increase in spontaneous electrical activity of RA neurons and the improved structure of songs that would normally occur as sparrows enter breeding condition. These results show that the incorporation of new neurons into the adult HVC is necessary for the recovery of both electrical activity and song behavior in breeding birds and demonstrate the value of the bird song system as a model for investigating adult neurogenesis at the level of long projection neural circuits.

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

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 ß-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.

Network analysis of microRNA and mRNA seasonal dynamics in a highly plastic sensorimotor neural circuit.

BACKGROUND: Adult neurogenesis and the incorporation of adult-born neurons into functional circuits requires precise spatiotemporal coordination across molecular networks regulating a wide array of processes, including cell proliferation, apoptosis, neurotrophin signaling, and electrical activity. MicroRNAs (miRs) - short, non-coding RNA sequences that alter gene expression by post-transcriptional inhibition or degradation of mRNA sequences - may be involved in the global coordination of such diverse biological processes. To test the hypothesis that miRs related to adult neurogenesis and related cellular processes are functionally regulated in the nuclei of the avian song control circuit, we used microarray analyses to quantify changes in expression of miRs and predicted target mRNAs in the telencephalic nuclei HVC, the robust nucleus of arcopallium (RA), and the basal ganglia homologue Area X in breeding and nonbreeding Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelli). RESULTS: We identified 46 different miRs that were differentially expressed across seasons in the song nuclei. miR-132 and miR-210 showed the highest differential expression in HVC and Area X, respectively. Analyzing predicted mRNA targets of miR-132 identified 33 candidate target genes that regulate processes including cell cycle control, calcium signaling, and neuregulin signaling in HVC. Likewise, miR-210 was predicted to target 14 mRNAs differentially expressed across seasons that regulate serotonin, GABA, and dopamine receptor signaling and inflammation. CONCLUSIONS: Our results identify potential miR-mRNA regulatory networks related to adult neurogenesis and provide opportunities to discover novel genetic control of the diverse biological processes and factors related to the functional incorporation of new neurons to the adult brain.

Seasonal changes in patterns of gene expression in avian song control brain regions.

Photoperiod and hormonal cues drive dramatic seasonal changes in structure and function of the avian song control system. Little is known, however, about the patterns of gene expression associated with seasonal changes. Here we address this issue by altering the hormonal and photoperiodic conditions in seasonally-breeding Gambel's white-crowned sparrows and extracting RNA from the telencephalic song control nuclei HVC and RA across multiple time points that capture different stages of growth and regression. We chose HVC and RA because while both nuclei change in volume across seasons, the cellular mechanisms underlying these changes differ. We thus hypothesized that different genes would be expressed between HVC and RA. We tested this by using the extracted RNA to perform a cDNA microarray hybridization developed by the SoNG initiative. We then validated these results using qRT-PCR. We found that 363 genes varied by more than 1.5 fold (>log(2) 0.585) in expression in HVC and/or RA. Supporting our hypothesis, only 59 of these 363 genes were found to vary in both nuclei, while 132 gene expression changes were HVC specific and 172 were RA specific. We then assigned many of these genes to functional categories relevant to the different mechanisms underlying seasonal change in HVC and RA, including neurogenesis, apoptosis, cell growth, dendrite arborization and axonal growth, angiogenesis, endocrinology, growth factors, and electrophysiology. This revealed categorical differences in the kinds of genes regulated in HVC and RA. These results show that different molecular programs underlie seasonal changes in HVC and RA, and that gene expression is time specific across different reproductive conditions. Our results provide insights into the complex molecular pathways that underlie adult neural plasticity.

Immediate and long-term effects of testosterone on song plasticity and learning in juvenile song sparrows.

Steroid sex hormones play critical roles in the development of brain regions used for vocal learning. It has been suggested that puberty-induced increases in circulating testosterone (T) levels crystallize a bird's repertoire and inhibit future song learning. Previous studies show that early administration of T crystallizes song repertoires but have not addressed whether new songs can be learned after this premature crystallization. We brought 8 juvenile song sparrows (Melospiza melodia) into the laboratory in the late summer and implanted half of them with subcutaneous T pellets for a two week period in October. Birds treated with T tripled their singing rates and crystallized normal songs in 2 weeks. After T removal, subjects were tutored by 4 new adults. Birds previously treated with T tended toward learning fewer new songs post T, consistent with the hypothesis that T helps to close the song learning phase. However, one T-treated bird proceeded to learn several new songs in the spring, despite singing perfectly crystallized songs in the fall. His small crystallized fall repertoire and initial lag behind other subjects in song development suggest that this individual may have had limited early song learning experience. We conclude that an exposure to testosterone sufficient for crystallization of a normal song repertoire does not necessarily prevent future song learning and suggest that early social experiences might override the effects of hormones in closing song learning.

Seasonal changes in androgen receptor mRNA in the brain of the white-crowned sparrow.

