Genomic loss of GPR108 disrupts AAV transduction in birds.

The G protein-coupled receptor 108 (GPR108) gene encodes a protein factor identified as critical for adeno-associated virus (AAV) entry into mammalian cells, but whether it is universally involved in AAV transduction is unknown. Remarkably, we have discovered that GPR108 is absent in the genomes of birds and in most other sauropsids, providing a likely explanation for the overall lower AAV transduction efficacy of common AAV serotypes in birds compared to mammals. Importantly, transgenic expression of human GPR108 and manipulation of related glycan binding sites in the viral capsid significantly boost AAV transduction in zebra finch cells. These findings contribute to a more in depth understanding of the mechanisms and evolution of AAV transduction, with potential implications for the design of efficient tools for gene manipulation in experimental animal models, and a range of gene therapy applications in humans.

Cell type specializations of the vocal-motor cortex in songbirds.

Identifying molecular specializations in cortical circuitry supporting complex behaviors, like learned vocalizations, requires understanding of the neuroanatomical context from which these circuits arise. In songbirds, the robust arcopallial nucleus (RA) provides descending cortical projections for fine vocal-motor control. Using single-nuclei transcriptomics and spatial gene expression mapping in zebra finches, we have defined cell types and molecular specializations that distinguish RA from adjacent regions involved in non-vocal motor and sensory processing. We describe an RA-specific projection neuron, differential inhibitory subtypes, and glia specializations and have probed predicted GABAergic interneuron subtypes electrophysiologically within RA. Several cell-specific markers arise developmentally in a sex-dependent manner. Our interactive apps integrate cellular data with developmental and spatial distribution data from the gene expression brain atlas ZEBrA. Users can explore molecular specializations of vocal-motor neurons and support cells that likely reflect adaptations key to the physiology and evolution of vocal control circuits and refined motor skills.

Bats possess the anatomical substrate for a laryngeal motor cortex.

Cortical neurons that make direct connections to motor neurons in the brainstem and spinal cord are specialized for fine motor control and learning [1, 2]. Imitative vocal learning, the basis for human speech, requires the precise control of the larynx muscles [3]. While much knowledge on vocal learning systems has been gained from studying songbirds [4], an accessible laboratory model for mammalian vocal learning is highly desirable. Evidence indicative of complex vocal repertoires and dialects suggests that bats are vocal learners [5, 6], however the circuitry that underlies vocal control and learning in bats is largely unknown. A key feature of vocal learning animals is a direct cortical projection to the brainstem motor neurons that innervate the vocal organ [7]. A recent study [8] described a direct connection from the primary motor cortex to medullary nucleus ambiguus in the Egyptian fruit bat (Rousettus aegyptiacus). Here we show that a distantly related bat, Seba's short-tailed bat (Carollia perspicillata) also possesses a direct projection from the primary motor cortex to nucleus ambiguus. Our results, in combination with Wirthlin et al. [8], suggest that multiple bat lineages possess the anatomical substrate for cortical control of vocal output. We propose that bats would be an informative mammalian model for vocal learning studies to better understand the genetics and circuitry involved in human vocal communication.

Recovery of a learned behavior despite partial restoration of neuronal dynamics after chronic inactivation of inhibitory neurons.

Maintaining motor skills is crucial for an animal's survival, enabling it to endure diverse perturbations throughout its lifespan, such as trauma, disease, and aging. What mechanisms orchestrate brain circuit reorganization and recovery to preserve the stability of behavior despite the continued presence of a disturbance? To investigate this question, we chronically silenced a fraction of inhibitory neurons in a brain circuit necessary for singing in zebra finches. Song in zebra finches is a complex, learned motor behavior and central to reproduction. This manipulation altered brain activity and severely perturbed song for around two months, after which time it was precisely restored. Electrophysiology recordings revealed abnormal offline dynamics, resulting from chronic inhibition loss, some aspects of which returned to normal as the song recovered. However, even after the song had fully recovered, the levels of neuronal firing in the premotor and motor areas did not return to a control-like state. Single-cell RNA sequencing revealed that chronic silencing of interneurons led to elevated levels of microglia and MHC I, which were also observed in normal juveniles during song learning. These experiments demonstrate that the adult brain can overcome extended periods of abnormal activity, and precisely restore a complex behavior, without recovering normal neuronal dynamics. These findings suggest that the successful functional recovery of a brain circuit after a perturbation can involve more than mere restoration to its initial configuration. Instead, the circuit seems to adapt and reorganize into a new state capable of producing the original behavior despite the persistence of some abnormal neuronal dynamics.

Motor cortex analogue neurons in songbirds utilize Kv3 channels to generate ultranarrow spikes.

