Julie Elizabeth Miller

So, L.Y, Munger, S.J. and Miller, J.E. (2019) Social Context-Dependent Singing Alters Molecular Markers of Dopaminergic and Glutamatergic Signaling in Finch Basal Ganglia Area X. Behav Brain Res. 360:103-112 doi: 10.1016/j.bbr.2018.12.004. PMID: 30521933

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.

Miller, J.E., Hafzalla, G.W., Burkett, Z.D, Fox, C.M. and White, S.A. (2015) Reduced Vocal Variability in a Zebra Finch Model of Dopamine Depletion: Implications for Parkinson disease. Physiol Rep, 3 (11), 2015, e12599, doi: 10.14814/phy2.12599. PMID: 26564062  

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.

*Hilliard, A., *Miller, J., Fraley, E., Horvath, S., & White, S. (2012). Molecular Microcircuitry Underlies Functional Specification in a Basal Ganglia Circuit Dedicated to Vocal Learning. Neuron, 73, 537-552.

*Equal authorship

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.

*Grant, L. M., *Richter, F., Miller, J., White, S., Fox, C., Zhu, C., Chesselet, M., & Ciucci, M. (2014). Vocalization deficits in mice over-expressing alpha-synuclein, a model of pre-manifest Parkinson's disease.. Behav Neurosci, 128, 110-121.

*Equal authorship

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.

Hilliard, A. T., Miller, J., Horvath, S., & White, S. (2012). Distinct Neurogenomic States in Basal Ganglia Subregions Relate Differently to Singing Behavior in Songbirds. PloS Comput Biol, 8(11), e1002773.

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.