Effect of Vitamin B12 Injection on the Vocal Performance of Professional Singers: A Randomized, Double-blind, Placebo-Controlled, Crossover Trial.

Importance: One-third of singers and vocal professionals report experiencing a benefit from empirical vitamin B12 injections for improvement of mild singing-related symptoms (eg, reduced stamina, vocal fatigue, and effort). However, there is no objective evidence to support or refute these claims. Objective: To assess the presence and magnitude of the effect of empirical vitamin B12 injection on the vocal performance of singers. Design, Setting, and Participants: A randomized, double-blind, placebo-controlled, crossover trial was conducted from November 7, 2017, to November 30, 2018, at an academic voice center among 20 active adult singers without dysphonia but with mild vocal symptoms. Individuals with known or suspected vitamin B12 deficiency or active or recent vitamin B12 treatment were excluded. Analysis was on a per-protocol basis. Interventions: Participants were randomized to receive an intramuscular (deltoid) injection of either vitamin B12 (1000 µg of cyanocobalmin) or placebo (0.9% sodium chloride). After a washout period of at least 4 weeks, participants were crossed over to receive the opposite injection. Both the investigators and participants were blinded to the order of injections. Main Outcomes and Measures: The participants completed the Singing Voice Handicap Index-10 (SVHI-10), the Voice Fatigue Index (VFI), and the Evaluation of the Ability to Sing Easily (EASE) before each injection and at intervals of 1 hour, 3 hours, 24 hours, 72 hours, and 1 week after the injection. The primary time point assessment was 72 hours after injection, and the SVHI-10 score was the primary outcome measure. Results: Twenty singers (10 men; median age, 22 years [range, 19-42 years]) were enrolled. The improvements after either placebo or vitamin B12 injections were comparable to each other. At 72 hours after the vitamin B12 injection, the median difference in the SVHI-10 score was 1 (95% CI, -1 to 2) compared with 3 (95% CI, 0-4) after placebo. The median difference between differences at 72 hours between placebo and vitamin B12 injections were 1.5 (95% CI, -2 to 5) for the SVHI-10, 1 (95% CI, -9 to 9) for the VFI, and -1 (95% CI, -3 to 2) for the EASE. The improvements after both injections failed to reach the estimated minimal clinically important difference. Of the 20 participants, 4 (20%) reached the estimated minimal clinically important difference in their SVHI-10 score after 72 hours for both vitamin B12 and placebo injections. Conclusions and Relevance: This randomized, double-blind, placebo-controlled, crossover trial found that after empirical vitamin B12 injection to improve mild voice-related symptoms, the improvement in self-reported voice measures in singers shows no meaningful difference compared with placebo. Trial Registration: ClinicalTrials.gov Identifier: NCT03437824.

Reversing song behavior phenotype: Testosterone driven induction of singing and measures of song quality in adult male and female canaries (Serinus canaria).

In songbirds, such as canaries (Serinus canaria), the song control circuit has been shown to undergo a remarkable change in morphology in response to exogenous testosterone (T). It is also well established that HVC, a telencephalic nucleus involved in song production, is significantly larger in males than in females. T regulates seasonal changes in HVC volume in males, and exposure to exogenous T in adult females increases HVC volume and singing activity such that their song becomes more male-like in frequency and structure. However, whether there are sex differences in the ability of T to modulate changes in the song system and song behavior has not been investigated in canaries. In this study, we compared the effects of increasing doses of T on singing and song control nuclei volumes in adult male and female American Singer canaries exposed to identical environmental conditions. Males were castrated and all birds were placed on short days (8L:16D) for 8 weeks. Males and females were implanted either with a 2, 6 or 12 mm long Silastic™ implant filled with crystalline T or an empty 12 mm implant as control. Birds were then housed individually in sound-attenuated chambers. Brains were collected from six birds from each group after 1 week or 3 weeks of treatment. Testosterone was not equally effective in increasing singing activity in both males and females. Changes in song quality and occurrence rate took place after a shorter latency in males than in females; however, females did undergo marked changes in a number of measures of song behavior if given sufficient time. Males responded with an increase in HVC volume at all three doses. In females, T-induced changes in HVC volume only had limited amplitude and these volumes never reached male-typical levels, suggesting that there are sex differences in the neural substrate that responds to T.

