Estrogen-inducible, sex-specific expression of brain-derived neurotrophic factor mRNA in a forebrain song control nucleus of the juvenile zebra finch

The expression of brain-derived neurotrophic factor (BDNF) mRNA is increased significantly within the high vocal center (HVc) of male but not female zebra finches from posthatching day 30-35 on. The population of HVc cells expressing BDNF mRNA included 35% of the neurons projecting to the nucleus robustus of the archistriatum (RA). In the RA and in RA-projecting neurons of the lateral portion of the magnocellular nucleus of the anterior neostriatum, BDNF mRNA was expressed at very low levels in both sexes. The BDNF-receptor trkB mRNA was expressed in the RA, in RA-projecting neurons of lateral portion of the magnocellular nucleus of the anterior neostriatum, and in the HVc, except in most of its RA-projecting neurons. Premature stimulation and an inhibitory effect on the normal increase of the BDNF mRNA expression in juvenile males occurred after treatments with 17beta-estradiol and the aromatase inhibitor fadrozole, respectively. The up-regulation of the BDNF expression in the HVc could be a mechanism by which estrogen triggers the differentiation of cells within and connected to the HVc of male zebra finches.     

Song-selective auditory circuits in the vocal control system of the zebra finch.

Birdsong is a learned behavior controlled by a distinct set of brain nuclei. The song nuclei known as area X, the medial nucleus of the dorsolateral thalamus (DLM), and the lateral magnocellular nucleus of the anterior neostriatum (L-MAN) form a pathway that plays an important but unknown role in song learning. One function served by this circuit might be auditory feedback, which is critical to normal song development. We used single unit recordings to demonstrate that all three of these nuclei contain auditory neurons in adult male zebra finches (Taeniopygia guttata). These neurons are song selective: they respond more robustly to the bird's own song than to songs of conspecific individuals, and they are sensitive to the temporal structure of song. Auditory neurons so highly specialized for song within a pathway required for song learning may play a role in the auditory feedback essential in song development. Recordings in the robust nucleus of the archistriatum (RA), the nucleus to which L-MAN projects, showed that RA also contains highly song-selective neurons. RA receives a direct projection from the caudal nucleus of the ventral hyperstriatum (HVc) as well as from L-MAN. We investigated the contributions of these two inputs to auditory responses of RA neurons by selectively inactivating one or both inputs. Our results suggest that there is a song-selective pathway directly from HVc to RA in addition to the circuit via L-MAN. Thus the songbird brain contains multiple auditory pathways specialized for song, and these circuits may vary in their functional importance at different stages of learning.     

Act locally and think globally: intracerebral testosterone implants induce seasonal-like growth of adult avian song control circuits

There is pronounced seasonal plasticity in the morphology of the neural circuits that regulate song behavior in adult songbirds, primarily in response to changes in plasma testosterone (T) levels. Most song nuclei have androgen receptors. Afferent input from the telencephalic nucleus HVc (also known as the "high vocal center") is necessary for seasonal growth of the direct efferent target nuclei RA and area X. We asked here whether T-stimulated growth of HVc is sufficient to induce growth of its efferent nuclei. Intracerebral T implants were placed unilaterally near HVc or RA in photosensitive adult male white-crowned sparrows for one month. The T implant near HVc produced significant growth of the ipsilateral (but not contralateral) HVc, RA, and area X, and increased neuronal number in the ipsilateral HVc. The T implant near RA did not produce selective growth of ipsilateral RA, HVc, or area X. Intracerebral T implants did not elevate plasma T levels, nor did they stimulate growth of two peripheral androgen sensitive targets, the syrinx and the cloacal protuberance. These results suggest that seasonal growth of the adult song circuits results from T acting directly on HVc, which then stimulates the growth of RA and area X transynaptically.     

Neuronal production, migration, and differentiation in a vocal control nucleus of the adult female canary brain.