In songbirds, neurons that regulate learned song behavior undergo extensive seasonal plasticity in their number and size in relation to the bird's reproductive status. Seasonal plasticity of these brain regions is primarily regulated by changes in circulating concentrations of testosterone. Androgen receptors are present in all of the major song nuclei, but it is unknown whether levels of androgen receptor mRNA in the telencephalic song regions HVC, the robust nucleus of the arcopallium, and the lateral magnocellular nucleus of the anterior nidopallium change as a function of season in white-crowned sparrows. To determine whether seasonal changes in levels of androgen receptor mRNA are specific to the song control system, we also measured levels of androgen receptor mRNA in a limbic nucleus, the lateral division of the bed nucleus of the stria terminalis (the lateral division of the bed nucleus of the stria terminalis). We found that levels of androgen receptor mRNA were higher in HVC and the lateral division of the bed nucleus of the stria terminalis of birds in the breeding condition compared with the nonbreeding condition; however, we observed no seasonal differences in levels of androgen receptor mRNA in either the robust nucleus of the arcopallium or the lateral magnocellular nucleus of the anterior nidopallium. These results are consistent with previous observations that seasonal plasticity of the song nuclei results from testosterone acting directly on HVC, which then exerts transsynaptic trophic effects on its efferent targets. The seasonal change in the expression of androgen receptor in HVC may be one component of the cellular mechanisms underlying androgenic effects on seasonal plasticity of the song control nuclei.

Steroid hormones act transsynaptically within the forebrain to regulate neuronal phenotype and song stereotypy.

Steroid sex hormones induce dramatic seasonal changes in reproductive related behaviors and their underlying neural substrates in seasonally breeding vertebrates. For example, in adult white-crowned sparrows, increased Spring photoperiod raises circulating testosterone, causing morphological and electrophysiological changes in song-control nuclei, which modify song behavior for the breeding season. We investigated how photoperiod and steroid hormones induce these changes in morphology, electrophysiology, and behavior. Neurons in a song premotor nucleus, the robust nucleus of the arcopallium (RA), show increased intrinsic spontaneous firing rate and soma size when birds are in breeding condition. Using combinations of systemic and unilateral local intracerebral hormonal manipulations, we show that long-day photoperiod accelerates the effects of systemic testosterone on RA neurons via the estradiol-synthesizing enzyme aromatase (CYP19A1); these changes require inputs from the afferent song control nucleus HVC (used as a proper name) and steroid receptor activation within HVC; local coactivation of androgen and estrogen receptors (ARs and ERs, respectively) within HVC, but not RA, is sufficient to cause neuronal changes in RA; activation of ARs in RA is also permissive. Using bilateral local intracerebral hormone-receptor blockade, we found that ARs and ERs in the song-control nucleus HVC mediate systemic testosterone-induced changes in song stereotypy but not rate. This novel transsynaptic effect of gonadal steroids on activity and morphology of RA neurons is part of a concerted change in key premotor nuclei, enabling stereotyped song.

Auditory feedback and song production do not regulate seasonal growth of song control circuits in adult white-crowned sparrows.

An important area of research in neuroscience is understanding what properties of brain structure and function are stimulated by sensory experience and behavioral performance. We tested the roles of experience and behavior in seasonal plasticity of the neural circuits that regulate learned song behavior in adult songbirds. Neurons in these circuits receive auditory input and show selective auditory responses to conspecific song. We asked whether auditory input or song production contribute to seasonal growth of telencephalic song nuclei. Adult male Gambel's white-crowned sparrows were surgically deafened, which eliminates auditory input and greatly reduces song production. These birds were then exposed to photoperiod and hormonal conditions that regulate the growth of song nuclei. We measured the volumes of the nuclei HVC, robust nucleus of arcopallium (RA), and area X at 7 and 30 d after exposure to long days plus testosterone in deafened and normally hearing birds. We also assessed song production and examined protein kinase C (PKC) expression because previous research reported that immunostaining for PKC increases transiently after deafening. Deafening did not delay or block the growth of the song nuclei to their full breeding-condition size. PKC activity in RA was not altered by deafening in the sparrows. Song continued to be well structured for up to 10 months after deafening, but song production decreased almost eightfold. These results suggest that neither auditory input nor high rates of song production are necessary for seasonal growth of the adult song control system in this species.

Act locally and think globally: intracerebral testosterone implants induce seasonal-like growth of adult avian song control circuits.

There is pronounced seasonal plasticity in the morphology of the neural circuits that regulate song behavior in adult songbirds, primarily in response to changes in plasma testosterone (T) levels. Most song nuclei have androgen receptors. Afferent input from the telencephalic nucleus HVc (also known as the "high vocal center") is necessary for seasonal growth of the direct efferent target nuclei RA and area X. We asked here whether T-stimulated growth of HVc is sufficient to induce growth of its efferent nuclei. Intracerebral T implants were placed unilaterally near HVc or RA in photosensitive adult male white-crowned sparrows for one month. The T implant near HVc produced significant growth of the ipsilateral (but not contralateral) HVc, RA, and area X, and increased neuronal number in the ipsilateral HVc. The T implant near RA did not produce selective growth of ipsilateral RA, HVc, or area X. Intracerebral T implants did not elevate plasma T levels, nor did they stimulate growth of two peripheral androgen sensitive targets, the syrinx and the cloacal protuberance. These results suggest that seasonal growth of the adult song circuits results from T acting directly on HVc, which then stimulates the growth of RA and area X transynaptically.