Deafening-induced vocal deterioration in adult songbirds is reversed by disrupting a basal ganglia-forebrain circuit.

Motor exploration can be an adaptive strategy when behavior fails to achieve an expected outcome. For example, like humans, adult songbirds change their vocal output when auditory feedback is altered or absent. Here, we show that the output of an anterior forebrain pathway (AFP) through the avian basal ganglia directly contributes to the expression of deafening-induced vocal changes in adulthood. Lesioning the output nucleus of this circuit in adult male zebra finches reverses moderate changes in song structure and variability caused by deafening. Furthermore, the results indicate that more severe deafening-induced changes in vocal behavior likely reflect altered function outside the AFP (e.g., within the vocal motor pathway). AFP lesions do not promote recovery if songs are severely deteriorated at the time of lesion even though previous work shows that the AFP is required for such deterioration to emerge. Thus, in birds, as in mammals, the contribution of basal ganglia-thalamic-cortical circuits to motor control may change when feedback is absent or unexpected and includes both "active" and "permissive" roles.

Increased Fos expression among midbrain dopaminergic cell groups during birdsong tutoring.

During avian vocal learning, birds memorize conspecific song patterns and then use auditory feedback to match their vocal output to this acquired template. Some models of song learning posit that during tutoring, conspecific visual, social and/or auditory cues activate neuromodulatory systems that encourage acquisition of the tutor's song and attach incentive value to that specific acoustic pattern. This hypothesis predicts that stimuli experienced during social tutoring activate cell populations capable of signaling reward. Using immunocytochemistry for the protein product of the immediate early gene c-Fos, we found that brief exposure of juvenile male zebra finches to a live familiar male tutor increased the density of Fos+ cells within two brain regions implicated in reward processing: the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). This activation of Fos appears to involve both dopaminergic and non-dopaminergic VTA/SNc neurons. Intriguingly, a familiar tutor was more effective than a novel tutor in stimulating Fos expression within these regions. In the periaqueductal gray, a dopamine-enriched cell population that has been implicated in emotional processing, Fos labeling also was increased after tutoring, with a familiar tutor again being more effective than a novel conspecific. As several neural regions implicated in song acquisition receive strong dopaminergic projections from these midbrain nuclei, their activation in conjunction with hearing the tutor's song could help to establish sensory representations that later guide motor sequence learning.

Androgens prevent normally occurring cell death in a sexually dimorphic spinal nucleus.

The spinal nucleus of the bulbocavernosus (SNB) contains many more motoneurons in adult male rats than in females. Androgens establish this sex difference during a critical perinatal period, which coincides with normally occurring cell death in the SNB region. Sex differences in SNB motoneuron number arise primarily because motoneuron loss is greater in females than in males during the early postnatal period. Perinatal androgen treatment in females attenuates cell death in the SNB region, reducing motoneuron loss to levels typical of males. The results suggest that steroid hormones determine sex differences in neuron number by regulating normally occurring cell death and that the timing of this cell death may therefore define critical periods for steroid effects on neuron number.

Characterization of CaMKII-expressing neurons within a striatal region implicated in avian vocal learning.

In songbirds, an anterior forebrain pathway has been implicated in vocal learning. Within Area X, the striatal-pallidal component of this forebrain pathway, early social tutoring dramatically increases the autophosphorylation of CaMKII (calcium-calmodulin-dependent protein kinase II). Activation of CaMKII often is associated with forms of synaptic plasticity (e.g. LTP) underlying learning and memory, and electrophysiological studies have demonstrated NMDA and dopamine (DA) receptor-dependent LTP among Area X medium spiny neurons [Ding, L., Perkel, D.J., 2002. Dopamine modulates excitability of spiny neurons in the avian Basal Ganglia. J. Neurosci. 22, 5210-5218]. Together, these data suggest that Area X neurons may help to encode a representation of song used for vocal mimicry. To identify which Area X neurons could participate in the CaMKII response to song tutoring, we used immunocytochemistry to assess the colocalization of CaMKII with several other biochemical markers that identify specific neuron classes within Area X. Virtually all (approximately 98%) Area X cells expressing CaMKII also expressed DARPP-32 (dopamine- and adenosine 3'5'-monophosphate-regulated phosphoprotein), a dopamine signaling protein enriched in medium spiny striatal neurons. The implication that medium spiny neurons are primary mediators of the pCaMKII response to tutoring is interesting in view of the established dopaminergic modulation of LTP in this cell type. Additionally, BrdU and DARPP-32 immunocytochemistry were combined to test whether medium spiny neurons are among the neurons generated and incorporated into Area X during song learning. Based upon their expression of DARPP-32, the majority of Area X neurons labeled by BrdU injections given on posthatch days 20-25 are medium spiny neurons.