The vocal control nucleus designated HVc (hyperstriatum ventrale, pars caudalis) of adult female canaries expands in response to systemic testosterone administration, which also induces the females to sing in a male-like manner. We became interested in the possibility of neurogenesis as a potential basis for this phenomenon. Intact adult female canaries were injected with [3H]thymidine over a 2-day period. Some birds were given testosterone implants at various times before thymidine. The birds were sacrificed 5 wk after hormone implantation, and their brains were processed for autoradiography. In parallel control experiments, some birds were given implants of cholesterol instead of testosterone. All birds showed considerable numbers of labeled neurons, glia, endothelia, and ventricular zone cells in and around HVc. Ultrastructural analysis confirmed the identity of these labeled neurons. Cholesterol- and testosterone-treated birds had similar neuronal labeling indices, which ranged from 1.8% to 4.0% in HVc. Thus, neurogenesis occurred in these adults independently of exogenous hormone treatment. Conversely, both glial and endothelial proliferation rates were markedly stimulated by exogenous testosterone treatment. We determined the origin of the thymidine-incorporating neurons by sacrificing two thymidine-treated females soon after their thymidine injections, precluding any significant migration of newly labeled cells. Analysis of these brains revealed no cells of neuronal morphology present in HVc but a very heavily labeled ventricular zone overlying HVc. We conclude that neuronal precursors exist in the HVc ventricular zone that incorporate tritiated thymidine during the S phase preceding their mitosis; after division these cells migrate into, and to some extent beyond, HVc. This ventricular zone neurogenesis seems to be a normally occurring phenomenon in intact adult female canaries.     

Antiestrogens fail to prevent the masculine ontogeny of the zebra finch song system

Early treatment with the antiestrogen, tamoxifen, fails to block the ontogeny of the male zebra finch song system which is hypothesized to occur as a result of early estradiol action. In Experiment 1, two other antiestrogens, LY117018 or CI628, or vehicle was administered daily to zebra finch chicks for the first 20 days after hatching at which time the males were castrated. Comparisons of experimental and control brains at 60 days revealed that neither antiestrogen prevented the masculinization of the song system in males. Rather, both compounds increased (hypermasculinized) neuronal soma area in male MAN (magnocellular nucleus of the anterior neostriatum), DLRA (dorsolateral portion of the robust nucleus of the archistriatum), and in HVc (caudal nucleus of the ventral hyperstriatum). In females both compounds masculinized by increasing neuronal soma area in HVc and inducing the formation of Area X. Experiment 2 showed that neither LY117018 nor CI628 was effective in preventing the masculinization of the song system typical of 25-day-old males when administered daily from hatching until sacrifice. Rather, both compounds masculinized females by inducing the formation of Area X, and LY117018 increased RA volume. LY117018 hypermasculinized males by increasing HVc volume and size of neuronal somata in MAN, HVc, and DLRA. CI628 also hypermasculinized males by increasing RA volume and neuronal soma size in HVc and RA. The failure of the present compounds to block masculinization of the song system and the paradox of hypermasculinization by antiestrogens are discussed with reference to the estradiol-masculinization hypothesis.     

Auditory representation of autogenous song in the song system of white-crowned sparrows

The HVc (hyperstriatum ventrale, pars caudale) is a forebrain nucleus in the motor pathway for the control of song. Neurons in the HVc also exhibit auditory responses. A subset of these auditory neurons in the white-crowned sparrow (Zonotrichia leucophrys) have been shown to be highly selective for the individual bird's own (autogenous) song. By using multiunit recording techniques to sample from a large population, we demonstrate that the entire population of auditory neurons in the HVc is selective for autogenous song. The selectivity of these neurons must reflect the song-learning process, for the acoustic parameters of a sparrow's song are acquired by learning. By testing with laboratory-reared birds, we show that HVc auditory neurons prefer autogenous song over the tutor model to which the birds were exposed early in life. Thus, these neurons must be specified at or after the time song crystallizes. Since song is learned by reference to auditory feedback, HVc auditory neurons may guide the development of the motor program for song. The maintenance of a precise auditory representation of autogenous song into adulthood can contribute to the ability to distinguish the fine differences among conspecific songs.     

Testosterone increases the recruitment and/or survival of new high vocal center neurons in adult female canaries.