Oxytocin-like receptors mediate pair bonding in a socially monogamous songbird.

Although many species form socially monogamous pair bonds, relevant neural mechanisms have been described for only a single species, the prairie vole (Microtus ochrogaster). In this species, pair bonding is strongly dependent upon the nonapeptides oxytocin (OT) and vasopressin, in females and males, respectively. Because monogamy has evolved many times in multiple lineages, data from additional species are required to determine whether similar peptide mechanisms modulate bonding when monogamy evolves independently. Here we test the hypothesis that OT-like receptor activation is required for pair bond formation in the socially monogamous zebra finch (Taeniopygia guttata). Males and females were administered chronic intracerebroventricular infusions of saline or an OT receptor antagonist and were observed twice daily for 3 days in a colony environment. A variety of affiliative, aggressive and other behaviours were quantified. The antagonist produced significant and selective effects on pair bonding (latency to pair; number of sessions paired; stable pairing) and the associated behaviour of allopreening. Importantly, findings for males follow the trends of females; this yields main effects of treatment in two-way ANOVAs, although within-sex analyses are significant only for females. These data provide evidence for both convergent evolution and species diversity in the neuroendocrine mechanisms of pair bonding.

Testosterone-dependency of male solo song in a duetting songbird--evidence from females.

For male songbirds of the temperate zone there is a tight link between seasonal song behaviour and circulating testosterone levels. Such a relationship does not seem to hold for tropical species where singing can occur year-round and breeding seasons are often extended. White-browed sparrow weavers (Plocepasser mahali) are cooperatively breeding songbirds with a dominant breeding pair and male and female subordinates found in eastern and southern Africa. Each group defends an all-purpose territory year-round. While all group members sing duets and choruses, the most dominant male additionally sings a solo song that comprises a distinct and large syllable repertoire. Previous studies suggested this type of song being associated with reproduction but failed to support a relationship with males' circulating testosterone levels. The present study aimed to investigate the steroid hormone sensitivity of the solo song in more detail. We found that dominant males had significantly higher circulating testosterone levels than subordinates during the early and late breeding seasons. No changes in solo song characteristics were found between both time points. Further, experimental implantation of captive adult females with exogenous testosterone induced solo singing within one week of treatment. Such females produced male-typical song regarding overall structure and syllable composition. Sex differences existed, however, concerning singing activity, repertoire size and temporal organisation of song. These results suggest that solo singing in white-browed sparrow weavers is under the control of gonadal steroid hormones. Moreover, the behaviour is not male-specific but can be activated in females under certain conditions.

Testosterone increases repertoire size in an open-ended learner: an experimental study using adult male European starlings (Sturnus vulgaris).

Song in songbirds is a learned secondary sexual behavior, first acquired during a sensitive phase of juvenile development, which is affected by hormones such as testosterone (T). While the latter has received much attention, the potential involvement of T in the adult repertoire changes observed in a number of species is much less understood. Yet, this may prove essential to understand the role of song as a sexually selected trait. We therefore performed a T-implantation experiment during the non-breeding season (when T is basal), using adult male European starlings (Sturnus vulgaris), a songbird species in which song repertoire size (and composition) changes seasonally and increases with age. Repertoire size increased rapidly in T-males, but not in control males, indicating a role for T in repertoire size changes. This increase resulted from a lower proportion of dropped song types in T-males than in control males, while the proportion of added song types did not differ between both groups. Interestingly, the observed repertoire turnover (adding and removing song types from the repertoire) in both groups, suggests that elevated plasma T levels were not essential for changes in repertoire composition (contrary to repertoire size). Finally, T-males (but not control males) significantly increased their song rate, while neither group showed a significant change in their song bout length and phrase repetition rate. Taken together, our results suggest a role for T in adult song learning and provide new insights into the information content of repertoire size and song bout length as sexually selected traits.

