Distinct neurogenomic states in basal ganglia subregions relate differently to singing behavior in songbirds.

Both avian and mammalian basal ganglia are involved in voluntary motor control. In birds, such movements include hopping, perching and flying. Two organizational features that distinguish the songbird basal ganglia are that striatal and pallidal neurons are intermingled, and that neurons dedicated to vocal-motor function are clustered together in a dense cell group known as area X that sits within the surrounding striato-pallidum. This specification allowed us to perform molecular profiling of two striato-pallidal subregions, comparing transcriptional patterns in tissue dedicated to vocal-motor function (area X) to those in tissue that contains similar cell types but supports non-vocal behaviors: the striato-pallidum ventral to area X (VSP), our focus here. Since any behavior is likely underpinned by the coordinated actions of many molecules, we constructed gene co-expression networks from microarray data to study large-scale transcriptional patterns in both subregions. Our goal was to investigate any relationship between VSP network structure and singing and identify gene co-expression groups, or modules, found in the VSP but not area X. We observed mild, but surprising, relationships between VSP modules and song spectral features, and found a group of four VSP modules that were highly specific to the region. These modules were unrelated to singing, but were composed of genes involved in many of the same biological processes as those we previously observed in area X-specific singing-related modules. The VSP-specific modules were also enriched for processes disrupted in Parkinson's and Huntington's Diseases. Our results suggest that the activation/inhibition of a single pathway is not sufficient to functionally specify area X versus the VSP and support the notion that molecular processes are not in and of themselves specialized for behavior. Instead, unique interactions between molecular pathways create functional specificity in particular brain regions during distinct behavioral states.

Vocal changes in a zebra finch model of Parkinson's disease characterized by alpha-synuclein overexpression in the song-dedicated anterior forebrain pathway.

Deterioration in the quality of a person's voice and speech is an early marker of Parkinson's disease (PD). In humans, the neural circuit that supports vocal motor control consists of a cortico-basal ganglia-thalamo-cortico loop. The basal ganglia regions, striatum and globus pallidus, in this loop play a role in modulating the acoustic features of vocal behavior such as loudness, pitch, and articulatory rate. In PD, this area is implicated in pathogenesis. In animal models of PD, the accumulation of toxic aggregates containing the neuronal protein alpha-synuclein (αsyn) in the midbrain and striatum result in limb and vocal motor impairments. It has been challenging to study vocal impairments given the lack of well-defined cortico-basal ganglia circuitry for vocalization in rodent models. Furthermore, whether deterioration of voice quality early in PD is a direct result of αsyn-induced neuropathology is not yet known. Here, we take advantage of the well-characterized vocal circuits of the adult male zebra finch songbird to experimentally target a song-dedicated pathway, the anterior forebrain pathway, using an adeno-associated virus expressing the human wild-type αsyn gene, SNCA. We found that overexpression of αsyn in this pathway coincides with higher levels of insoluble, monomeric αsyn compared to control finches. Impairments in song production were also detected along with shorter and poorer quality syllables, which are the most basic unit of song. These vocal changes are similar to the vocal abnormalities observed in individuals with PD.

Social context-dependent singing alters molecular markers of synaptic plasticity signaling in finch basal ganglia Area X.

Vocal communication is a crucial skill required throughout life. However, there is a critical gap in our understanding of the underlying molecular brain mechanisms, thereby motivating our use of the zebra finch songbird model. Adult male zebra finches show differences in neural activity patterns in song-dedicated brain nuclei when they sing in two distinct social contexts: a male singing by himself (undirected, UD) and a male singing to a female (female-directed, FD). In our prior work, we showed that in song-dedicated basal ganglia Area X, protein levels of a N-methyl-D-aspartate receptor subtype 2B (NMDAR2B) increased with more UD song and decreased with more FD song. We hypothesized that molecules downstream of this receptor would show differential protein expression levels in Area X between UD and FD song. Specifically, we investigated calcium/calmodulin dependent protein kinase II beta (CaMKIIB), homer scaffold protein 1 (HOMER1), serine/threonine protein kinase (Akt), and mechanistic target of rapamycin kinase (mTOR) following singing and non-singing states in Area X. We show relationships between social context and protein levels. HOMER1 protein levels decreased with time spent singing FD song, and mTOR protein levels decreased with the amount of and time spent singing FD song. For both HOMER1 and mTOR, there were no differences with the amount of UD song. With time spent singing UD, CaMKIIB protein levels trended in a U-shaped curve whereas Akt protein levels trended down. Both molecules showed no change with FD song. Our results support differential involvement of molecules in synaptic plasticity pathways between UD and FD song behaviors.