New neurons are added to the high vocal center (HVC) of adult male and female canaries. Exogenous testosterone induces a marked increase in HVC size in adult female canaries, though the mechanisms responsible for this increase remain unknown. To understand the mechanisms, we analyzed the effects of testosterone on neuronal recruitment in the female HVC. Intact adult female canaries received Silastic implants that were empty or filled with testosterone. Birds in the short-survival group received the Silastic implant, followed by a single injection of [3H]thymidine 2 days later, and were killed on the following day. Birds in the long-survival group were injected once a day for 5 days with [3H]thymidine and received the Silastic implant 20 and 40 days later. These birds were killed 60 days after the first injection of [3H]thymidine. The number of 3H-labeled ventricular zone cells above, rostral, or caudal to HVC was not affected by the hormone treatment in the short-survival birds, suggesting that testosterone did not affect neuronal production. However, the number of 3H-labeled HVC neurons that projected to robust nucleus of the archistriatum (RA) in the long-survival birds was three times greater in the hormone-treated than in the control group, though the total number of RA-projecting cells did not change significantly. Testosterone also induced an increase in the size of the HVC cells that project to RA. Thus, these experiments suggest that testosterone affects the recruitment and/or survival of newly generated RA-projecting HVC neurons but does not affect their production.     

Interaction between auditory and motor activities in an avian song control nucleus.

Discrete telencephalic nuclei HVc (hyperstriatum ventrale, pars caudale) and RA (nucleus robustus archistriatalis) have been implicated by lesion studies in the control of vocalization in songbirds. We demonstrate directly the role of HVc in vocalization by presenting neuronal recordings taken from HVc of singing birds. Intracellular recordings from anesthetized birds have shown that many neurons in HVc respond to auditory stimuli. We confirm this result in the extracellular recordings from awake-behaving birds and further demonstrate responses of HVc neurons to playback of the bird's own song. The functional significance of these responses is not yet clear, but behavioral studies show that auditory feedback plays a crucial role in the development of normal song. We show that the song-correlated temporal pattern of neural activity persists even in the deaf bird. Furthermore, we show that in the normal bird, the activity pattern correlated with production of certain song elements can be clearly distinguished from the pattern of auditory responses to the same song elements. This result implies that an interaction occurs in HVc of the singing bird between motor and auditory activity. Through experiments involving playback of sound while the bird is singing, we show that the interaction consists of motor inhibition of auditory activity in HVc and that this inhibition decays slowly over a period of seconds after the song terminates.     

Two distinct inputs to an avian song nucleus activate different glutamate receptor subtypes on individual neurons

Although neural circuits mediating various simple behaviors have been delineated, those generating more complex behaviors are less well described. The discrete structure of avian song control nuclei promises that circuits controlling complex behaviors, such as birdsong, can also be understood. To this end, we developed an in vitro brain slice preparation containing the robust nucleus of the archistriatum (RA), a forebrain song control nucleus, and its inputs from two other song nuclei, the caudal nucleus of the ventral hyperstriatum (HVc) and the lateral part of the magnocellular nucleus of the anterior neostriatum (L-MAN). Using intracellular recordings, we examined the pharmacological properties of the synapses made on RA neurons by L-MAN and HVc axons. Electrical stimulation of the L-MAN and the HVc fiber tracts evoked excitatory postsynaptic potentials (EPSPs) from >70% of RA neurons when slices were prepared from male birds of 40-90 days of age, suggesting that many individual RA neurons receive excitatory input from L-MAN and HVc axons. The "L-MAN" EPSPs were blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist D-(-)-2-amino-5-phosphonovaleric acid (D-APV) as well as the broad-spectrum glutamate receptor antagonist kynurenic acid but were relatively unaffected by the non-NMDA receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In contrast, "HVc" EP-SPs were relatively insensitive to D-APV but almost completely abolished by CNQX. These experiments suggest that L-MAN and HVc axons provide pharmacologically distinct types of excitatory input to many of the same RA neurons.     

