Estradiol decreases the excitability of RA projection neurons in adult male zebra finches.

Zebra finches are essential animal models for studying learned vocal signals. The robust nucleus of the arcopallium (RA) plays an important role in regulating singing behavior. Our previous study showed that castration inhibited the electrophysiological activity of RA projection neurons (PNs) in male zebra finches, demonstrating that testosterone modulates the excitability of RA PNs. Testosterone can be converted into estradiol (E2) in the brain through aromatase; however, the physiological functions of E2 in RA are still unknown. This study aimed to investigate the electrophysiological activities of E2 on the RA PNs of male zebra finches through patch-clamp recording. E2 rapidly decreased the rate of evoked and spontaneous action potentials (APs) of RA PNs, hyperpolarized the resting membrane potential, and decreased the membrane input resistance. Moreover, the G-protein-coupled membrane-bound estrogen receptor (GPER) agonist G1 decreased both the evoked and spontaneous APs of RA PNs. Furthermore, the GPER antagonist G15 had no effect on the evoked and spontaneous APs of RA PNs; E2 and G15 together also had no effect on the evoked and spontaneous APs of RA PNs. These findings suggested that E2 rapidly decreased the excitability of RA PNs and its binding to GPER suppressed the excitability of RA PNs. These pieces of evidence helped us fully understand the principle of E2 signal mediation via its receptors to modulate the excitability of RA PNs in songbirds.

BDNF enhances electrophysiological activity and excitatory synaptic transmission of RA projection neurons in adult male zebra finches.

The singing of songbirds is a complex vocal behavior. It was reported that brain-derived neurotrophic factor (BDNF), a key neurotrophic factor involved in neuronal survival and activity, plays an important role in regulation of songbirds' song behavior. In all song-related nuclei, the electrophysiological activity of robust nucleus of the arcopallium (RA) in the forebrain of songbirds is directly related to birdsong output. Whether BDNF regulates the electrophysiological activity and synaptic transmission of RA causing the change of song behavior need be further explored. In this study, the effects of BDNF on the electrophysiological activity and excitatory synaptic transmission of RA projection neurons (PNs) in adult male zebra finches were investigated using whole-cell patch clamp recordings in vitro. Our results showed that BDNF increased the firing of evoked action potentials in RA PNs and decreased the membrane input resistance and membrane time constant of RA PNs, indicating that BDNF can promote RA PNs excitability by reducing membrane input resistance and membrane time constant. Meanwhile, BDNF increased the frequency rather than amplitude of miniature excitatory postsynaptic currents (mEPSCs) in RA PNs. Moreover, the effects of BDNF on the excitability, intrinsic membrane properties and mEPSCs of RA PNs were blocked by its receptor TrkB antagonist K252a. These results indicate that BDNF via TrkB enhances the excitability and excitatory synaptic transmission of RA PNs in adult male songbirds through presynaptic mechanisms, suggesting a possible cellular mechanism by which BDNF regulates song behavior.

Sex differences and similarities in the neural circuit regulating song and other reproductive behaviors in songbirds.

In the 1970s, Nottebohm and Arnold reported marked male-biased sex differences in the volume of three song control nuclei in songbirds. Subsequently a series of studies on several songbird species suggested that there is a positive correlation between the degree to which there is a sex difference in the volume of these song control nuclei and in song behavior. This correlation has been questioned in recent years. Furthermore, it has become clear that the song circuit is fully integrated into a more comprehensive neural circuit that regulates multiple courtship and reproductive behaviors including song. Sex differences in songbirds should be evaluated in the context of the full complement of behaviors produced by both sexes in relation to reproduction and based on the entire circuit in order to understand the functional significance of variation between males and females in brain and behavior. Variation in brain and behavior exhibited among living songbird species provides an excellent opportunity to understand the functional significance of sex differences related to social behaviors.

Dopamine Modulates Excitatory Synaptic Transmission by Activating Presynaptic D1-like Dopamine Receptors in the RA Projection Neurons of Zebra Finches.

