NR2B downregulation in a forebrain region required for avian vocal learning is not sufficient to close the sensitive period for song learning

The neural changes that limit the sensitive period for avian song development are unknown, but neurons in a forebrain region critical for song learning, the lMAN, exhibit experience-driven changes in NMDAR subunit expression that could regulate sensitive period closure. Specifically, NR2B levels in lMAN decrease during song acquisition, potentially reducing synaptic plasticity by decreasing NMDAR EPSC duration and/or affecting NMDAR-coupled intracellular cascades. While rearing birds in isolation extends the sensitive period and also delays the developmental changes in NR2B expression and NMDAR physiology, recent work indicates that a transition to faster NMDAR currents does not preclude further song learning. However, NR2B mRNA expression in isolates remains elevated beyond the age at which NMDAR currents shorten, leaving open the possibility that NR2B levels regulate closure of the sensitive period through effects other than those mediated by NMDAR current duration. To determine whether the experience-driven decrease in NR2B expression in lMAN closes the sensitive period, we promoted this change in gene expression either by treating isolation-reared zebra finches briefly with testosterone (T-isolates) or by allowing males limited access to conspecific song (pre-exposed isolates). We then assessed if these birds could acquire song from tutors after the normal close of the sensitive period. Despite a normal decline in NR2B expression, T-isolate and pre-exposed isolate birds learned tutor songs heard from d65-90, while normally reared birds did not. These findings suggest that the normal decline in NR2B expression with lMAN is not sufficient for sensitive period closure.     

Dissociation between extension of the sensitive period for avian vocal learning and dendritic spine loss in the song nucleus lMAN

Several instances of early learning coincide with significant rearrangements of neural connections in regions contributing to these behaviors. In fact developmentally restricted learning may be constrained temporally by the opportunity for experience to selectively maintain appropriate synapses amidst the elimination of exuberant connections. Consistent with this notion, during the normal sensitive period for vocal learning in zebra finches (Taenopygia guttata), there is a decline in the density of dendritic spines within a region essential for song development, the lateral magnocellular nucleus of the anterior nidopallium (lMAN). Moreover, in birds isolated from conspecific song shortly after hatching, both the closure of the sensitive period for vocal learning and the pruning of spines from lMAN neurons is delayed. Here, we employed a more subtle form of deprivation to delay the close of the sensitive period for song learning, and found that late song learning occurred without obvious alterations in the pruning of dendritic spines on lMAN neurons. At posthatch day (PHD) 65 (beyond the end of the normal sensitive period for song memorization in zebra finches), birds isolated from song beginning on PHD30 did not differ from normally reared birds in measures of dendritic spine density on Golgi-Cox stained lMAN neurons. Moreover, tutor exposure from PHD65 to 90 did not increase spine elimination in these isolates (who memorized new song material) relative to controls (who did not). Thus, we conclude that the extent of normally occurring lMAN spine loss is not sufficient to account for the timing of the sensitive period for zebra finch song learning.     

A Brain of Her Own: A Neural Correlate of Song Assessment in a Female Songbird

The song control region in the avian forebrain is a series of discrete, interconnected nuclei mediating song learning and production. It has been studied in males or in species where both sexes sing. Little is known about the neural correlates of song perception in nonsinging females, often the intended recipients of song. We studied cowbirds (Molothrus ater), a species in which only males sing but in which females discriminate between males on the basis of song. We focused on nucleus lMAN because it has been implicated in early song acquisition, a stage relevant to both sexes to choose among competing acoustic models. We found that volume of lMAN was monomorphic in cowbirds. Moreover, the volume and neuronal number of female lMAN were positively correlated with selectivity of copulatory responding. The results provide strong evidence of nonsinging female's use of “song” control nuclei for song perception without the possibility of song production.     

