Remodeling of Dendritic Spines in the Avian Vocal Motor Cortex Following Deafening Depends on the Basal Ganglia Circuit.

Deafening elicits a deterioration of learned vocalization, in both humans and songbirds. In songbirds, learned vocal plasticity has been shown to depend on the basal ganglia-cortical circuit, but the underlying cellular basis remains to be clarified. Using confocal imaging and electron microscopy, we examined the effect of deafening on dendritic spines in avian vocal motor cortex, the robust nucleus of the arcopallium (RA), and investigated the role of the basal ganglia circuit in motor cortex plasticity. We found rapid structural changes to RA dendritic spines in response to hearing loss, accompanied by learned song degradation. In particular, the morphological characters of RA spine synaptic contacts between 2 major pathways were altered differently. However, experimental disruption of the basal ganglia circuit, through lesions in song-specialized basal ganglia nucleus Area X, largely prevented both the observed changes to RA dendritic spines and the song deterioration after hearing loss. Our results provide cellular evidence to highlight a key role of the basal ganglia circuit in the motor cortical plasticity that underlies learned vocal plasticity.

Living high training low induces physiological cardiac hypertrophy accompanied by down-regulation and redistribution of the renin-angiotensin system.

AIM: Living high training low" (LHTL) is an exercise-training protocol that refers living in hypoxia stress and training at normal level of O2. In this study, we investigated whether LHTL caused physiological heart hypertrophy accompanied by changes of biomarkers in renin-angiotensin system in rats. METHODS: Adult male SD rats were randomly assigned into 4 groups, and trained on living low-sedentary (LLS, control), living low-training low (LLTL), living high-sedentary (LHS) and living high-training low (LHTL) protocols, respectively, for 4 weeks. Hematological parameters, hemodynamic measurement, heart hypertrophy and plasma angiotensin II (Ang II) level of the rats were measured. The gene and protein expression of angiotensin-converting enzyme (ACE), angiotensinogen (AGT) and angiotensin II receptor I (AT1) in heart tissue was assessed using RT-PCR and immunohistochemistry, respectively. RESULTS: LLTL, LHS and LHTL significantly improved cardiac function, increased hemoglobin concentration and RBC. At the molecular level, LLTL, LHS and LHTL significantly decreased the expression of ACE, AGT and AT1 genes, but increased the expression of ACE and AT1 proteins in heart tissue. Moreover, ACE and AT1 protein expression was significantly increased in the endocardium, but unchanged in the epicardium. CONCLUSION: LHTL training protocol suppresses ACE, AGT and AT1 gene expression in heart tissue, but increases ACE and AT1 protein expression specifically in the endocardium, suggesting that the physiological heart hypertrophy induced by LHTL is regulated by region-specific expression of renin-angiotensin system components.

Deafening-induced rapid changes to spine synaptic connectivity in the adult avian vocal basal ganglia.

The basal ganglia have been implicated in auditory-dependent vocal learning and plasticity in human and songbirds, but the underlying neural phenotype remains to be clarified. Here, using confocal imaging and three-dimensional electron microscopy, we investigated striatal structural plasticity in response to hearing loss in Area X, the avian vocal basal ganglia, in adult male zebra finch (Taeniopygia guttata). We observed a rapid elongation of dendritic spines, by approximately 13%, by day 3 after deafening, and a considerable increase in spine synapse density, by approximately 61%, by day 14 after deafening, compared with the controls with an intact cochlea. These findings reveal structural sensitivity of Area X to auditory deprivation and suggest that this striatal plasticity might contribute to deafening-induced changes to learned vocal behavior.

Song control nuclei in male and female large-billed crows (Corvus macrorhynchos).

We show that the learned vocalizations of male and female large-billed crows (Corvus macrorhynchos) are similar and that their functions and physical features show significant differences from those of other oscine species. We investigate whether the song control nuclei of crows show any sexual differences in size, reflecting differences in their singing behavior, and whether these nuclei are different from those of other songbirds in terms of neural connectivity size and relative to the forebrain. Our Nissl staining results reveal that 1) of the four song nuclei examined (HVC; the robust nucleus of the arcopallium [RA]; Area X; and the dorsolateral medial nucleus [DLM]), HVC, RA, and Area X volumes are significantly larger in males than in females, but DLM volume and body and brain weights show no significant gender differences; and 2) the sizes of song nuclei relative to the forebrain are within the range of other oscines. By injecting a neural tract tracer (DiI) into various song nuclei in brain slices, we found that, as in other songbirds, HVC projects to RA and Area X, while Area X projects to the lateral magnocellular nucleus of the anterior nidopallium (IMAN) and DLM, DLM to IMAN, and IMAN to RA. Our results Indicate that, although the crow has songs very different from those of other oscine species, Its song nuclei and the connections between them are not obviously different.