Songbirds are useful vertebrate study models for vocal learning and memory. The robust nucleus of the arcopallium (RA) receives synaptic inputs from both the posterior and anterior pathways of the song control system in songbirds. Hence, RA plays an important role in the control of singing. RA receives dopaminergic (DArgic) inputs that increase the excitability of RA projection neurons (PNs). However, the effects of DA on excitatory synaptic transmission are yet to be deciphered. In this study, the effects of DA on the excitatory synaptic transmission of the PNs in the RA of adult male zebra finches were investigated using a whole-cell patch-clamp recording. We observed that DA decreased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The effects of DA were mimicked by the D1-like DA receptor (D1R) agonist, SKF-38393, but not the D2-like DA receptor (D2R) agonist, Quinpirole. Also, the effects of DA were blocked by D1R antagonist, SCH-23390, but not the D2R antagonist, Sulpiride. These results demonstrate that DA modulates excitatory synaptic transmission by acting on D1R in the RA of adult male zebra finches.

Sexual dimorphism of inhibitory synaptic transmission in RA projection neurons of songbirds.

The song control system in the brain of songbirds is important for the production and acquisition of song and exhibits some of the largest neural sex differences observed in vertebrates. The robust nucleus of the arcopallium (RA) is a premotor nucleus, playing a key role in controlling singing. RA projection neurons (PNs) receives denser synapse inputs including excitatory in males than in females. However, the inhibitory synaptic transmission in the RA has not been reported. In the present study, using whole-cell voltage-clamp recording, spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) of the males and females were recorded. The average frequency and amplitude of sIPSCs/mIPSCs in males were higher than females. These results demonstrate the sexually dimorphic of the inhibitory synaptic transmission in the RA PNs and the RA PNs in males receive more inhibitory synaptic transmission. These findings contribute to further illuminate the neural mechanisms under the sexually dimorphism song production of adult zebra finches.

Activation of D1-like dopamine receptors increases the NMDA-induced gain modulation through a PKA-dependent pathway in the premotor nucleus of adult zebra finches.

Interaction between dopamine (DA) and N-methyl-d-aspartate (NMDA) in the brain plays an important role in learning and memory. In the songbirds, the premotor robust nucleus of the arcopallium (RA) receives excitatory glutamatergic inputs from the high vocal center (HVC) and lateral magnocellular nucleus of the anterior nidopallium (LMAN), as well as dopaminergic inputs mostly from the periaqueductal gray (PAG) and ventral tegmental area (VTA). In zebra finch, DA potentiates the excitability of projection neurons in the RA through activation of D1-like dopamine receptors (D1 receptors). The relationship between D1 receptors and NMDA in the RA projection neurons is essentially unknown. Our previous work showed that NMDA can induce gain modulation in the RA projection neurons. Here, using the whole-cell current-clamp recording from brain slices of male zebra finches, we observed whether D1 receptors regulate the NMDA-induced gain modulation in the RA projection neurons. Our results showed that activation of D1 receptors further increased the slope (gain) of the firing frequency-injected current (f-I) relationship induced by NMDA in the RA projection neurons. Blocking D1 receptors had no effect on the NMDA-induced gain modulation in the RA projection neurons. The enhanced effects of D1 receptors agonists were blocked by protein kinase A (PKA) inhibitors. Our results suggest that activation of D1 receptors can increase the NMDA-induced gain modulation through a PKA-dependent pathway.

Sex differences of excitatory synaptic transmission in RA projection neurons of adult zebra finches.