Wireless battery free fully implantable multimodal recording and neuromodulation tools for songbirds.

Wireless battery free and fully implantable tools for the interrogation of the central and peripheral nervous system have quantitatively expanded the capabilities to study mechanistic and circuit level behavior in freely moving rodents. The light weight and small footprint of such devices enables full subdermal implantation that results in the capability to perform studies with minimal impact on subject behavior and yields broad application in a range of experimental paradigms. While these advantages have been successfully proven in rodents that move predominantly in 2D, the full potential of a wireless and battery free device can be harnessed with flying species, where interrogation with tethered devices is very difficult or impossible. Here we report on a wireless, battery free and multimodal platform that enables optogenetic stimulation and physiological temperature recording in a highly miniaturized form factor for use in songbirds. The systems are enabled by behavior guided primary antenna design and advanced energy management to ensure stable optogenetic stimulation and thermography throughout 3D experimental arenas. Collectively, these design approaches quantitatively expand the use of wireless subdermally implantable neuromodulation and sensing tools to species previously excluded from in vivo real time experiments.

Middle age, a key time point for changes in birdsong and human voice.

Voice changes caused by natural aging and neurodegenerative diseases are prevalent in the aging population and diminish quality of life. Most treatments involve behavioral interventions that target the larynx because of a limited understanding of central brain mechanisms. The songbird offers a unique entry point into studying age-related changes in vocalizations because of a well-characterized neural circuitry for song that shares homology to human vocal control areas. Previously we established a translational dictionary for evaluating acoustic features of birdsong in the context of human voice measurements. In the present study, we conduct extensive analyses of birdsongs from young, middle-aged, and old male zebra finches. Our findings show that birdsongs become louder with age, and changes in periodic energy occur at middle age but are transient; songs appear to stabilize in old birds. Furthermore, faster songs are detected in finches at middle age compared with young and old finches. Vocal disorders in humans emerge at middle age, but the underlying brain pathologies are not well identified. The current findings will motivate future investigations using the songbird model to identify possible brain mechanisms involved in human vocal disorders of aging. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Social context-dependent singing alters molecular markers of dopaminergic and glutamatergic signaling in finch basal ganglia Area X.

Dopamine (DA) is an important neuromodulator of motor control across species. In zebra finches, DA levels vary in song nucleus Area X depending upon social context. DA levels are high and song output is less variable when a male finch sings to a female (female directed, FD) compared to when he is singing by himself (undirected, UD). DA modulates glutamatergic input onto cortico-striatal synapses in Area X via N-methyl-d-aspartate (NMDA) and DA receptor mechanisms, but the relationship to UD vs. FD song output is unclear. Here, we investigate the expression of molecular markers of dopaminergic and glutamatergic synaptic transmission (tyrosine hydroxylase - TH, alpha-synuclein - α-syn) and plasticity (NMDA 2B receptor - GRIN2B) following singing (UD vs. FD) and non-singing states to understand the molecular mechanisms driving differences in song output. We identified relationships between protein levels for these biomarkers in Area X based on singing state and the amount of song, measured as the number of motifs and time spent singing. UD song amount drove increases in TH, α-syn, and NMDA 2B receptor protein levels. By contrast, the amount of FD song did not alter TH and NMDA 2B receptor expression. Levels of α-syn showed differential expression patterns based on UD vs. FD song, consistent with its role in modulating synaptic transmission. We propose a molecular pathway model to explain how social context and amount of song are important drivers of molecular changes required for synaptic transmission and plasticity.

Common Terminology and Acoustic Measures for Human Voice and Birdsong.