Complex motor skills in vertebrates require specialized upper motor neurons with precise action potential (AP) firing. To examine how diverse populations of upper motor neurons subserve distinct functions and the specific repertoire of ion channels involved, we conducted a thorough study of the excitability of upper motor neurons controlling somatic motor function in the zebra finch. We found that robustus arcopallialis projection neurons (RAPNs), key command neurons for song production, exhibit ultranarrow spikes and higher firing rates compared to neurons controlling non-vocal somatic motor functions (dorsal intermediate arcopallium [AId] neurons). Pharmacological and molecular data indicate that this striking difference is associated with the higher expression in RAPNs of high threshold, fast-activating voltage-gated Kv3 channels, that likely contain Kv3.1 (KCNC1) subunits. The spike waveform and Kv3.1 expression in RAPNs mirror properties of Betz cells, specialized upper motor neurons involved in fine digit control in humans and other primates but absent in rodents. Our study thus provides evidence that songbirds and primates have convergently evolved the use of Kv3.1 to ensure precise, rapid AP firing in upper motor neurons controlling fast and complex motor skills.

Emergence of sex-specific transcriptomes in a sexually dimorphic brain nucleus.

We present the transcriptomic changes underlying the development of an extreme neuroanatomical sex difference. The robust nucleus of the arcopallium (RA) is a key component of the songbird vocal motor system. In zebra finch, the RA is initially monomorphic and then atrophies in females but grows up to 7-fold larger in males. Mirroring this divergence, we show here that sex-differential gene expression in the RA expands from hundreds of predominantly sex chromosome Z genes in early development to thousands of predominantly autosomal genes by the time sexual dimorphism asymptotes. Male-specific developmental processes include cell and axonal growth, synapse assembly and activity, and energy metabolism; female-specific processes include cell polarity and differentiation, transcriptional repression, and steroid hormone and immune signaling. Transcription factor binding site analyses support female-biased activation of pro-apoptotic regulatory networks. The extensive and sex-specific transcriptomic reorganization of RA provides insights into potential drivers of sexually dimorphic neurodevelopment.

Resurgent Na+ currents promote ultrafast spiking in projection neurons that drive fine motor control.

The underlying mechanisms that promote precise spiking in upper motor neurons controlling fine motor skills are not well understood. Here we report that projection neurons in the adult zebra finch song nucleus RA display robust high-frequency firing, ultra-narrow spike waveforms, superfast Na+ current inactivation kinetics, and large resurgent Na+ currents (INaR). These properties of songbird pallial motor neurons closely resemble those of specialized large pyramidal neurons in mammalian primary motor cortex. They emerge during the early phases of song development in males, but not females, coinciding with a complete switch of Na+ channel subunit expression from Navß3 to Navß4. Dynamic clamping and dialysis of Navß4's C-terminal peptide into juvenile RA neurons provide evidence that Navß4, and its associated INaR, promote neuronal excitability. We thus propose that INaR modulates the excitability of upper motor neurons that are required for the execution of fine motor skills.

Molecular specializations of deep cortical layer analogs in songbirds.

How the evolution of complex behavioral traits is associated with the emergence of novel brain pathways is largely unknown. Songbirds, like humans, learn vocalizations via tutor imitation and possess a specialized brain circuitry to support this behavior. In a comprehensive in situ hybridization effort, we show that the zebra finch vocal robust nucleus of the arcopallium (RA) shares numerous markers (e.g. SNCA, PVALB) with the adjacent dorsal intermediate arcopallium (AId), an avian analog of mammalian deep cortical layers with involvement in motor function. We also identify markers truly unique to RA and thus likely linked to modulation of vocal motor function (e.g. KCNC1, GABRE), including a subset of the known shared markers between RA and human laryngeal motor cortex (e.g. SLIT1, RTN4R, LINGO1, PLXNC1). The data provide novel insights into molecular features unique to vocal learning circuits, and lend support for the motor theory for vocal learning origin.

Serotonin modulates response properties of neurons in the dorsal cochlear nucleus of the mouse.

The neurochemical serotonin (5-hydroxytryptamine, 5-HT) is involved in a variety of behavioral functions including arousal, reward, and attention, and has a role in several complex disorders of the brain. In the auditory system, 5-HT fibers innervate a number of subcortical nuclei, yet the modulatory role of 5-HT in nearly all of these areas remains poorly understood. In this study, we examined spiking activity of neurons in the dorsal cochlear nucleus (DCN) following iontophoretic application of 5-HT. The DCN is an early site in the auditory pathway that receives dense 5-HT fiber input from the raphe nuclei and has been implicated in the generation of auditory disorders marked by neuronal hyperexcitability. Recordings from the DCN in awake mice demonstrated that iontophoretic application of 5-HT had heterogeneous effects on spiking rate, spike timing, and evoked spiking threshold. We found that 56% of neurons exhibited increases in spiking rate during 5-HT delivery, while 22% had decreases in rate and the remaining neurons had no change. These changes were similar for spontaneous and evoked spiking and were typically accompanied by changes in spike timing. Spiking increases were associated with lower first spike latencies and jitter, while decreases in spiking generally had opposing effects on spike timing. Cases in which 5-HT application resulted in increased spiking also exhibited lower thresholds compared to the control condition, while cases of decreased spiking had no threshold change. We also found that the 5-HT2 receptor subtype likely has a role in mediating increased excitability. Our results demonstrate that 5-HT can modulate activity in the DCN of awake animals and that it primarily acts to increase neuronal excitability, in contrast to other auditory regions where it largely has a suppressive role. Modulation of DCN function by 5-HT has implications for auditory processing in both normal hearing and disordered states.