Blockade of NMDA Receptors in the Anterior Forebrain Impairs Sensory Acquisition in the Zebra Finch (Poephila guttata)

Juvenile zebra finches (Poephila guttata) learn song in two stages: during sensory acquisition, they memorize the song of an adult tutor, and during sensorimotor learning, they alter their vocalizations to match the stored song model. Like many other forms of neural plasticity and memory formation, vocal learning in zebra finches is impaired by pharmacological blockade of NMDA receptors, but the relevant NMDA receptors have not yet been localized. During song development, one neural region that has been implicated specifically in song learning, the lMAN, exhibits an increased density of NMDA receptors as well as decreased binding affinity for the NMDA antagonist MK-801. To test the hypothesis that sensory acquisition requires activation of NMDA receptors in or near the lMAN we infused the NMDA receptor antagonist amino-5-phosphonopentanoic acid (AP5; 2.5 μg in 0.1 μl) directly into the anterior forebrain. Birds receiving AP5 infusions prior to each of 10 tutoring sessions copied significantly less of their tutor's song than did sham-operated birds, saline-infused birds, birds that received AP5 infusions on nontutoring days, or birds that received AP5 infusions into the cerebellum. Furthermore, infusions of AP5 in the anterior forebrain did not impair young birds’ ability to discriminate zebra finch from canary song. These findings are consistent with the hypothesis that NMDA receptor activation in the anterior forebrain is necessary for the memorization of song material during avian vocal learning. This is also the first report that song-related regions of the anterior forebrain contribute to sensory acquisition specifically.     

Developmental and seasonal changes in canary song and their relation to changes in the anatomy of song-control nuclei

Young male canaries become sexually mature in late winter, 8-12 months after hatching. During the months between hatching and sexual maturity they develop adult song. The successive stages in the development of adult song are subsong, plastic song, and stable or full song. Once stable song is achieved it lasts for the duration of the breeding season. After the end of the breeding season there is a recurrence of song instability during summer and early fall. This plastic song is followed, once more, by stable song. New song syllables are added to the song of adult male canaries and some of the earlier syllables disappear. The song repertoire sung at 2 years of age is substantially larger, and different, from that sung during the first breeding season, when the birds were 1 year old. A comparable change occurs between the second and third breeding seasons. Most of the syllables acquired by adult males are formed during the summer-fall period of song instability. Developmental and seasonal changes in song are accompanied by anatomical changes in two forebrain nuclei known to be involved in song control, the hyperstriatum ventralis, pars caudalis (HVc), and the robust nucleus of the archistriatum (RA). HVc and RA grow during the subsong and plastic song periods of song development. These nuclei reach adult size by the time stable adult song is first produced, and retain this size during the breeding season. However, the size of HVc and RA diminishes by late summer, when it becomes comparable to that of a 3- to 4-month-old bird. This reduction in size is temporary and has been corrected by the following breeding season. It is suggested that these seasonal changes in volume reflect circuit changes which are under hormonal control, and that these changes are related to processes of learning and, possibly, forgetting. Despite earlier reports of left hemispheric dominance in canary song production, we failed to find any evidence of right-left systematic differences in the size of HVc and RA during development or in adulthood. Various hypotheses relating song learning to changes in the underlying anatomy are offered.     

Enlargement of Neuronal Somata in the LMAN Coincides with the Onset of Sensorimotor Learning for Song

The lateral magnocellular nucleus of the anterior neostriatum (LMAN) in the zebra finch (Taeniopygia guttata) has been shown to play a developmentally restricted role that is essential during song-learning processes. Dendritic spine frequencies and synapse numbers in LMAN have been reported to decline in males during early vocal motor learning and thereafter, but not in females, who do not sing. Nissl staining has shown the LMAN volume to be very similar in both sexes, however. To gain more insight into the development of sex-specific differences in LMAN, the size of neuronal somata and cell nuclei were analyzed in 1-μm semithin sections. Cell somata and nuclei were similar in males and females during the initial phases of sensory memory formation for song, but during early vocal motor learning cell size increased in males and decreased in females. Sex differences in neuronal somata size were present at 50 days and remained throughout life. This sex difference may be indicative of a difference in protein biosynthesis in LMAN, arising as a consequence of vocal learning in males.     