A relationship between behavior, neurotrophin expression, and new neuron survival

The high vocal center (HVC) controls song production in songbirds and sends a projection to the robust nucleus of the archistriatum (RA) of the descending vocal pathway. HVC receives new neurons in adulthood. Most of the new neurons project to RA and replace other neurons of the same kind. We show here that singing enhances mRNA and protein expression of brain-derived neurotrophic factor (BDNF) in the HVC of adult male canaries, Serinus canaria. The increased BDNF expression is proportional to the number of songs produced per unit time. Singing-induced BDNF expression in HVC occurs mainly in the RA-projecting neurons. Neuronal survival was compared among birds that did or did not sing during days 31-38 after BrdUrd injection. Survival of new HVC neurons is greater in the singing birds than in the nonsinging birds. A positive causal link between pathway use, neurotrophin expression, and new neuron survival may be common among systems that recruit new neurons in adulthood.     

Two separate areas of the brain differentially guide the development of a song control nucleus in the zebra finch.

A brain nucleus that is important for the generation of song in the adult male zebra finch (Poephila guttata), the robust nucleus of the archistriatum (RA), receives dual inputs from two other telencephalic song nuclei: the hyperstriatum ventrale pars caudale (HVc) and the lateral magnocellular nucleus of the anterior neostriatum (L-MAN). We lesioned each of these afferent inputs to the RA early in development, either by themselves or both at the same time in the same side of the brain, to determine what influences each of these nuclei exerts on the normal development of the RA into adulthood. We found that lesioning the HVc in a 20-day-old male zebra finch prevents the later increase in RA volume and soma size that would normally occur around 35 days post-hatching. MAN lesions at this same early age, on the other hand, had a large effect on reducing the volume and cell number of RA neurons, without affecting soma size. Lesioning both inputs early in development induced considerable RA neuronal cell death and atrophy of the nucleus. This study shows that the development of the RA is affected differently by each of its two input nuclei.     

Mechanisms underlying the sensitivity of songbird forebrain neurons to temporal order.

Neurons in the songbird forebrain area HVc (hyperstriatum ventrale pars caudale or high vocal center) are sensitive to the temporal structure of the bird's own song and are capable of integrating auditory information over a period of several hundred milliseconds. Extracellular studies have shown that the responses of some HVc neurons depend on the combination and temporal order of syllables from the bird's own song, but little is known about the mechanisms underlying these response properties. To investigate these mechanisms, we recorded intracellular responses to a set of auditory stimuli designed to assess the degree of dependence of the responses on temporal context. This report provides evidence that HVc neurons encode information about temporal structure by using a variety of mechanisms including syllable-specific inhibition, excitatory postsynaptic potentials with a range of different time courses, and burst-firing nonlinearity. The data suggest that the sensitivity of HVc neurons to temporal combinations of syllables results from the interactions of several cells and does not arise in a single step from afferent inputs alone.     

Auditory representation of the vocal repertoire in a songbird with multiple song types

Neural mechanisms for representing complex communication sounds must solve the problem of encoding multiple and potentially overlapping signals. Birdsong provides an excellent model for such processing, in that many songbird species produce multiple song types. Although auditory song representations in single song type species have been studied, how song is represented in the brains of species that sing multiple song types remains unknown. Here we examine song type representations in swamp sparrows (Melospiza georgiana), a multiple song type species, by making in vivo intracellular recordings from the telencephalic nucleus HVc, the major auditory-vocal interface in the songbird brain. These recordings show that single HVc relay neurons often generate action potentials to playback of only a single song type, even though synaptic inputs on these cells can be activated by playback of other song types in the bird's repertoire and songs of other birds. These subthreshold response patterns suggest that the song evoked action potential discharge of a single relay neuron is more selective than its presynaptic network. One component of this presynaptic network is likely to be in HVc, because multiple recordings from single birds show that different relay neurons can respond best to different song types, whereas single interneurons can generate action potentials to all song types in the bird's repertoire. These results show that single HVc neurons can generate song type-specific action potential responses, a feature that may facilitate the selective auditory encoding of multiple learned vocalizations in a single brain area.     

Androgen receptor, estrogen receptor alpha, and estrogen receptor beta show distinct patterns of expression in forebrain song control nuclei of European starlings.