Zebra finches are ideal animals to investigate sex difference in songbirds. Only males can sing. The brain nuclei controlling song learning and production in males are considerably larger than in females. The robust nucleus of the arcopallium (RA) is a premotor nucleus, playing a key role in controlling singing. RA receives denser synapse inputs in males than in females. Sex differences of excitatory synaptic transmission in the RA projection neurons (PNs) have not been reported. In the present study, using whole-cell voltage-clamp recording, spontaneous EPSCs (sEPSCs) and miniature EPSCs (mEPSCs) of RA PNs in the intact males and females were recorded. The average frequency and amplitude of sEPSCs/mEPSCs in the intact males were higher than females. The half-width and decay time of sEPSCs/mEPSCs in the intact males were longer than females. In order to verify whether these sex differences related to sex steroids, males were castrated. The average frequency of sEPSCs/mEPSCs in castrated males was lower than intact males and was similar to in females; the amplitude was not changed after castrating. These results demonstrate the sexually dimorphic of the excitatory synaptic transmission in the RA PNs, the RA PNs in males receive more excitatory synaptic transmission and these sex differences were partly affected by sex hormones. These findings contribute to further illuminate the neural mechanisms under the sexually dimorphism in song production of adult zebra finches.

Castration modulates singing patterns and electrophysiological properties of RA projection neurons in adult male zebra finches.

Castration can change levels of plasma testosterone. Androgens such as testosterone play an important role in stabilizing birdsong. The robust nucleus of the arcopallium (RA) is an important premotor nucleus critical for singing. In this study, we investigated the effect of castration on singing patterns and electrophysiological properties of projection neurons (PNs) in the RA of adult male zebra finches. Adult male zebra finches were castrated and the changes in bird song assessed. We also recorded the electrophysiological changes from RA PNs using patch clamp recording. We found that the plasma levels of testosterone were significantly decreased, song syllable's entropy was increased and the similarity of motif was decreased after castration. Spontaneous and evoked firing rates, membrane time constants, and membrane capacitance of RA PNs in the castration group were lower than those of the control and the sham groups. Afterhyperpolarization AHP time to peak of spontaneous action potential (AP) was prolonged after castration.These findings suggest that castration decreases song stereotypy and excitability of RA PNs in male zebra finches.

Developmental changes in BDNF protein in the song control nuclei of zebra finches.

The zebra finch song system provides an excellent model to study the mechanisms underlying the development of sex difference in brain structure and function. Only male zebra finches sing and the brain nuclei controlling song learning and production are considerably larger than in females. Sexual differentiation may in part be regulated by estrogen, but other molecules including neurotrophic factors likely also affect masculinization. Brain derived neurotrophic factor (BDNF) plays a crucial role in numerous aspects of vertebrate brain development and function, including neurogenesis, cell survival, growth of axonal projections, synaptogenesis and processes linked to learning and memory. The current study investigated the expression of BDNF protein in juvenile males and females at four ages, as well as in adults, to begin to evaluate the potential roles of endogenous BDNF in particular stages of structural and functional development of the song system. In both HVC and the robust nucleus of the arcopallium (RA), males had more BDNF+ cells than females. The number of immunopositive cells increased in males and decreased in females as they matured, in a pattern generally consistent with a role for BDNF in sensorimotor integration of song learning. In addition, in HVC (but not RA) the ratio of mature BDNF compared to its precursor proBDNF was greater in adult males than those at post-hatching day 25, indicating a region-specific shift in the relative availability of the two forms. Collectively, the data suggest that changes in BDNF protein expression across development may be associated with song system maturation, particularly during the sensorimotor integration of masculine vocalizations.

Auditory signal processing in communication: perception and performance of vocal sounds.

Learning and maintaining the sounds we use in vocal communication require accurate perception of the sounds we hear performed by others and feedback-dependent imitation of those sounds to produce our own vocalizations. Understanding how the central nervous system integrates auditory and vocal-motor information to enable communication is a fundamental goal of systems neuroscience, and insights into the mechanisms of those processes will profoundly enhance clinical therapies for communication disorders. Gaining the high-resolution insight necessary to define the circuits and cellular mechanisms underlying human vocal communication is presently impractical. Songbirds are the best animal model of human speech, and this review highlights recent insights into the neural basis of auditory perception and feedback-dependent imitation in those animals. Neural correlates of song perception are present in auditory areas, and those correlates are preserved in the auditory responses of downstream neurons that are also active when the bird sings. Initial tests indicate that singing-related activity in those downstream neurons is associated with vocal-motor performance as opposed to the bird simply hearing itself sing. Therefore, action potentials related to auditory perception and action potentials related to vocal performance are co-localized in individual neurons. Conceptual models of song learning involve comparison of vocal commands and the associated auditory feedback to compute an error signal that is used to guide refinement of subsequent song performances, yet the sites of that comparison remain unknown. Convergence of sensory and motor activity onto individual neurons points to a possible mechanism through which auditory and vocal-motor signals may be linked to enable learning and maintenance of the sounds used in vocal communication. This article is part of a Special Issue entitled "Communication Sounds and the Brain: New Directions and Perspectives".