Purpose The zebra finch is used as a model to study the neural circuitry of auditory-guided human vocal production. The terminology of birdsong production and acoustic analysis, however, differs from human voice production, making it difficult for voice researchers of either species to navigate the literature from the other. The purpose of this research note is to identify common terminology and measures to better compare information across species. Method Terminology used in the birdsong literature will be mapped onto terminology used in the human voice production literature. Measures typically used to quantify the percepts of pitch, loudness, and quality will be described. Measures common to the literature in both species will be made from the songs of 3 middle-age birds using Praat and Song Analysis Pro. Two measures, cepstral peak prominence (CPP) and Wiener entropy (WE), will be compared to determine if they provide similar information. Results Similarities and differences in terminology and acoustic analyses are presented. A core set of measures including frequency, frequency variability within a syllable, intensity, CPP, and WE are proposed for future studies. CPP and WE are related yet provide unique information about the syllable structure. Conclusions Using a core set of measures familiar to both human voice and birdsong researchers, along with both CPP and WE, will allow characterization of similarities and differences among birds. Standard terminology and measures will improve accessibility of the birdsong literature to human voice researchers and vice versa. Supplemental Material https://doi.org/10.23641/asha.7438964.

Reduced vocal variability in a zebra finch model of dopamine depletion: implications for Parkinson disease.

Midbrain dopamine (DA) modulates the activity of basal ganglia circuitry important for motor control in a variety of species. In songbirds, DA underlies motivational behavior including reproductive drive and is implicated as a gatekeeper for neural activity governing vocal variability. In the zebra finch, Taeniopygia guttata, DA levels increase in Area X, a song-dedicated subregion of the basal ganglia, when a male bird sings his courtship song to a female (female-directed; FD). Levels remain stable when he sings a less stereotyped version that is not directed toward a conspecific (undirected; UD). Here, we used a mild dose of the neurotoxin 6-hydroxydopamine (6-OHDA) to reduce presynaptic DA input to Area X and characterized the effects on FD and UD behaviors. Immunoblots were used to quantify levels of tyrosine hydroxylase (TH) as a biomarker for DA afferent loss in vehicle- and 6-OHDA-injected birds. Following 6-OHDA administration, TH signals were lower in Area X but not in an adjacent subregion, ventral striatal-pallidum (VSP). A postsynaptic marker of DA signaling was unchanged in both regions. These observations suggest that effects were specific to presynaptic afferents of vocal basal ganglia. Concurrently, vocal variability was reduced during UD but not FD song. Similar decreases in vocal variability are observed in patients with Parkinson disease (PD), but the link to DA loss is not well-understood. The 6-OHDA songbird model offers a unique opportunity to further examine how DA loss in cortico-basal ganglia pathways affects vocal control.

Vocalization deficits in mice over-expressing alpha-synuclein, a model of pre-manifest Parkinson's disease.

Communication and swallowing deficits are common in Parkinson's disease (PD). Evidence indicates that voice and speech dysfunction manifest early, prior to motor deficits typically associated with striatal dopamine loss. Unlike deficits in the extremities, cranial sensorimotor deficits are refractory to standard dopamine-related pharmacological and surgical interventions, thus the mechanisms underlying vocal deficits are unclear. Although neurotoxin models have provided some insight, they typically model nigrostriatal dopamine depletion and are therefore limited. Widespread alpha-synuclein (aSyn) pathology is common to familial and sporadic PD, and transgenic mouse models based on aSyn overexpression present a unique opportunity to explore vocalization deficits in relation to extrastriatal, nondopaminergic pathologies. Specifically, mice overexpressing human wild-type aSyn under a broad neuronal promoter (Thy1-aSyn) present early, progressive motor and nonmotor deficits starting at 2-3 months, followed by parkinsonism with dopamine loss at 14 months. We recorded ultrasonic vocalizations from Thy1-aSyn mice and wild-type (WT) controls at 2-3, 6-7, and 9 months. Thy1-aSyn mice demonstrated early, progressive vocalization deficits compared with WT. Duration and intensity of calls were significantly reduced and call profile was altered in the Thy1-aSyn mice, particularly at 2-3 months. Call rate trended toward a more drastic decrease with age in the Thy1-aSyn mice compared with WT. Alpha-synuclein pathology is present in the periaqueductal gray and may underlie the manifestation of vocalization deficits. These results indicate that aSyn overexpression can induce vocalization deficits at an early age in mice and provides a new model for studying the mechanisms underlying cranial sensorimotor deficits and treatment interventions for PD.

Molecular microcircuitry underlies functional specification in a basal ganglia circuit dedicated to vocal learning.