Selective impairment of song learning following lesions of a forebrain nucleus in the juvenile zebra finch

Area X, a large sexually dimorphic nucleus in the avian ventral forebrain, is part of a highly discrete system of interconnected nuclei that have been implicated in either song learning or adult song production. Previously, this nucleus has been included in the song system because of its substantial connections with other vocal control nuclei, and because its volume is positively correlated with the capacity for song. In order to directly assess the role of Area X in song behavior, this nucleus was bilaterally lesioned in both juvenile and adult zebra finches, using ibotenic acid. We report here that lesioning Area X disrupts normal song development in juvenile birds, but does not affect the production of stereotyped song by adult birds. Although juvenile-lesioned birds were consistently judged as being in earlier stages of vocal development than age-matched controls, they continued to produce normal song-like vocalizations. Thus, unlike the lateral magnocellular nucleus of the anterior neostriatum, another avian forebrain nucleus implicated in song learning, Area X does not seem to be necessary for sustaining production of juvenile song. Rather, the behavioral results suggest Area X is important for either the acquisition of a song model or the improvement of song through vocal practice.     

Differences in the complexity of song tutoring cause differences in the amount learned and in dendritic spine density in a songbird telencephalic song control nucleus

In our search for relations between vocal learning and neuron structure in the song control nuclei of songbird forebrains, we tested whether differential experience that leads to differences in adult song repertoire would affect dendritic spine density in HVc (also called high vocal center) and RA (robustus archistriatalis). We tape-tutored juvenile Eastern marsh wrens (Cistothorus palustris) with either 5 or 45 song types. As adults, the small repertoire group had learned mostly 5 or 6 song types, and the large repertoire group had learned 36 to 47. Wrens that learned the large song repertoires had a greater dendritic spine density for the most spiny neurons present in HVc (mean difference, 36%), but not in RA. Recent physiological evidence describes HVc as a premotor area coding syllables, motifs, and higher-order song patterns, and our data now clearly reveal that differences in the size of the song repertoire that is experienced lead to differences both in song learning and in the density of dendritic spines in HVc. In the forebrain song nuclei of these songbirds, as in some other vertebrate systems, differences in learning and performance are associated with differences in synaptic anatomy specifically in the region that organizes the learned pattern.     

Altered daylength affects dendritic structure in a song-related brain region in red-winged blackbirds.

Substantial neural and behavioral plasticity occurs in the avian song system in adulthood. Changes in the volume of one of the song control nuclei, robustus archistriatalis (RA), have been associated with seasonal changes in singing behavior in adult canaries (Serinus canarius) and red-winged blackbirds (Agelaius phoeniceus). The present work assessed the effects of changed daylength on dendritic morphology in RA in adult male red-winged blackbirds. Brains from hand-reared red-winged blackbirds maintained on long days or long days followed by short days were stained with a Golgi-Cox procedure. Dendritic morphology and spine density of type IV neurons from nucleus RA were compared between long and short day birds. Neurons from short day birds have smaller dendritic fields than neurons from long day birds, with the difference greatest for distal dendrites. In addition, the density of dendritic spines is significantly smaller for neurons from short day birds. Together, these changes result in the loss of approximately 40% of the spines on this neuron class. In previous work in adult female canaries, external testosterone administration has been shown to be associated with increases in dendritic field size and synapse number. The similarity of the neuronal changes in RA that are associated with the two sorts of manipulations suggest that some consequences of altered daylength are mediated by changes in the levels of gonadal steroids.     