Neurogenic development of the visual areas in the Chinese softshell turtle (Pelodiscus sinensis) and evolutionary implications.

To characterize the neurogenic development of the visual areas of the turtle (Pelodiscus sinensis) during embryogenesis, a single dose of [(3)H]-thymidine (10 microCi) was injected into egg yolks from stages S11-12 to S21. At hatching, localization of [(3)H]-thymidine incorporation was examined, and led to three main observations. (1) Neurogenesis occurred in the stratum griseum centrale of the tectum opticum from S11-12 to S16 with a peak at S12. No obvious gradients of neurogenesis were observed. (2) Neurogenesis in the nucleus rotundus (Rot) and in the dorsal lateral geniculate nucleus (GLd) occurred from S11-12 to S15. Gradients of neurogenesis were detected along ventral-dorsal and lateral-medial axes in the Rot, but only the latter neurogenic gradient occurred in the GLd. (3) In the visual region of the dorsal ventricular ridge, neurogenesis lasted from S11-12 to S16. Similarly, neurogenesis occurred from S11-12 to S16-17 in the dorsal cortex, with a peak at S12 for both telencephalic visual regions. Neurogenesis followed a ventrolateral to dorsomedial gradient in the visual region of the dorsal ventricular ridge, and a superficial to deep gradient in the caudal dorsal cortex. A significant number of neurons in the rostral dorsal cortex followed a deep (earlier arising) to superficial (later arising) pattern of neurogenesis, similar to that in the avian Wulst or in the mammalian isocortex. Finally, we compared the timing and development of neurogenesis in the turtle with birds and mammals to understand the evolutionary implications of these processes.

Comparative analysis of neurogenesis between the core and shell regions of auditory areas in the chick (Gallus gallus domesticus).

Early embryogenesis can reflect constituting organizations and evolutionary origins of brain areas. To determine whether a clear core-versus-shell distinction of neurogenesis that occurs from the auditory midbrain to the telencephalon in the reptile also appears in the bird, a single dose of [(3)H]-thymidine was injected into chick (Gallus gallus domesticus) eggs at some successive embryonic days (E) (from E3 to E10). Towards the end of hatching, [(3)H]-thymidine labeling was examined, and the results were as follows: 1) Neuronal generation in the nucleus intercollicularis (ICo) (shell region) began at E3, whereas neurogenesis began at E4 in the nucleus mesencephalicus lateralis pars dorsalis (MLd) (core region); 2) Neurogenesis initiated at E3 in the nucleus ovoidalis (Ov) shell, but initiated at E4 in the rostral Ov core. In the medial or caudal Ov core, the percentage of heavily-labeled neurons with [(3)H]-thymidine was significantly lower at E3 age group than that in the Ov shell; 3) In field L1 and L3, two flanking regions of the primary telencephalic auditory area (field L2a), neurogenesis started at E5, but started at E6 in field L2a. These data indicate that the onset of embryogenesis began earlier in the auditory shell areas than in the core areas from the midbrain to the telencephalon. These findings provide insight into the organization of auditory nuclei and their evolution in amniotes.

Comparative analyses of song complexity and song-control nuclei in fourteen oscine species.

Most studies on the relationship between measures of song behavior and the sizes of song control nuclei have focused on one or two oscine species, and often show inconsistent results. To address this issue, we first measured four variables for song complexity, i.e., song repertoire size, syllable repertoire size, the mean number of syllables per phrase (MNS) and the number of syllables in the longest phrase (NSLP), and the sizes of three song control nuclei, i.e., HVC, RA (the robust nucleus of the arcopallium), and Area X in 14 oscine species from eight families. To tackle the problem of statistical non-independence that probably existed among the closely related species, we reconstructed the phylogeny of the species studied using mitochondrial cytochrome b DNA sequences from GenBank. By using the methods adopted in most previous reports, we tested the relationship between song complexity and the sizes of the song control nuclei. We found that: 1) the absolute sizes of RA and Area X, but not of HVC, were positively correlated to the three measures of song complexity, but that only the residual size of RA in regard to telencephalon size was significantly correlated to the song measures; 2) independent contrasts analysis showed RA and Area X to be significantly associated with NSLP. Our results indicated that the relationship between song behavior and its neural structures varied among song nuclei, suggesting that each song control nucleus may play a different role in song behavior.

