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.

Variation in sequence dynamics improves maintenance of stereotyped behavior in an example from bird song.

Performing a stereotyped behavior successfully over time requires both maintaining performance quality and adapting efficiently to environmental or physical changes affecting performance. The bird song system is a paradigmatic example of learning a stereotyped behavior and therefore is a good place to study the interaction of these two goals. Through a model of bird song learning, we show how instability in neural representation of stable behavior confers advantages for adaptation and maintenance with minimal cost to performance quality. A precise, temporally sparse sequence from the premotor nucleus HVC is crucial to the performance of song in songbirds. We find that learning in the presence of sequence variations facilitates rapid relearning after shifts in the target song or muscle structure and results in decreased error with neuron loss. This robustness is due to the prevention of the buildup of correlations in the learned connectivity. In the absence of sequence variations, these correlations grow, due to the relatively low dimensionality of the exploratory variation in comparison with the number of plastic synapses. Our results suggest one would expect to see variability in neural systems executing stereotyped behaviors, and this variability is an advantageous feature rather than a challenge to overcome.

Brain-Derived Neurotrophic Factor Has a Transsynaptic Trophic Effect on Neural Activity in an Adult Forebrain Circuit.

While hormone-driven plasticity in the adult brain is well studied, the underlying cellular and molecular mechanisms are less well understood. One example of this is seasonal plasticity in the avian brain, where song nuclei exhibit hormonally driven changes in response to changing photoperiod and circulating sex steroid hormones. Hormone receptor activation in song nucleus HVC (proper name) elicits a robust change in activity in target nucleus RA (robust nucleus of the arcopallium), but the molecular signal responsible for this is unknown. This study addressed whether brain-derived neurotrophic factor (BDNF) mediates a transsynaptic effect from HVC to RA in male Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii). In situ hybridization confirmed an increase in BDNF expression in HVC neurons of birds switched to a long-day (LD) photoperiod plus systemically elevated testosterone (T) levels, compared with short-day (SD) conditions. BDNF expression was virtually absent in RA neurons of SD birds, increasing to barely detectable levels in a small subset of cells in LD+T birds. Infusion of BDNF protein adjacent to the RA of SD birds caused an increase in the spontaneous neuron firing rate. Conversely, the infusion of ANA12, a specific antagonist of the tyrosine-related kinase B (TrkB) for BDNF, prevented the increase in RA neuron firing rate in LD+T birds. These results indicate that BDNF is sufficient, and TrkB receptor activation is necessary, for the transsynaptic trophic effect exerted by HVC on RA. The dramatic change in the activity of RA neurons during the breeding season provides a clear example of transsynaptic BDNF effects in the adult brain in a functionally relevant circuit.SIGNIFICANCE STATEMENT Sex steroid hormones drive changes in brain circuits in all vertebrates, both within specific neurons and on their synaptic targets. Such changes can lead to profound changes in behavior, but little is known about the precise molecular mechanisms that underlie this process. We addressed this question in a seasonally breeding songbird and found that the trophic effects of one forebrain song nucleus on its target are mediated transsynaptically by the neurotrophin BDNF. This suggests that, in addition to their role in development, neurotrophins have critical roles in adult brain plasticity.

Dopaminergic modulation of basal ganglia output through coupled excitation-inhibition.

Learning and maintenance of skilled movements require exploration of motor space and selection of appropriate actions. Vocal learning and social context-dependent plasticity in songbirds depend on a basal ganglia circuit, which actively generates vocal variability. Dopamine in the basal ganglia reduces trial-to-trial neural variability when the bird engages in courtship song. Here, we present evidence for a unique, tonically active, excitatory interneuron in the songbird basal ganglia that makes strong synaptic connections onto output pallidal neurons, often linked in time with inhibitory events. Dopamine receptor activity modulates the coupling of these excitatory and inhibitory events in vitro, which results in a dynamic change in the synchrony of a modeled population of basal ganglia output neurons receiving excitatory and inhibitory inputs. The excitatory interneuron thus serves as one biophysical mechanism for the introduction or modulation of neural variability in this circuit.

Postsynaptic neural activity regulates neuronal addition in the adult avian song control system.

A striking feature of the nervous system is that it shows extensive plasticity of structure and function that allows animals to adjust to changes in their environment. Neural activity plays a key role in mediating experience-dependent neural plasticity and, thus, creates a link between the external environment, the nervous system, and behavior. One dramatic example of neural plasticity is ongoing neurogenesis in the adult brain. The role of neural activity in modulating neuronal addition, however, has not been well studied at the level of neural circuits. The avian song control system allows us to investigate how activity influences neuronal addition to a neural circuit that regulates song, a learned sensorimotor social behavior. In adult white-crowned sparrows, new neurons are added continually to the song nucleus HVC (proper name) and project their axons to its target nucleus, the robust nucleus of the arcopallium (RA). We report here that electrical activity in RA regulates neuronal addition to HVC. Decreasing neural activity in RA by intracerebral infusion of the GABAA receptor agonist muscimol decreased the number of new HVC neurons by 56%. Our results suggest that postsynaptic electrical activity influences the addition of new neurons into a functional neural circuit in adult birds.