Song learning and cognitive ability are not consistently related in a songbird

Learned aspects of song have been hypothesized to signal cognitive ability in songbirds. We tested this hypothesis in hand-reared song sparrows (Melospiza melodia) that were tutored with playback of adult songs during the critical period for song learning. The songs developed by the 19 male subjects were compared to the model songs to produce two measures of song learning: the proportion of notes copied from models and the average spectrogram cross-correlation between copied notes and model notes. Song repertoire size, which reflects song complexity, was also measured. At 1 year of age, subjects were given a battery of five cognitive tests that measured speed of learning in the context of a novel foraging task, color association, color reversal, detour-reaching, and spatial learning. Bivariate correlations between the three song measures and the five cognitive measures revealed no significant associations. As in other studies of avian cognition, different cognitive measures were for the most part not correlated with each other, and this result remained true when 22 hand-reared female song sparrows were added to the analysis. General linear mixed models controlling for effects of neophobia and nest of origin indicated that all three song measures were associated with better performance on color reversal and spatial learning but were associated with worse performance on novel foraging and detour-reaching. Overall, the results do not support the hypothesis that learned aspects of song signal cognitive ability.     

Experience with songs in adulthood reduces song-induced gene expression in songbird auditory forebrain

Male songbirds learn to produce song within a limited phase early in life; however they continue to learn to recognize songs in adulthood. Studies looking at Zenk activation after exposure to songs learned early in life for song production and songs learned in adulthood show opposite patterns of activation, suggesting distinct neural mechanisms may be involved in these two forms of learning. In this study, we look at IEG Zenk activation in auditory regions NCM and CMM of song sparrows (Melospiza melodia) to see whether recent exposure to song in adulthood leads to greater or decreased Zenk activation upon hearing that song versus a novel song. We found significantly lower activation in birds exposed to previously heard songs versus novel songs in vNCM but not dNCM, though further analysis suggest an overall trend in NCM. We found no significant difference in the amount of activation to previously heard songs vs. novel songs in CMM. These results support previous findings suggesting that activation is reduced to learned stimuli; we discuss possible implications of these findings in relation to song production learning early in life and song recognition learning in adulthood.     

Changes in stereotyped central motor patterns controlling vocalization are induced by peripheral nerve injury.

Adult male zebra finches (Taeniopygia guttata), as closed-ended learners, normally crystallize their songs at 90 days of age, and the song remains fixed throughout life (Price, 1979). We show that injuring the tracheosyringeal nerve(s) (each of which innervates the ipsilateral half of the syrinx, the avian vocal organ) results in a short-term deficit in the syllables forming adult male song; this deficit disappears after ts nerve regeneration. However, when adult males were followed for a period of several weeks after unilateral tracheosyringeal nerve injury, long-term changes occurred in the temporal patterning of song. Syllables were deleted, remaining portions of the song were linked, and new syllables were added. Syllables with call-like morphology were less likely to be deleted from and more likely to be added to the song. Deletions were most often contiguous chunks of syllables. Changes in the temporal patterning of song occurred during specific periods following nerve injury, were completed within 100 days after nerve transection, and were not dependent upon regeneration of the ts nerve. The resulting newly formed song patterns were stable, remaining unchanged up to 1 year later. The ability of adult male zebra finches to make specific types of changes to crystallized song indicates that some form of vocal plasticity remains even after song learning is completed, though this plasticity may be restricted to a subset of song characteristics. The limitations on the types of changes that are possible may reflect how song is centrally organized.     

Increase in Syntaxin 1B mRNA in Hippocampal and Cortical Circuits During Spatial Learning Reflects a Mechanism of Trans-synaptic Plasticity Involved in Establishing a Memory Trace

It has long been proposed that the cellular and molecular mechanisms responsible for LTP may well involve the mechanisms that lead to the type of synaptic modification that occurs during learning. However, it is also known that a single memory trace is encoded in spatially distributed networks; implying that alterations of synaptic strength occur at multiple sites along circuits of connected cells. Recent evidence suggests that regulation of the gene encoding syntaxin 1B, a presynaptic protein involved in exocytosis, plays an important role in the mediation of trans-synaptic LTP, a candidate mechanism for the propagation of plasticity in neural circuits during learning. Using in situ hybridization to measure the mRNA levels at different time points after learning a spatial working or reference memory task, we show that expression of the gene encoding this protein in the hippocampal and corticoprefrontal circuits increases linearly with performance at a critical window of learning when rats are reaching between 75% and 100% of their maximal performance. No changes were observed during the early phases of learning or when rats where overtrained. The correlational analysis indicates that coordinated increases in syntaxin 1B expression occurs in hippocampal circuits during working memory and in more widespread hippocampocortical circuits during reference memory. These results suggest that a form of trans-synaptic plasticity mediated in part by regulation of the expression of syntaxin 1B may play an active role in configuring specific spatially distributed circuits during the laying down of memories.     