Distinction of neurochemistry between the cores and their shells of auditory nuclei in tetrapod species.

The distribution of Met-enkephalin (ENK), substance P (SP) and serotonin (5-HT) differs between the core and shell regions of the mesencephalic and diencephalic auditory nuclei of the turtle [Belekhova et al., 2002]. These neurochemical distinctions are also found in other tetrapods (mammals, birds and amphibians). The distribution of ENK, SP and 5-HT was examined in the core and shell regions of both mesencephalic and diencephalic auditory nuclei, and in the telencephalic auditory areas of Bengalese finches (Lonchura striata) and mice (Mus musculus), as well as in corresponding auditory areas in toads (Bufo bufo). ENK, SP and 5-HT immunoreactive fibers and perikarya were largely absent from the core regions of both mesencephalic and diencephalic auditory nuclei, in comparison with the shell regions of mice and Bengalese finches. In the toad, however, this pattern was observed in the mesencephalic auditory nucleus, but not in the diencephalic auditory areas. ENK and SP immunoreactive perikarya were detected in the telencephalic auditory area of mice, whereas no ENK, SP or 5-HT immunolabeling was observed in the telencephalic auditory area (Field L) of Bengalese finches. These findings are discussed in terms of the evolution of the core-and-shell organization of auditory nuclei of tetrapods.

Differences in neurogenesis differentiate between core and shell regions of auditory nuclei in the turtle (Pelodiscus sinensis): evolutionary implications.

There is a clear core-versus-shell distinction in cytoarchitecture, electrophysiological properties and neural connections in the mesencephalic and diencephalic auditory nuclei of amniotes. Determining whether the embryogenesis of auditory nuclei shows a similar organization is helpful for further understanding the constituent organization and evolution of auditory nuclei. Therefore in the present study, we injected [(3)H]-thymidine into turtle embryos (Pelodiscus sinensis) at various stages of development. Upon hatching, [(3)H]-thymidine labeling was examined in both the core and shell auditory regions in the midbrain, diencephalon and dorsal ventricular ridge. Met-enkephalin and substance P immunohistochemistry was used to distinguish the core and shell regions. In the mesencephalic auditory nucleus, the occurrence of heavily labeled neurons in the nucleus centralis of the torus semicircularis reached its peak at embryonic day 9, one day later than the surrounding shell. In the diencephalic auditory nucleus, the production of heavily labeled neurons in the central region of the reuniens (Re) was highest at embryonic day (E) 8, one day later than that in the shell region of reuniens. In the region of the dorsal ventricular ridge that received inputs from the central region of Re, the appearance of heavily labeled neurons also reached a peak one day later than that in the area receiving inputs from the shell region of reuniens. Thus, there is a core-versus-shell organization of neuronal generation in reptilian auditory areas.

Dynamic changes of apoptosis and expression of Bcl-2 family members in the posthatch hippocampus of Bengalese finches.

The hippocampus of songbirds plays an important role in spatial memory, and probably in song learning. Although prolonged neuronal generation and apoptosis are thought to be closely correlated with memory function, natural changes of the number of neurons and in apoptosis in the hippocampus of songbirds have not been fully investigated during development and in the adult. In the current study, we examined developmental changes in the volume and the number of neurons and apoptotic cells in the hippocampus of songbirds (Lonchura striata) from posthatch day (P5) to adulthood. Apoptotic cells were determined by Nissl staining and immunohistochemistry for cleaved caspase-3, a key apoptotic caspase executioner. The expression levels of Bcl-2 family member mRNA and protein, including Bcl-2, Bcl-xL and Bax, were also investigated. Our results indicated that: (1) the hippocampus volume significantly increased from P5 to P60, although the number of neurons remained stable in all studied stages; (2) the number of apoptotic cells was highest at P45, based either on the Nissl staining or on the immunohistochemistry for caspase-3; (3) Bcl-2 mRNA expression was high from P5 to adulthood, while Bax mRNA declined abruptly from P5 to adulthood, and Bcl-x mRNA was high after P45. Bcl-2 protein was only detected at P5 and P15, while detection of Bcl-xL and Bax proteins paralleled levels of mRNA expression. Our study provides detailed changes of apoptosis in the posthatch songbird hippocampus, suggesting an important role for caspase-3 and Bcl-2 family members in hippocampus apoptosis.