Blocking estradiol synthesis affects memory for songs in auditory forebrain of male zebra finches.

Estradiol (E2) has recently been shown to modulate sensory processing in an auditory area of the songbird forebrain, the caudomedial nidopallium (NCM). When a bird hears conspecific song, E2 increases locally in NCM, where neurons express both the aromatase enzyme that synthesizes E2 from precursors and estrogen receptors. Auditory responses in NCM show a form of neuronal memory: repeated playback of the unique learned vocalizations of conspecific individuals induces long-lasting stimulus-specific adaptation of neural responses to each vocalization. To test the role of E2 in this auditory memory, we treated adult male zebra finches (n=16) with either the aromatase inhibitor fadrozole (FAD) or saline for 8 days. We then exposed them to 'training' songs and, 6 h later, recorded multiunit auditory responses with an array of 16 microelectrodes in NCM. Adaptation rates (a measure of stimulus-specific adaptation) to playbacks of training and novel songs were computed, using established methods, to provide a measure of neuronal memory. Recordings from the FAD-treated birds showed a significantly reduced memory for the training songs compared with saline-treated controls, whereas auditory processing for novel songs did not differ between treatment groups. In addition, FAD did not change the response bias in favor of conspecific over heterospecific song stimuli. Our results show that E2 depletion affects the neuronal memory for vocalizations in songbird NCM, and suggest that E2 plays a necessary role in auditory processing and memory for communication signals.

Relationship between blood mercury levels and components of male song in Nelson's sparrows (Ammodramus nelsoni).

Mercury (Hg) adversely affects the health and behavior of exposed wildlife; however, behavioral effects remain largely unknown. Changes in avian singing behavior may affect a male's fitness because song reveals male quality and thus influences female mate choice and male territory-holding ability. Nelson's sparrows (Ammodramus nelsoni) live exclusively on salt marshes and risk high levels of Hg exposure and bioaccumulation. We recorded songs of male Nelson's sparrows at two locations with different Hg exposure to determine if total blood Hg concentration was related to song characteristics, as previously reported for other species. Males with higher blood Hg levels sang at higher maximum tonal frequency, but blood Hg and site location did not influence low tonal frequency and bout duration, contrary to predictions based on other species. Within the contaminated site, Hg levels were related to bouts per minute and gap duration, such that males at that site sang faster songs. Hg influences hormones and alters brain development, raising questions about specific effects on the brains and singing behavior of male Nelson's sparrows.

17ß-estradiol regulates the sexually dimorphic expression of BDNF and TrkB proteins in the song system of juvenile zebra finches.

Mature brain derived neurotrophic factor (BDNF) plays critical roles in development of brain structure and function, including neurogenesis, axon growth, cell survival and processes associated with learning. Expression of this peptide is regulated by estradiol (E2). The zebra finch song system is sexually dimorphic - only males sing and the brain regions controlling song are larger and have more cells in males compared to females. Masculinization of this system is partially mediated by E2, and earlier work suggests that BDNF with its high affinity receptor TrkB may also influence this development. The present study evaluated expression of multiple forms of both BDNF and TrkB in the developing song system in juvenile males and females treated with E2 or a vehicle control. Using immunohistochemistry and Western blot analysis, BDNF was detected across the song nuclei of 25-day-old birds. Westerns allowed the pro- and mature forms of BDNF to be individually identified, and proBDNF to be quantified. Several statistically significant effects of sex existed in both the estimated total number of BDNF+ cells and relative concentration of proBDNF, varying across the regions and methodologies. E2 modulated BDNF expression, although the specific nature of the regulation depended on brain region, sex and the technique used. Similarly, TrkB (both truncated and full-length isoforms) was detected by Western blot in the song system of juveniles of both sexes, and expression was regulated by E2. In the context of earlier research on these molecules in the developing song system, this work provides a critical step in describing specific forms of BDNF and TrkB, and how they can be mediated by sex and E2. As individual isoforms of each can have opposing effects on mechanisms, such as cell survival, it will now be important to investigate in depth their specific functions in song system maturation.