In songbirds, singing behavior is controlled by a discrete network of interconnected brain nuclei known collectively as the song control system. Both the development of this system and the expression of singing behavior in adulthood are strongly influenced by sex steroid hormones. Although both androgenic and estrogenic steroids have effects, androgen receptors (AR) are more abundantly and widely expressed in song nuclei than are estrogen receptors (ER alpha). The recent cloning of a second form of the estrogen receptor in mammals, ER beta, raises the possibility that a second receptor subtype is present in songbirds and that estrogenic effects in the song system may be mediated via ER beta. We therefore cloned the ER beta complementary DNA (cDNA) from a European starling preoptic area-hypothalamic cDNA library and used in situ hybridization histochemistry to examine its expression in forebrain song nuclei, relative to the expression of AR and ER alpha messenger RNA (mRNA), in the adjacent brain sections. The starling ER beta cDNA has an open reading frame of 1662-bp, predicted to encode a protein of 554 amino acids. This protein shares greater than 70% sequence identity with ER beta in other species. We report that starling ER beta is expressed in a variety of tissues, including brain, pituitary, skeletal muscle, liver, adrenal, kidney, intestine, and ovary. Similar to reports in other songbird species, we detected AR mRNA-containing cells in several song control nuclei, including the high vocal center (HVc), the medial and lateral portions of the magnocellular nucleus of the anterior neostriatum, and the robust nucleus of the archistriatum. We detected ER alpha expression in the medial portion of HVc (also called paraHVc) and along the medial border of the caudal neostriatum. ER beta was not expressed in HVc, in the medial and lateral portions of the magnocellular nucleus of the anterior neostriatum, in the robust nucleus of the archistriatum, or in area X. In contrast, ER beta mRNA-containing cells were detected in the caudomedial neostriatum and medial preoptic area in a pattern reminiscent of P450 aromatase expression in the same brain regions in other songbirds. These data suggest that estrogenic effects on the song system are not mediated via ER beta-producing cells within song nuclei. Nonetheless, the overlapping expression of ER beta- and aromatase-producing cells in the caudomedial neostriatum suggests that locally synthesized estrogens may act via ER beta, in addition to ER alpha, to mediate seasonal or developmental effects on nearby song nuclei (e.g. HVc).     

Transient expression and transport of brain-derived neurotrophic factor in the male zebra finch’s song system during vocal development

The distribution of brain-derived neurotrophic factor (BDNF) in the song system of male zebra finches changes with posthatching age. At day 20, the hyperstriatum ventrale, pars caudale is the only song nucleus in which neurons showed BDNF immunoreactivity. At day 45, the staining in hyperstriatum ventrale, pars caudale was denser than at day 20 and the robust nucleus of the archistriatum, another song nucleus, showed BDNF labeling. By day 65, two additional song nuclei, area X and the lateral magnocellular nucleus of the anterior neostriatum, have become immunoreactive. In the adult, however, the amount of BDNF labeling in all of these brain nuclei is sharply reduced. These sequential events, the anatomical connections between these song nuclei, and the labeling of relevant axons and terminals suggest anterograde transport of BDNF. Furthermore, the timing of BDNF expression coincident with the development of singing behavior suggests that this neurotrophin may be directly involved with the differentiation of the song system.     

Feedback circuitry within a song-learning pathway.

The song system of birds consists of several neural pathways. One of these, the anterior forebrain pathway, is necessary for the acquisition but not for the production of learned song in zebra finches. It has been shown that the anterior forebrain pathway sequentially connects the following nuclei: the high vocal center, area X of lobus parolfactorius, the medial portion of the dorsolateral thalamic nucleus, the lateral magnocellular nucleus of anterior neostriatum (IMAN), and the robust nucleus of the archistriatum (RA). We now show in zebra finches (Taeniopygia guttata) that IMAN cells that project to RA also project to area X, forming a feedback loop within the anterior forebrain pathway. The axonal endings of the IMAN projection into area X form cohesive and distinct domains. Small injections of tracer in subregions of area X backfill a spatially restricted subset of cells in IMAN, that, in turn, send projections to RA that are arranged in horizontal layers, which may correspond to the functional representation of vocal tract muscles demonstrated by others. We infer from our data that there is a myotopic representation throughout the anterior forebrain pathway. In addition, we suggest that the parcellation of area X into smaller domains by the projection from IMAN highlights a functional architecture within X, which might correspond to units of motor control, to the representation of acoustic features of song, or both.     