Song recognition in zebra finches: Are there sensitive periods for song memorization?

Male zebra finches learn to sing songs that they hear between 25 and 65 days of age, the sensitive period for song learning. In this experiment, male and female zebra finches were exposed to zebra finch songs either before (n = 9) or during (n = 4) the sensitive period. Following song exposure, recognition memory for the songs was assessed with an operant discrimination between familiar and novel songs. Zebra finches that were exposed to songs between 22 and 30 days of age discriminated between familiar and novel songs; zebra finches exposed to songs from 9 to 17 days of age did not. Failure to memorize songs heard prior to the sensitive period may contribute to the exclusion of those songs from the repertoire of songbirds.     

Mouse vocal communication system: Are ultrasounds learned or innate?

Mouse ultrasonic vocalizations (USVs) are often used as behavioral readouts of internal states, to measure effects of social and pharmacological manipulations, and for behavioral phenotyping of mouse models for neuropsychiatric and neurodegenerative disorders. However, little is known about the neurobiological mechanisms of rodent USV production. Here we discuss the available data to assess whether male mouse song behavior and the supporting brain circuits resemble those of known vocal non-learning or vocal learning species. Recent neurobiology studies have demonstrated that the mouse USV brain system includes motor cortex and striatal regions, and that the vocal motor cortex sends a direct sparse projection to the brainstem vocal motor nucleus ambiguous, a projection previously thought be unique to humans among mammals. Recent behavioral studies have reported opposing conclusions on mouse vocal plasticity, including vocal ontogeny changes in USVs over early development that might not be explained by innate maturation processes, evidence for and against a role for auditory feedback in developing and maintaining normal mouse USVs, and evidence for and against limited vocal imitation of song pitch. To reconcile these findings, we suggest that the trait of vocal learning may not be dichotomous but encompass a broad spectrum of behavioral and neural traits we call the continuum hypothesis, and that mice possess some of the traits associated with a capacity for limited vocal learning.     

Seasonal changes in gonadal hormone levels of adult male canaries and their relation to song

Samples of song and blood levels of three gonadal hormones, T, DHT, and E2, were taken at monthly intervals from six adult male canaries over a period of 1 year, as these birds went from 12 to 24 months of age. Song variability and addition of new syllable types were maximal during the summer and early fall, with a peak in September. A secondary peak in new syllable acquisition occurred in March. Blood T levels were particularly low during July-August and during February. Thus, a lowering of blood T levels preceded by about 1 month the two marked peaks in new syllable acquisition. Blood levels of the two other hormones were related less obviously to song learning peaks. It is hypothesized that a concurrence of hormonal, neural, and behavioral changes facilitates song learning in adulthood.     

Synaptic plasticity in the hypoglossal nucleus of female canaries: structural correlates of season, hemisphere, and testosterone treatment

The caudal portion of the hypoglossal nucleus (nXIIts) contains the motor neurons that control the syrinx in songbirds. In canaries, song occurs seasonally, is principally produced by males, and appears to be produced predominantly by muscles on the left side of the syrinx. The present study measures the effect of seasonal change and manipulation of testosterone levels on synapse number and morphology in nXIIts in adult female canaries. We find that synapse density is lower in testosterone-treated birds than in control birds and lower in fall than in spring. The number of vesicles per presynaptic profile increases in the spring as a result of a general increase in this measure in all synapses. The number of vesicles per presynaptic profile also increases with testosterone treatment, primarily due to an increase in the proportion of synapses associated with unusually high vesicle counts. Together, these changes suggest that large reserves of neurotransmitter may be necessary to sustain singing. Several ultrastructural differences between hemispheres are found. Postsynaptic thickenings are longer, and postsynaptic processes are larger on the left side than on the right side. In the spring, there are more vesicles per synapse on the left than on the right, but this lateralization is reversed in the fall. Thus, lateralization of song production is associated with lateral asymmetries in synapse morphology. These hemispheric differences are relatively small, like those seen at the light microscope level, encouraging further consideration of peripheral as well as CNS sources of functional lateralization. The seasonal and testosterone-induced changes in synapse number and morphology may be components of the periodic reorganization of canary vocalization.     