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.

HTR2 receptors in a songbird premotor cortical-like area modulate spectral characteristics of zebra finch song.

Serotonin [5-hydroxytryptamine (5-HT)] is involved in modulating an array of complex behaviors including learning, depression, and circadian rhythms. Additionally, HTR2 receptors on layer V pyramidal neurons are thought to mediate the actions of psychedelic drugs; the native function of these receptors at this site, however, remains unknown. Previously, we found that activation of HTR2 receptors in the zebra finch forebrain song premotor structure the robust nucleus of the arcopallium (RA) led to increased excitation, and that endogenous 5-HT could roughly double spontaneous firing rate. Here, using in vivo single-unit recordings, we found that direct application of 5-HT to these same RA projection neurons, which are analogous to layer V cortical pyramidal neurons, caused a significant increase in the number of action potentials per song-related burst, and a dramatic decrease in signal-to-noise ratio. Injection of the serotonergic neurotoxin 5,7-dihydroxytryptamine into the third ventricle greatly reduced telencephalic 5-HT and resulted in decreased fundamental frequency of harmonic syllables as well as increased goodness of pitch. Both of these results can be explained by the observed actions of 5-HT on RA projection neurons, and both effects recovered to baseline within 2 weeks following the toxin injection. These results show that 5-HT is involved in modulating spectral properties of song, likely via effects on RA projection neurons, but that adult zebra finches can partially compensate for this deficit within 7 d.

Engineered deafness reveals that mouse courtship vocalizations do not require auditory experience.

Auditory experience during development is necessary for normal language acquisition in humans. Although songbirds, some cetaceans, and maybe bats may also be vocal learners, vocal learning has yet to be well established for a laboratory mammal. Mice are potentially an excellent model organism for studying mechanisms underlying vocal communication. Mice vocalize in different social contexts, yet whether they learn their vocalizations remains unresolved. To address this question, we compared ultrasonic courtship vocalizations emitted by chronically deaf and normal hearing adult male mice. We deafened CBA/CaJ male mice, engineered to express diphtheria toxin (DT) receptors in hair cells, by systemic injection of DT at postnatal day 2 (P2). By P9, almost all inner hair cells were absent and by P16 all inner and outer hair cells were absent in DTR mice. These mice did not show any auditory brainstem responses as adults. Wild-type littermates, also treated with DT at P2, had normal hair cells and normal auditory brainstem responses. We compared the temporal structure of vocalization bouts, the types of vocalizations, the patterns of syllables, and the acoustic features of each syllable type emitted by hearing and deaf males in the presence of a female. We found that almost all of the vocalization features we examined were similar in hearing and deaf animals. These findings indicate that mice do not need auditory experience during development to produce normal ultrasonic vocalizations in adulthood. We conclude that mouse courtship vocalizations are not acquired through auditory feedback-dependent learning.

Premotor synaptic plasticity limited to the critical period for song learning.

Synaptic plasticity has been hypothesized to underlie learning and memory. Understanding of how such plasticity might produce motor learning is limited, in part because of the paucity of model systems with a tractable learned behavior under control of a discrete neural circuit. Songbirds possess both of these traits, thereby providing an excellent model for studying vertebrate motor learning. We report unique evidence of long-term depression (LTD) in the juvenile songbird premotor robust nucleus of the arcopallium (RA). LTD induction at RA recurrent collateral synapses requires NMDA receptors, postsynaptic depolarization, and postsynaptic calcium, and can be reversed by high-frequency stimulation. In adult birds, which have exited the critical period for sensorimotor learning and cannot modify their song, we were no longer able to induce LTD at RA collateral synapses. Furthermore, testosterone-induced premature maturation of song in juveniles abolishes LTD. LTD in nucleus RA therefore makes an excellent candidate mechanism to mediate song learning during development and is well-suited to provide insight into other forms of vertebrate motor learning.

Dynamics of odor-source localization: Insights from real-time odor plume recordings and head-motion tracking in freely moving mice.

Animals navigating turbulent odor plumes exhibit a rich variety of behaviors, and employ efficient strategies to locate odor sources. A growing body of literature has started to probe this complex task of localizing airborne odor sources in walking mammals to further our understanding of neural encoding and decoding of naturalistic sensory stimuli. However, correlating the intermittent olfactory information with behavior has remained a long-standing challenge due to the stochastic nature of the odor stimulus. We recently reported a method to record real-time olfactory information available to freely moving mice during odor-guided navigation, hence overcoming that challenge. Here we combine our odor-recording method with head-motion tracking to establish correlations between plume encounters and head movements. We show that mice exhibit robust head-pitch motions in the 5-14Hz range during an odor-guided navigation task, and that these head motions are modulated by plume encounters. Furthermore, mice reduce their angles with respect to the source upon plume contact. Head motions may thus be an important part of the sensorimotor behavioral repertoire during naturalistic odor-source localization.