Conserved mechanisms of vocalization coding in mammalian and songbird auditory midbrain.

The ubiquity of social vocalizations among animals provides the opportunity to identify conserved mechanisms of auditory processing that subserve communication. Identifying auditory coding properties that are shared across vocal communicators will provide insight into how human auditory processing leads to speech perception. Here, we compare auditory response properties and neural coding of social vocalizations in auditory midbrain neurons of mammalian and avian vocal communicators. The auditory midbrain is a nexus of auditory processing because it receives and integrates information from multiple parallel pathways and provides the ascending auditory input to the thalamus. The auditory midbrain is also the first region in the ascending auditory system where neurons show complex tuning properties that are correlated with the acoustics of social vocalizations. Single unit studies in mice, bats and zebra finches reveal shared principles of auditory coding including tonotopy, excitatory and inhibitory interactions that shape responses to vocal signals, nonlinear response properties that are important for auditory coding of social vocalizations and modulation tuning. Additionally, single neuron responses in the mouse and songbird midbrain are reliable, selective for specific syllables, and rely on spike timing for neural discrimination of distinct vocalizations. We propose that future research on auditory coding of vocalizations in mouse and songbird midbrain neurons adopt similar experimental and analytical approaches so that conserved principles of vocalization coding may be distinguished from those that are specialized for each species. This article is part of a Special Issue entitled "Communication Sounds and the Brain: New Directions and Perspectives".

Sexually dimorphic and developmentally regulated expression of tubulin-specific chaperone protein A in the LMAN of zebra finches.

Sex differences in brain and behavior exist across vertebrates, but the molecular factors regulating their development are largely unknown. Songbirds exhibit substantial sexual dimorphisms. In zebra finches, only males sing, and the brain areas regulating song learning and production are much larger in males. Recent data suggest that sex chromosome genes (males ZZ; females ZW) may play roles in sexual differentiation. The present studies tested the hypothesis that a Z-gene, tubulin-specific chaperone protein A (TBCA), contributes to sexual differentiation of the song system. This taxonomically conserved gene is integral to microtubule synthesis, and within the song system, its mRNA is specifically increased in males compared to females in the lateral magnocellular nucleus of the anterior nidopallium (LMAN), a region critical for song learning and plasticity. Using in situ hybridization, Western blot analysis, and immunohistochemistry, we observed effects of both age and sex on TBCA mRNA and protein expression. The transcript is increased in males compared to females at three juvenile ages, but not in adults. TBCA protein, both the number of immunoreactive cells and relative concentration in LMAN, is diminished in adults compared to juveniles. The latter was also increased in males compared to females at post-hatching day 25. With double-label immunofluorescence and retrograde tract tracing, we also document that the majority of TBCA+ cells in LMAN are neurons, and that they include robust nucleus of the arcopallium-projecting cells. These results indicate that TBCA is both temporally and spatially primed to facilitate the development of a sexually dimorphic neural pathway critical for song.

[Lateral differences in the forebrain and midbrain control of learned vocalizations in adult male Zebra Finch (Taeniopygia guttata)].

Learned vocalizations (long call and song) of adult male songbirds start from the high vocal center (HVC), and are integrated and output by the robust nucleus of the arcopallium (RA), which connects synaptic relationships with the dorsomedial nucleus of the intercollicular complex (DM). To determine the effect on learned vocalization of the unilateral forebrain and midbrain in adult male zebra finch, electrolytic lesions and acoustic analysis technology were used. The results indicated that RA and DM nuclei are involved in the control of learned vocalization, and the right side is dominant in the forebrain and midbrain.