Molecular and behavioral changes associated with adult hippocampus-specific SynGAP1 knockout

The synaptic Ras/Rap-GTPase-activating protein (SynGAP1) plays a unique role in regulating specific downstream intracellular events in response to N-methyl-D-aspartate receptor (NMDAR) activation. Constitutive heterozygous loss of SynGAP1 disrupts NMDAR-mediated physiological and behavioral processes, but the disruptions might be of developmental origin. Therefore, the precise role of SynGAP1 in the adult brain, including its relative functional significance within specific brain regions, remains unexplored. The present study constitutes the first attempt in achieving adult hippocampal-specific SynGAP1 knockout using the Cre/loxP approach. Here, we report that this manipulation led to a significant numerical increase in both small and large GluA1 and NR1 immunoreactive clusters, many of which were non-opposed to presynaptic terminals. In parallel, the observed marked decline in the amplitude of spontaneous excitatory currents (sEPSCs) and inter-event intervals supported the impression that SynGAP1 loss might facilitate the accumulation of extrasynaptic glutamatergic receptors. In addition, SynGAP1-mediated signaling appears to be critical for the proper integration and survival of newborn neurons. The manipulation impaired reversal learning in the probe test of the water maze and induced a delay-dependent impairment in spatial recognition memory. It did not significantly affect anxiety or reference memory acquisition but induced a substantial elevation in spontaneous locomotor activity in the open field test. Thus, the present study demonstrates the functional significance of SynGAP1 signaling in the adult brain by capturing several changes that are dependent on NMDAR and hippocampal integrity.     

Behavioral consequences of song learning: Discrimination of song types by male white-crowned sparrows

The functional significance of learned population differences in male song in the white-crowned sparrow was explored in natural populations using playback tests. Laboratory results have shown that learning of the population-specific song seems to take place in early life and is strongly dependent upon the nature of the auditory experience at that time. However, the varied results of recent studies make it difficult to reach a confident conclusion about the ecological functions of song learning. The present research took advantage of naturally occurring variation in the differences between songs of adjacent populations to determine a function relating degree of difference in song to intensity of territorial singing elicited. Applying a typological evaluation of syllable structure to the four segments of the song allowed a crude quantitative ranking of the differences between local songs and playback stimuli. These results, together with those of other studies, suggest a unimodal aggressive response function of males to songs of other males. A maximum response to songs slightly different from the local song environment suggests that male exclusion based upon acquired song components may contribute to the maintenance of discrete and stable song dialects.     

Song perception during the sensitive period of song learning in zebra finches (Taeniopygia guttata)

The sensitive period is a special time for auditory learning in songbirds. However, little is known about perception and discrimination of song during this period of development. The authors used a go/no-go operant task to compare discrimination of conspecific song from reversed song in juvenile and adult zebra finches (Taeniopygia guttata), and to test for possible developmental changes in perception of syllable structure and syllable syntax. In Experiment 1, there were no age or sex differences in the ability to learn the discrimination, and the birds discriminated the forward from reversed song primarily on the basis of local syllable structure. Similar results were found in Experiment 2 with juvenile birds reared in isolation from song. Experiment 3 found that juvenile zebra finches could discriminate songs on the basis of syllable order alone, although this discrimination was more difficult than one based on syllable structure. The results reveal well-developed song discrimination and song perception in juvenile zebra finches, even in birds with little experience with song.