[Effect of gardenin on gene expression profile in brain of rats with focal cerebral ischemia].

OBJECTIVE: To explore the pharmacologic mechanism of gardenin in treating cerebral ischemia, by studying its effect on gene expression profile in brain of rats with focal cerebral ischemia (FCI). METHODS: Total RNAs were isolated from rats with FCI and those treated with gardenin. The mRNAs were reversely transcribed to cDNA with incorporation of fluorescent Cy5- or Cy3-dUTP to prepare hybridization probes. The PCR products of 4096 genes were spotted on the chip after a serial treatment. The mixed probes were hybridized to the cDNA microarray. Axon Genepix 4000B and GenePixPro 3.0 software were used to scan and analyze the fluorescent signals. RESULTS: In the group treated with gardenin, there were 70 genes had expression profiles different to that in the model group in the focal cerebral ischemic brain tissue, in which 68 were up-regulated and 2 down-regulated. CONCLUSION: Gardenin has regulatory effect on the gene expression in rats with focal cerebral ischemia, which elucidates part of the pharmacologic mechanism of Qingkailing in molecular level.

Sexual Differences in Cell Loss during the Post-Hatch Development of Song Control Nuclei in the Bengalese Finch.

Birdsongs and the regions of their brain that control song exhibit obvious sexual differences. However, the mechanisms underlying these sexual dimorphisms remain unknown. To address this issue, we first examined apoptotic cells labeled with caspase-3 or TUNEL in Bengalese finch song control nuclei - the robust nucleus of the archopallium (RA), the lateral magnocellular nucleus of the anterior nidopallium (LMAN), the high vocal center (HVC) and Area X from post-hatch day (P) 15 to 120. Next, we investigated the expression dynamics of pro-apoptotic (Bid, Bad and Bax) and anti-apoptotic (Bcl-2 and Bcl-xL) genes in the aforementioned nuclei. Our results revealed that the female RA at P45 exhibited marked cell apoptosis, confirmed by low densities of Bcl-xL and Bcl-2. Both the male and female LMAN exhibited apoptotic peaks at P35 and P45, respectively, and the observed cell loss was more extensive in males. A corresponding sharp decrease in the density of Bcl-2 after P35 was observed in both sexes, and a greater density of Bid was noted at P45 in males. In addition, we observed that RA volume and the total number of BDNF-expressing cells decreased significantly after unilateral lesion of the LMAN or HVC (two areas that innervate the RA) and that greater numbers of RA-projecting cells were immunoreactive for BDNF in the LMAN than in the HVC. We reasoned that a decrease in the amount of BDNF transported via HVC afferent fibers might result in an increase in cell apoptosis in the female RA. Our data indicate that cell apoptosis resulting from different pro- and anti-apoptotic agents is involved in generating the differences between male and female song control nuclei.

Immunohistochemistry and neural connectivity of the Ov shell in the songbird and their evolutionary implications.

The neuropeptide immunohistochemistry and neural connectivity of areas surrounding the thalamic auditory nucleus (the nucleus ovoidalis [Ov]), as well as the areas to which it is connected, were investigated in a songbird, the Bengalese finch. The results showed that met-enkephalin was present in the Ov shell and most of the areas connected to it, but not in the Ov core. Anterograde and retrograde tracing studies showed that the Ov shell was more widely connected than the Ov core. The Ov shell was mainly connected to: 1). areas flanking the primary telencephalic auditory field (i.e., fields L2b, L1, and L3) and areas surrounding the robust nucleus of the archistriatum (RA); 2). several hypothalamic areas such as the nucleus ventromedialis hypothalami (VMN) and the nucleus anterior medialis hypothalami (AM). Some of these areas connected to the Ov shell are thought to be involved in auditory mediated neurosecretory activities. These results, which are similar to those reported previously in non-songbirds, suggest that the Ov shell and other surrounding areas of auditory and song-control nuclei are conserved in birds. These findings are discussed in terms of the evolution of the core-and-surround organization of auditory and song-control nuclei.