Contrasting patterns of adaptive sequence convergence among echolocating mammals.

Several recent studies have described genes demonstrating adaptive sequence convergence between echolocating bats and dolphin, suggesting that common selective pressures can induce common molecular changes, even in distantly related species. However, in the case of the auditory genes Otoferlin (Otof), Cadherin 23 (Cdh23) and Protocadherin 15 (Pcdh15), the reported sequence convergence was supported only by incongruent gene and species trees and counts of convergent substitutions. Therefore, it remains unclear whether echolocating bats and dolphin really do demonstrate evidence of adaptive sequence convergence, or whether there is simply a high level of random background convergence in these genes. To address this question, we estimated the number of convergent and divergent amino acid substitutions along all independent branches of a sufficiently deep phylogeny containing between 22 and 32 mammals for each gene, and compared convergence between the two proposed suborders of bat, Yangochiroptera and Yinpterochiroptera, and dolphin. We find no support for convergence between bats and dolphin in the gene Pcdh15. For the gene Otof we report minimal evidence for convergent evolution only between the Yinpterochiroptera and dolphin. Cdh23 displayed a high level of convergence between dolphin and the Yinpterochiroptera. In addition, dolphin and certain members of the Yangochiroptera that emit high frequency echolocation calls shared several unique convergent substitutions. These results indicate that the convergent evolution of Cdh23 was likely driven by selection for hearing above a certain frequency threshold. Moreover, the contrasting patterns of convergence between the two bat suborders and dolphin in all auditory genes studied thus far suggest echolocation may have evolved independently in the Yinpterochiroptera and Yangochiroptera.

Dopaminergic projections of the subparafascicular thalamic nucleus to the auditory brainstem.

Neuromodulators can alter the response properties of sensory neurons, including those in the auditory system. Dopamine, which plays a major role in reward and movement, has been shown to alter neural responses in the auditory brainstem and midbrain. Recently we identified the subparafascicular thalamic nucleus (SPF), part of the A11 dopaminergic cell group, as the source of dopamine to the inferior colliculus (IC). The superior olivary complex (SOC) is also a likely target of dopaminergic projections from the SPF because it receives projections from the SPF and contains fibers and terminals immunoreactive for tyrosine hydroxylase, the rate limiting enzyme in dopamine synthesis. However, it is unknown if the projections from the SPF to SOC are dopaminergic, and if single neurons in the SPF project to both the IC and SOC. Using anterograde tracing combined with fluorescent immunohistochemistry, we found that the SPF sends dopaminergic projections to the superior paraolivary nucleus and the medial nucleus of the trapezoid body, but not the lateral superior olive. We confirmed these projections using a retrograde tracer. By making dual retrograde deposits in the IC and SOC, we found that individual dopaminergic cells innervate both the IC and SOC. These results suggest dopaminergic innervation, likely released in a context dependent manner, occurs at multiple levels of the auditory pathway.

Dopaminergic Input to the Inferior Colliculus in Mice.

The response of sensory neurons to stimuli can be modulated by a variety of factors including attention, emotion, behavioral context, and disorders involving neuromodulatory systems. For example, patients with Parkinson's disease (PD) have disordered speech processing, suggesting that dopamine alters normal representation of these salient sounds. Understanding the mechanisms by which dopamine modulates auditory processing is thus an important goal. The principal auditory midbrain nucleus, the inferior colliculus (IC), is a likely location for dopaminergic modulation of auditory processing because it contains dopamine receptors and nerve terminals immunoreactive for tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. However, the sources of dopaminergic input to the IC are unknown. In this study, we iontophoretically injected a retrograde tracer into the IC of mice and then stained the tissue for TH. We also immunostained for dopamine beta-hydroxylase (DBH), an enzyme critical for the conversion of dopamine to norepinephrine, to differentiate between dopaminergic and noradrenergic inputs. Retrogradely labeled neurons that were positive for TH were seen bilaterally, with strong ipsilateral dominance, in the subparafascicular thalamic nucleus (SPF). All retrogradely labeled neurons that we observed in other brain regions were TH-negative. Projections from the SPF were confirmed using an anterograde tracer, revealing TH-positive and DBH-negative anterogradely labeled fibers and terminals in the IC. While the functional role of this dopaminergic input to the IC is not yet known, it provides a potential mechanism for context dependent modulation of auditory processing.