Similarities between speech and birdsong make songbirds advantageous for investigating the neurogenetics of learned vocal communication--a complex phenotype probably supported by ensembles of interacting genes in cortico-basal ganglia pathways of both species. To date, only FoxP2 has been identified as critical to both speech and birdsong. We performed weighted gene coexpression network analysis on microarray data from singing zebra finches to discover gene ensembles regulated during vocal behavior. We found ∼2,000 singing-regulated genes comprising three coexpression groups unique to area X, the basal ganglia subregion dedicated to learned vocalizations. These contained known targets of human FOXP2 and potential avian targets. We validated biological pathways not previously implicated in vocalization. Higher-order gene coexpression patterns, rather than expression levels, molecularly distinguish area X from the ventral striato-pallidum during singing. The previously unknown structure of singing-driven networks enables prioritization of molecular interactors that probably bear on human motor disorders, especially those affecting speech.

Song practice promotes acute vocal variability at a key stage of sensorimotor learning.

BACKGROUND: Trial by trial variability during motor learning is a feature encoded by the basal ganglia of both humans and songbirds, and is important for reinforcement of optimal motor patterns, including those that produce speech and birdsong. Given the many parallels between these behaviors, songbirds provide a useful model to investigate neural mechanisms underlying vocal learning. In juvenile and adult male zebra finches, endogenous levels of FoxP2, a molecule critical for language, decrease two hours after morning song onset within area X, part of the basal ganglia-forebrain pathway dedicated to song. In juveniles, experimental 'knockdown' of area X FoxP2 results in abnormally variable song in adulthood. These findings motivated our hypothesis that low FoxP2 levels increase vocal variability, enabling vocal motor exploration in normal birds. METHODOLOGY/PRINCIPAL FINDINGS: After two hours in either singing or non-singing conditions (previously shown to produce differential area X FoxP2 levels), phonological and sequential features of the subsequent songs were compared across conditions in the same bird. In line with our prediction, analysis of songs sung by 75 day (75d) birds revealed that syllable structure was more variable and sequence stereotypy was reduced following two hours of continuous practice compared to these features following two hours of non-singing. Similar trends in song were observed in these birds at 65d, despite higher overall within-condition variability at this age. CONCLUSIONS/SIGNIFICANCE: Together with previous work, these findings point to the importance of behaviorally-driven acute periods during song learning that allow for both refinement and reinforcement of motor patterns. Future work is aimed at testing the observation that not only does vocal practice influence expression of molecular networks, but that these networks then influence subsequent variability in these skills.

Birdsong decreases protein levels of FoxP2, a molecule required for human speech.

Cognitive and motor deficits associated with language and speech are seen in humans harboring FOXP2 mutations. The neural bases for FOXP2 mutation-related deficits are thought to reside in structural abnormalities distributed across systems important for language and motor learning including the cerebral cortex, basal ganglia, and cerebellum. In these brain regions, our prior research showed that FoxP2 mRNA expression patterns are strikingly similar between developing humans and songbirds. Within the songbird brain, this pattern persists throughout life and includes the striatal subregion, Area X, that is dedicated to song development and maintenance. The persistent mRNA expression suggests a role for FoxP2 that extends beyond the formation of vocal learning circuits to their ongoing use. Because FoxP2 is a transcription factor, a role in shaping circuits likely depends on FoxP2 protein levels which might not always parallel mRNA levels. Indeed our current study shows that FoxP2 protein, like its mRNA, is acutely downregulated in mature Area X when adult males sing with some differences. Total corticosterone levels associated with the different behavioral contexts did not vary, indicating that differences in FoxP2 levels are not likely attributable to stress. Our data, together with recent reports on FoxP2's target genes, suggest that lowered FoxP2 levels may allow for expression of genes important for circuit modification and thus vocal variability.

Steroid hormone activation of wandering in the isolated nervous system of Manduca sexta.

Steroid hormones modulate motor circuits in both vertebrates and invertebrates. The insect Manduca sexta, with its well-characterized developmental and endocrinological history, is a useful model system in which to study these effects. Wandering is a stage-specific locomotor behavior triggered by the steroid hormone 20-hydroxyecdysone (20E), consisting of crawling and burrowing movements as the animal searches for a pupation site. This study was undertaken to determine whether the wandering motor pattern is activated by direct action of 20E on the CNS. 20E acts on the isolated larval nervous system to induce a fictive motor pattern showing features of crawling and burrowing. The latency of the response to 20E is long, suggestive of a genomic mechanism of action. The abdominal motoneurons or segmental pattern generating circuits are unlikely to be the primary targets of 20E action in inducing fictive wandering. Exposure of the segmental ganglia alone to hormone did not evoke fictive wandering. Therefore, as suggested by an earlier study, the likely site of 20E action is within the brain.