The impact of deafness to the survival of the newborn cells in the brain of juvenile white-rumped munia, Lonchura striata.

In white-rumped munia, early auditory experience is critical for normal song development. New neurons are constantly added to the telencephalon in juveniles. We examined the potential role of auditory experience in regulating the developmental changes in the song nuclei and the survival of newborn cells. We chose two special days, postnatal day 23 and 37, at which we deafened the birds through bilateral cochlea removal. All birds were injected with the cell birth marker BrdU two weeks before the lesion surgeries, and then were killed two weeks or one month later. The BrdU-positive cells were distributed throughout the brain, including the high vocal center (HVC), Lobus parolfactorius and the ventricle zone (VZ) in telencephalon, the granular cell layer (GCL) of cerebellum. Moreover, these BrdU-positive cells in the GCL could self-renew. However, the nucleus robustus archistriatalis (RA) did not sprout new neurons in juvenile. In telencephalon except the VZ, 41 percent of BrdU-positive cells were NeuN-positive, too. Deafness had no significant effect on development of HVC and RA, the distribution of new cells, and the survival of new cells in telencephalon. From these data, we propose that auditory deprivation could not affect the survival of new cells of telencephalon within one month. Surprisingly, we found deafness had a complex and dramatic effect on the number of new cells in cerebellum. Deafness at postnatal day 23 could increase the number of new cells in the GCL, while deafness at postnatal day 37 decreased the number.

Sexual differences in cell proliferation in the ventricular zone, cell migration and differentiation in the HVC of juvenile Bengalese finch.

Song control nuclei have distinct sexual differences and thus are an ideal model to address how brain areas are sexually differentiated. Through a combination of histological analysis and electrical lesions, we first identified the ventricle site for HVC progenitor cells. We then found that there were significant sex differences in the cellular proliferation activity in the ventricular zone of the HVC, the number of migrating cells along the radial cells (positive immunoreactions to vimentin) and differentiation towards neurons. Through co-culturing of male and female slices containing the developing HVC in the same well, we found that the male slices could produce diffusible substances to masculinize the female HVC. By adding estrogen, an estrogen antagonist, brain-derived neurotrophic factor (BDNF) or its antibody into the culture medium, separately or in combination, we found that these diffusible substances may include estrogen and BDNF. Finally, we found that 1) estrogen-induced BDNF upregulation could be detected 48 hr after estrogen treatment and could not be blocked by a vascular endothelial growth factor (VEGF) receptor inhibitor and 2) the amount of VEGF mRNA expressed in the developing HVC and its adjacent area did not display any significant sex differences, as did the distribution of VEGF and laminin-expressing endothelial cells in the developing HVC. Because these findings are largely different from previous reports on the adult female HVC, it is suggested that our estrogen-induced BDNF up-regulation and the resultant sexual differentiation might not be mediated by VEGF and endothelial cells, but instead, may result from the direct effects of estrogen on BDNF.

Alteration of CaBP expression pattern in the nucleus magnocellularis following unilateral cochlear ablation in adult zebra finches.

Songbirds have the rare ability of auditory-vocal learning and maintenance. Up to now, the organization and function of the nucleus magnocellularis (NM), the first relay of the avian ascending auditory pathway is largely based on studies in non-vocal learning species, such as chickens and owls. To investigate whether NM exhibits different histochemical properties associated with auditory processing in songbirds, we examined the expression patterns of three calcium-binding proteins (CaBPs), including calretinin (CR), parvalbumin (PV) and calbindin-D28k (CB), and their relations to auditory inputs in NM in adult zebra finches. We found enriched and co-localized immunostaining of CR, PV and CB in the majority of NM neurons, without neuronal population preference. Furthermore, they were sensitive to adult deafferentation with differential plasticity patterns. After unilateral cochlear removal, CR staining in the ipsilateral NM decreased appreciably at 3 days after surgery, and continued to decline thereafter. PV staining showed down-regulation first at 3 days, but subsequently recovered slightly. CB staining did not significantly decrease until 7 days after surgery. Our findings suggest that the three CaBPs might play distinct roles in association with auditory processing in zebra finches. These results are in contrast to the findings in the NM of chickens where CR is the predominant CaBP and deafferentation had no apparent effect on its expression. Further extended studies in other avian species are required to establish whether the difference in CaBP patterns in NM is functionally related to the different auditory-vocal behaviors.

