Ernest J. Nordeen

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.

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.

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.

Anatomical and synaptic substrates for avian song learning.

In songbirds, vocal learning occurs during periods of major cellular and synaptic change. This neural reorganization includes massive synaptogenesis associated with the addition of new neurons into the vocal motor pathway, as well as pruning of connections between song regions. These observations, coupled with behavioral evidence that song development requires NMDA receptor activation in specific song nuclei, suggest that experiences associated with vocal learning are encoded by activity driven, Hebbianlike processes of synaptic change akin to those implicated in many other forms of developmental plasticity and learning. In this review we discuss the hypothesis that develpmental and/or seasonal changes in NMDA receptor function and the availability of new synapses may modulate thresholds for plasticity and thereby define sensitive periods for vocal learning.

Long-term maintenance of song in adult zebra finches is not affected by lesions of a forebrain region involved in song learning

The long-term maintenance of stable song patterns produced by adult male zebra finches depends upon auditory feedback. It is not known which song-related brain areas process this auditory information, in part because previous functional studies in adult birds have not been of sufficient duration to detect behavioral changes that might result from interference with auditory feedback mechanisms. In the present study, adult males were subjected to bilateral lesions of a nucleus known to be necessary for auditory-based song learning, the lateral portion of the magnocellular nucleus of the anterior neostriatum (IMAN). Songs were then recorded over a period of time during which deficits due to removal of auditory feedback become apparent. Our data suggest that the IMAN does not mediate the stabilizing influence of auditory feedback on adult song production. That is, while deafening produces alterations in song structure within 16 weeks, bilateral destruction of the IMAN did not produce any substantial changes in song over this same period. These results extend a previous report by Bottjer, Miesner, and Arnold, Science, 224, 901-903 (1985) and suggest that the pathways through which auditory feedback affects adult song behavior do not involve the IMAN.

Sexual Dimorphisms in the Neural Vocal Control System in Song Birds: Ontogeny and Phylogeny

Sex differences in the neural song system in oscine song birds develop in response to estradiol secreted during early periods of development. Estradiol produces sex differences in cell number and in the proportion of cells which are steroid targets. The pattern of development of these sex differences varies in different brain regions, suggesting that the mechanisms of estradiol regulation of neural development may also vary. The magnitude of sexual dimorphism in the neural song system varies across species, and is generally correlated with the magnitude of sexual dimorphism in vocal ability. Large species differences in neural structure can potentially be explained by small differences in the ontogenetic pattern of estradiol secretion, as is suggested by studies of neural development.

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)

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.

Early sensory and hormonal experience modulate age-related changes in NR2B mRNA within a forebrain region controlling avian vocal learning

Male zebra finches are most apt to mimic songs heard between posthatch days (PHD) 35 and 65, and this vocal learning depends, in part, on the activation of N-methyl-D-aspartate receptors (NMDAR) within a discrete forebrain circuit that includes the lateral magnocellular nucleus of the anterior neostriatum (lMAN) and area X. Using in situ hybridization, we show that transcripts for both the constitutive NMDAR subunit NR1 and the modulatory subunit NR2B decrease abruptly in the lMAN between PHD20 and 40. This downregulation corresponds to the onset of song learning and a transition from slow to faster NMDAR currents in lMAN neurons. In area X, NR1 mRNA increases as NR2B mRNA decreases during song development. To understand how these changes in NMDAR mRNA might regulate song learning, we next investigated how manipulations that influence song development affect NMDAR mRNA expression. Early isolation from conspecific song (which delays closure of the sensitive period for song learning) selectively increases NR2B, but not NR1 mRNA, within lMAN at PHD60. In contrast, exposure to testosterone beginning at PHD20 (which impairs song development and hastens the developmental transition to faster NMDAR current kinetics within lMAN) accelerates the decline in NR2B mRNA in lMAN, again without affecting NR1 transcript levels. Neither manipulation significantly effects NR1 or NR2B mRNA levels in area X. Our data suggest that developmental changes in the expression of specific NMDAR subunits may regulate periods of neural and behavioral plasticity and that flexibility in the timing of these sensitive periods may be achieved through experience and/or hormone-dependent modulation of NMDAR gene expression.

Developmental regulation of NMDA receptor 2B subunit mRNA and ifenprodil binding in the zebra finch anterior forebrain

In passerine songbirds, song learning often is restricted to an early sensitive period and requires the participation of several discrete regions within the anterior forebrain. Activation of N-methyl-D-aspartate (NMDA) receptors is implicated in song learning and in one forebrain song region, the lateral magnocellular nucleus of the anterior neostriatum (IMAN), NMDA receptors decrease in density, their affinity for the antagonist MK-801 increases, and their currents decay more quickly as young male zebra finches lose the ability to imitate new song elements. These developmental changes in NMDA receptor pharmacology and physiology suggest that the subunit composition of NMDA receptors changes developmentally. Here, we have used in situ hybridization and [3H]ifenprodil receptor autoradiography to study the developmental regulation of the NMDA receptor 2B subunit (NR2B) within the anterior forebrain of male zebra finches. NR2B mRNA expression within the IMAN was twice as great in 30-day-old males (early in the sensitive period for song learning) as in adult males, and this developmental decrease in NR2B mRNA expression was mirrored by a decrease in high-affinity (NR2B-associated) [3H]ifenprodil binding within this song region. In another anterior forebrain song region, Area X, NR2B mRNA also declined significantly after 30 days posthatch, but this decline was not accompanied by a significant decrease in [3H]ifenprodil binding. The results are consistent with the hypothesis that developmental changes in NMDA receptor function mediated by regulation of subunit composition contribute to the sensitive period for vocal learning in birds.