Neurogenesis and sensitive periods in avian song learning.

In many species of birds the propensity to learn songs from conspecifics is greatest during one or more distinct periods in life. These 'sensitive' learning periods, together with our detailed knowledge of the neural circuitry controlling avian song, have facilitated the discovery of radical neuroanatomical changes that accompany vocal development. One of the most remarkable of these changes is the production and incorporation of new, song-related neurons. The neurogenesis of specific cell types during song development helps create and recreate motor pathways for song production and provides synaptic plasticity that may both encourage and temporally constrain learning.

Sexual differentiation of androgen accumulation within the zebra finch brain through selective cell loss and addition.

In zebra finches males, but not females, respond to gonadal androgens with the production of a stereotyped courtship song. Corresponding to this dimorphism in hormone sensitivity, two song-related neural regions, magnocellular nucleus of the anterior neostriatum (MAN) and hyperstriatum ventralis pars caudalis (HVc), have more cells that accumulate androgens in males than in females. The early hormonal environment establishes these sex differences in the brain and behavior such that females treated with estradiol (E2) shortly after hatching have male-typical levels of androgen accumulation in MAN and HVc, and respond to adult androgen stimulation with male-typical song. Using autoradiographic procedures employing the androgen 3[H]-dihydrotestosterone (DHT), the present study focused on whether E2 masculinizes androgen target cell numbers in MAN and HVc by inducing, or rather preserving, a male-typical number of androgen target cells. First, it was demonstrated that E2 treatment of females as late as 20 days after hatching increases the number of MAN and HVc cells accumulating 3[H]-DHT or its metabolites in adulthood to levels typical of males. Then the number of androgen target cells present within these nuclei in 20-day-old females was compared with the number present in adult females previously masculinized with E2 treatment on day 20. In MAN, E2 masculinizes androgen accumulation by preserving androgen target cell numbers in the face of massive overall cell loss, a result that suggests that E2 promotes the survival of androgen target cells during sexual differentiation. In HVc, however, E2 treatment on day 20 promotes an addition of androgen target cells, suggesting that during adolescence, E2 regulates the proliferation, migration, and/or survival of these cells.

Ascending auditory projections to the inferior colliculus in the adult gerbil, Meriones unguiculatus.

Ascending auditory projections to the inferior colliculus (IC) of the adult gerbil were studied using the retrograde transport of horseradish peroxidase. Our results indicate that in gerbils, the IC receives afferent projections from most brainstem auditory nuclei. A strong contralateral projection originates in the cochlear nuclear complex (CN). A smaller but consistent projection from all three divisions of ipsilateral CN is also present. The medial superior olive (MSO), superior parolivary nucleus, and ventral nucleus of the lateral lemniscus all maintain ipsilateral projections to the IC. Bilateral projections arise from the lateral superior olive, lateral nucleus of the trapezoid body, and dorsal nucleus of the lateral lemniscus. Previous investigations in other mammalian species provide conflicting data concerning the magnitude of a direct ipsilateral projection from CN to the IC. Our quantitative data indicate that the ipsilateral projection from CN in the gerbil is nearly one third as large as the projection from ipsilateral MSO. The projection from contralateral CN is six times larger than the MSO projection. The distribution of labeled cells across the rostrocaudal extent of MSO and the three divisions of the cochlear nuclear complex are presented.

Ascending projections to the inferior colliculus following unilateral cochlear ablation in the neonatal gerbil, Meriones unguiculatus.