Ultrastructural and electrophysiological analysis of Area X in the untutored and deafened Bengalese finch in relation to normally reared birds.

Birdsong learning bears many similarities to human speech acquisition. Although the anterior forebrain pathway (AFP) is believed to be involved in birdsong learning, the underlying neural mechanisms are unclear. We produced two types of abnormal song learning: young birds untutored from adult "song tutors", or birds deafened by bilateral cochlear removal before the onset of sensory learning. We then studied how ultrastructure and electrophysiological activity changed in an AFP nucleus, Area X, among these birds at adulthood. Our results showed that, although the size of Area X did not change significantly, the numbers of synapses per unit area and compound synapses and the percent of concave synapses increased significantly in the untutored or deafened birds. The percent of perforated synapses or axo-spinous synapses decreased compared to the normally reared birds, suggesting a decreased efficiency of synaptic transmission in the untutored or deafened birds. We then identified several types of spontaneously firing cells in Area X. Cells with fast and slow firing rates did not show significant electrophysiological differences among the groups, but cells with moderate firing rates, most likely DLM-projecting neurons, fired at significantly lower rates in the untutored and deafened birds. In addition, cells firing irregularly were only found in the deafened birds. Thus, the decreased or irregular electrophysiological activity in the untutored or deafened birds, together with the corresponding ultrastructural findings, could be implicated in the abnormal song production in these two types of birds.

Changes in ultra-structures and electrophysiological properties in HVC of untutored and deafened Bengalese finches relation to normally reared birds: implications for song learning.

Songbirds are increasingly used as an experimentally tractable system to study the neurobiological underpinnings of vocal learning. To gain additional insights into how birdsongs are learned, we compared the size of HVC, the high vocal center for song production, and its ultrastructural or electrophysiological properties between the normally reared Bengalese finches, and the untutored or deafened ones before the onset of sensory learning (around post-hatching day 20). Our results showed that HVC had more synapses and concave synaptic curvature, but fewer perforated synapse, in the untutored or deafened birds in comparison with those in the normally reared birds. Although there was no significant difference of the ratio of straight or compound synapses, there was an increasing tendency for the untutored and deafened birds to possess more straight and compound synapses. These data revealed that synapses in the isolated or deafened birds had lower synapse activity in relation to those with normal hearing. This was confirmed by our electrophysiological results to show significant decreases in the firing rates of spike or burst in the isolated or deafened birds in the three types of HVC neurons i.e., putative X-projecting neurons, RA-projecting neurons and interneurons. In addition, low firing frequency (<10Hz) occurred much more in the above three types of HVC neurons in the tutored or deafened birds than in the normally reared birds. These data suggest that all the three putative types of neurons in HVC might be involved in the activity of the production of adult normal songs.

Comparative study on song behavior, and ultra-structural, electrophysiological and immunoreactive properties in RA among deafened, untutored and normal-hearing Bengalese finches.

To gain additional insight into how a birdsong is learned, we compared the songs of Bengalese finch males that were deafened early in development or raised without tutors to control finches that learned songs from adult models. Fewer note types and a more variable number of notes per bout were observed in untutored male songs, and no audible songs were detected in deafened males. We then investigated the ultrastructural, immunohistological, and electrophysiological correlates of the outcomes of song learning within the robust nucleus of the archopallium (RA), a forebrain nucleus for song production. In comparison to control birds, untutored and deafened birds had more synapses per unit volume, fewer vesicles per synapse, longer postsynaptic densities, and a lower proportion of perforated synapses, which suggest lower activity or decreased efficiency of synaptic transmission within the RA of the treated birds. For anesthetized birds, neurons within the RA of untutored and deafened males had lower spontaneous firing rates, fewer and shorter bursts, and higher coefficient of variation of the instantaneous firing rate than the normally reared males. Compared with controls, the untutored and deafened males had higher staining intensities within the RA of GABA and the GABA(A) receptor, less staining of tyrosine hydroxylase and no difference in the staining of NMDA receptors. Thus, both the ultrastructural and immunohistochemical results could explain for the stronger electrophysiological activities in normally reared birds. Because RA is involved in generating the motor commands, these data might account for the deficits in birds with abnormal song learning.