We evaluated the consequences of neonatal cochlear destruction upon ascending projections to the inferior colliculi. Unilateral cochlear ablations were performed in both neonatal and adult gerbils. Four to 12 months later, the inferior colliculus (IC) was examined physiologically and injected unilaterally with horseradish peroxidase (HRP). The number of labeled cells was determined bilaterally in all three divisions of cochlear nucleus (CN) and in the medial superior olive (MSO). In both experimental groups, transneuronal changes within the deafferented CN were greater in the ventral divisions than in the dorsal division. On the unoperated side the magnitude of projections from CN to the inferior colliculi was altered in animals lesioned as neonates. Following HRP injections into the IC on the unoperated side, the number of ipsilaterally labeled cells in CN (unoperated side) was significantly greater in the neonatal experimental group than in adult experimental and control animals. These anatomical changes were accompanied by increased ipsilaterally evoked excitatory activity recorded in the IC on the unoperated side. Following HRP injections into the IC on the ablated side, the number of contralaterally labeled cells in CN (unoperated side) was significantly reduced in animals lesioned as neonates as compared with control animals. The number of labeled cells in ipsilateral MSO was not significantly different across groups. Our interpretation is that unilateral cochlear ablation in neonatal gerbils results in an increase in the magnitude of ipsilateral projections and a decrease in the magnitude of contralateral projections from CN on the unoperated side to the inferior colliculi. These data suggest that the normal pattern of innervation of the IC results, in part, from interactions among afferent projections.

Hormonal control of neuron number in sexually dimorphic spinal nuclei of the rat: III. Differential effects of the androgen dihydrotestosterone.

The spinal cord of the rat contains two sexually dimorphic nuclei: the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN). These nuclei and the perineal muscles they innervate are present in males but reduced or absent in females. The sex difference in motoneuron number in these nuclei is due to an androgen-regulated motoneuron death. Developing females treated with the androgen testosterone propionate (TP) have a fully masculine number of SNB and DLN motoneurons and retain the perineal muscles they would normally have lost. Paradoxically, females treated prenatally with the androgen dihydrotestosterone propionate (DHTP) also retain the perineal musculature but as adults lack the SNB motoneurons which would normally innervate them. The SNB target muscles retained by DHTP females are anomalously innervated by motoneurons in the DLN. Counts of motoneurons and degenerating cells in the developing SNB of DHTP-treated females showed that their feminine number is the result of a failure of DHTP to prevent the death of SNB motoneurons. Furthermore, the peak number of SNB motoneurons was below that of normal females, suggesting that DHTP treatment may also have inhibited motoneuronal migration. However, DHTP treatment fully masculinized both motoneuron number and degenerating cell counts in the DLN of these females, and it is this masculinized DLN that gives rise to the anomalous projection. Taken together, these results suggest that the effects of different androgens during development are specific and complex, involving the regulation of motoneuron death, migration, and specification of peripheral projections.

Neural correlates of sensitive periods in avian song learning.

A wide range of neural events takes place during avian song learning, including changes in receptor systems and synaptic organization and the wholesale production and growth of new cells and their connections. Any (or all) of these events may be involved in song learning, and therefore their timing may restrict when auditory memories are laid down in the song system and when these memories are used to pattern vocal behavior. In particular, mounting evidence indicates that NMDA receptor activation participates in the acquisition of auditory memories during song learning, and developmental changes in the number and function of NMDA receptors within the song system may regulate the capacity for song learning. These developmental changes in NMDA receptors occur in conjunction with a period of synapse elimination, and NMDA receptor activation during song learning may regulate this regressive event. In addition, both developmental and seasonal periods of vocal learning correlate well with neuronal addition and turnover within the vocal motor pathway. This temporal overlap provides the opportunity for auditory experience to shape the initial organization of naive motor circuitry. Thus, a variety of cellular substrates present themselves as candidates for providing the neural plasticity necessary for song learning and may together regulate how and when experience is stored in the nervous system.

Characterization of neurons born and incorporated into a vocal control nucleus during avian song learning.

In male zebra finches, song learning is accompanied by the addition of new neurons to Area X, a nucleus necessary for normal song development. We examined whether Area X neurons born after 15 days post-hatch are recruited into the efferent pathway from Area X to the dorsolateral nucleus of the medial anterior thalamus (DLM). Our data suggest that the majority of these new Area X neurons are interneurons and that DLM-projecting neurons are incorporated into Area X prior to song learning.

Projections of androgen-accumulating neurons in a nucleus controlling avian song.

In zebra finches, androgens stimulate song production and promote growth of the neural regions controlling song. Early exposure to estrogen establishes this sensitivity to androgens and increases the number of androgen-accumulating cells in two song regions, the hyperstriatum ventralis pars caudalis (HVc) and the magnocellular nucleus of the anterior neostriatum. To examine if these regional changes in androgen accumulation could directly influence androgen responsiveness elsewhere in the song system, we combined autoradiographic and retrograde tracing techniques to determine if androgen-accumulating HVc neurons project to other vocal control nuclei. We report here that both major efferent projections from HVc (to Area X and the nucleus robustus of the archistriatum) include a substantial proportion of androgen-accumulating neurons. These data are consistent with the hypothesis that the extent of androgen accumulation in HVc may in turn regulate the androgenic sensitivity of other song regions.

Anatomical and ontogenetic factors producing variation in HVc neuron number in zebra finches.

The volume of nucleus HVc of the avian song system varies greatly both among and within songbird species, and is positively correlated to song complexity in many species. Moreover, the number of neurons in HVc predicts the ability of individual zebra finches to imitate song accurately. To better understand this brain/behavior relationship, we used the retrograde tracer Fast Blue to assess how specific HVc neuronal subpopulations contribute to variation in overall HVc neuron number in adult male zebra finches. We also investigated whether sibling order predicts the number of HVc neurons and/or yolk levels of testosterone, a hormone that might regulate the development of HVc. We report that total HVc neuron number is consistently and independently predicted by the size of each of its two projection populations, suggesting that the proportional makeup of HVc is tightly regulated at least in male zebra finches. Also, while we failed to detect a significant effect of sibling order on either the number of neurons in HVc or yolk testosterone concentration, we found that clutch of origin made a large contribution to variation in both early hormone levels and HVc neuron number.

Estrogen stimulates the incorporation of new neurons into avian song nuclei during adolescence.

In zebra finches the song control nuclei hyperstriatum ventralis pars caudalis (HVc) and area X of the lobus parolfactorius (LPO), continue to add new neurons during the juvenile period of song learning. Normally, males add many more of these new cells than do females (who do not sing), leading to pronounced sexual dimorphism within these regions. Exposing females to estradiol (E2) shortly after hatching masculinizes the HVc and area X and such females sing in response to later androgen stimulation. We investigated whether exposing female hatchlings to E2 stimulates the incorporation of HVc and area X neurons born during the juvenile period, and whether later androgen stimulation further influences addition of these late-generated neurons. Females were implanted with E2 on day 3 and received either empty or dihydrotestosterone (DHT)-filled capsules on day 25. These females, and normal males and females received [3H]thymidine daily between 20 and 40 days and were killed at 65 days. Autoradiographic analyses of HVc and area X-LPO revealed that neuron number, as well as the incidence and number of thymidine-labeled neurons was increased in E2-treated females to levels approaching those typical of males. DHT did not further influence these measures in females. These data indicate that E2 promotes either the production, migration, or survival of HVc and area X neurons born during the juvenile period.

Ontogeny of sex differences among newly-generated neurons of the juvenile avian brain.

In zebra finches, only males sing and brain regions controlling song exhibit sex differences in neuron number that stem from actions of estrogen during a critical developmental period. In certain song nuclei, these dimorphisms emerge long after neurogenesis and migration are complete, and estrogen promotes masculinization by preventing the death of well-differentiated neurons. But in another region, the higher vocal center (HVC), cellular mechanisms underlying sex differences in neuron number are not so well understood. In the HVC, neurogenesis continues throughout the post-hatch period of sexual differentiation, and sex differences arise during this time because neuron number increases in males but not females. We used [3H]thymidine autoradiography to establish when sex differences in neuron number first develop among a small group of HVC neuronal cohorts. We report that HVC neurons labeled by [3H]thymidine on days 15 and 16 after hatching are sexually dimorphic in number within 10 days of their birth, even before all cells in this cohort complete their migration and/or differentiation. This suggests that the cellular mechanisms contributing to sex differences in neuron number in the HVC may differ from those in other sexually dimorphic neural regions of the vertebrate nervous system. In addition, we found that although many thymidine-labeled HVC neurons ultimately project to the robust nucleus of the archistriatum (RA), a sexually dimorphic target, sex differences in their number develop before this efferent projection is established. These results have important implications regarding the site(s) of hormone action, since they suggest that sexual differentiation acts on certain HVC neurons before they establish their efferent projections, and perhaps even before they arrive within the HVC.

Estrogen promotes neuron addition to an avian song-control nucleus by regulating post-mitotic events.

Only male zebra finches sing and several telencephalic song control regions exhibit sex differences in neuron number that presumably reflect effects of estrogen (E2) exerted during the first few posthatch weeks. That is, implanting females with E2 during this time masculinizes neuron number and instills the capacity for vocal behavior. In certain song regions, E2 masculinizes neuron number by preventing the naturally-occurring death of neurons, long after their production, migration and process outgrowth are complete. However, in the Higher Vocal Center (HVC), the cellular mechanisms by which E2 establishes sex differences in neuron number are poorly understood. In contrast with other song regions, HVC neurogenesis overlaps with sexual differentiation and the incorporation of new neurons is greater in young males and E2-treated females, than in normal females. However, it is not known whether E2 promotes the addition of HVC neurons by stimulating their production, specification, and/or survival. To address this issue we injected males and females with [3H]thymidine on days 15 and 16 to label a small group of sexually dimorphic HVC neuronal cohorts born during sexual differentiation. Afterwards, on day 17, females were implanted with Silastic pellets filled with estradiol benzoate (EB) or left empty. We report here that EB exposure on day 17 masculinized (increased) the number of neurons in the HVC at day 35 that were labeled by [3H]thymidine injections on days 15/16. Thus, EB was able to increase cell number among at least some HVC neuronal cohorts after their final division, implying estrogenic regulation of post-mitotic events.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Sex and regional differences in the incorporation of neurons born during song learning in zebra finches.

In zebra finches only males sing, and several nuclei controlling song contain more neurons in adult males than in females. The ontogeny of sex differences in neuron number differs across song regions and overlaps with song learning in males. We examined the development of neuron number in several song regions in both sexes. We then determined whether neurons are born and incorporated into song nuclei as sex differences in neuron number emerge, and whether sex or regional differences in the insertion of such neurons may account for differences in the development of these areas. Males add neurons to hyperstriatum ventralis pars caudalis (HVc) and Area X between 20 and 55 d of age. In females there is no change in HVc neuron number during this time, and Area X never appears as a distinct nucleus. In both sexes, 3H-thymidine administration between 20 and 30 d results in neuronal labeling at 55 d in HVc and the region of Area X. However, in these areas the incidence of labeled neurons is higher in males than in females. In contrast to HVc and Area X, sex differences in neuron number in the robustus nucleus of the archistriatum (RA) and the magnocellular nucleus of the neostriatum (MAN) emerge because males retain neurons that are lost in females between 20 and 55 d of age. Accordingly, RA and MAN neurons are not labeled following 3H-thymidine administration between 20 and 30 d of age. These data indicate that sex and regional differences in the ontogeny of song nuclei are related to differences in the incorporation of neurons born during song learning.

Auditory feedback is necessary for the maintenance of stereotyped song in adult zebra finches.

Although songbirds rely on auditory input for normal song development, many species eventually attain adult song patterns that are thought to be maintained without reference to auditory feedback. In such species, it is believed that a central motor program for song is established when the stereotyped adult song pattern is achieved. However, we report here that in the Australian zebra finch, stereotyped song patterns gradually change in adult males following bilateral cochlear removal. By 16 weeks after surgery, deaf birds accurately reproduced only 36% of the song syllables produced prior to surgery. Moreover, on average, the phonology of over 50% of the syllables produced by deaf birds was either only slightly similar or unlike the phonology of any syllable produced prior to surgery. In contrast, control birds accurately retained over 90% of their syllables over a comparable time period and less than 5% of their syllables was unmatched or only slightly similar in phonology to previously recorded syllables. In many of the deafened birds, changes in song patterns were not evident until 6-8 weeks after surgery. These data indicate that continued auditory input is necessary to maintain the patterns of neural organization supporting learned song in zebra finches and raise questions concerning the neural sites and cellular mechanisms that mediate this feedback control.

Projection neurons within a vocal motor pathway are born during song learning in zebra finches.

Many birds learn song during a restricted 'sensitive' period. Juveniles memorize a song model, and then learn the pattern of muscle contractions necessary to reproduce the song. Of the neural changes accompanying avian song learning, perhaps the most remarkable is the production of new neurons which are inserted into the hyperstriatum ventralis pars caudalis (HVc), a region critical for song production. We report here that in young male zebra finches many of the new neurons incorporated into the HVc innervate the robust nucleus of the archistriatum (RA) which projects to motor neurons controlling the vocal musculature. Furthermore, far fewer of these new neurons are incorporated into the HVc of either adult males that are beyond the sensitive learning period, or young females (who do not develop song). Thus, a major portion of the vocal motor pathway is actually created during song learning. This may enable early sensory experience and vocal practice to not only modify existing neuronal circuits, but also shape the insertion and initial synaptic contacts of neurons controlling adult song.