Growth and atrophy of neurons labeled at their birth in a song nucleus of the zebra finch.

The robust nucleus of the archistriatum (RA) is one of the forebrain nuclei that control song production in birds. In the zebra finch (Poephila guttata), this nucleus contains more and larger neurons in the male than in the female. A single injection of tritiated thymidine into the egg on the 6th or 7th day of incubation resulted in labeling of many RA neurons with tritium. The size of tritium-labeled neurons and the tissue volume containing them did not differ between the sexes at 15 days after hatching. In the adult brain, tritium-labeled neurons and the tissue volume containing them were much larger in the male than in the female. Also, tritium-labeled RA neurons were large in females which received an implant of estrogen immediately after hatching. The gender differences in the neuron size and nuclear volume of the zebra finch RA are, therefore, due not to the replacement of old neurons by new ones during development but to the growth and atrophy of neurons born before hatching. Similarly, the masculinizing effects of estrogen on the female RA are due not to neuronal replacement but to the prevention of atrophy and promotion of growth in preexisting neurons.     

Postsynaptic neural activity regulates neuronal addition in the adult avian song control system

A striking feature of the nervous system is that it shows extensive plasticity of structure and function that allows animals to adjust to changes in their environment. Neural activity plays a key role in mediating experience-dependent neural plasticity and, thus, creates a link between the external environment, the nervous system, and behavior. One dramatic example of neural plasticity is ongoing neurogenesis in the adult brain. The role of neural activity in modulating neuronal addition, however, has not been well studied at the level of neural circuits. The avian song control system allows us to investigate how activity influences neuronal addition to a neural circuit that regulates song, a learned sensorimotor social behavior. In adult white-crowned sparrows, new neurons are added continually to the song nucleus HVC (proper name) and project their axons to its target nucleus, the robust nucleus of the arcopallium (RA). We report here that electrical activity in RA regulates neuronal addition to HVC. Decreasing neural activity in RA by intracerebral infusion of the GABA_A receptor agonist muscimol decreased the number of new HVC neurons by 56%. Our results suggest that postsynaptic electrical activity influences the addition of new neurons into a functional neural circuit in adult birds.The ongoing birth and incorporation of neurons into functional neural circuits in the central nervous system of higher vertebrates was first demonstrated conclusively in songbirds and subsequently in rodents, nonhuman primates, and humans (reviewed in ref. 1). Because new neuron generation continues throughout adulthood, the fundamental importance and the clinical implications of neurogenesis are clear. The mechanisms by which new neurons integrate into functional neural circuits, however, are not well understood.Songbirds provide a tractable model for understanding the mechanisms that regulate new neuron addition into functional circuits. Song is a learned sensorimotor behavior that is important for songbird reproduction. Song learning and production are regulated by a discrete, well-characterized neural circuit that includes HVC (proper name) and its target nucleus, the robust nucleus of the arcopallium (RA), both located in the avian forebrain (Fig. 1A) (2). In the adult Gambel’s white-crowned sparrow (WCS), the song control system shows extreme seasonal neuroplasticity (reviewed in ref. 3). Early in the breeding season, HVC and RA of WCS nearly double in volume. The increase in HVC volume results largely from an increase in new neuron incorporation, whereas the increase in RA volume results from increases in neuron size and spacing, but not number. RA neurons also show increased spontaneous electrical activity in the breeding season (4, 5). WCS typically produce only one song type that is longer and more stereotyped in structure during the breeding season (6, 7).Open in a separate windowFig. 1.Inhibition of RA neural activity by muscimol infusion decreases HVC neuronal addition. Birds were injected with BrdU to label adult-born neurons, and muscimol, a GABA_A receptor agonist, was infused unilaterally near RA to inhibit its neural activity. (A) A coronal schematic of the song-control system showing major song system projections. Black arrows show projections in the motor output circuit; red arrows show bilateral projections; blue arrows show recursive projections; and green dashed arrow shows a weak projection from RA to HVC. (B) Experimental timeline. (C) A representative image of BrdU and Hu immunolabeling in HVC. BrdU is shown in red, and Hu, a neuronal marker, is shown in green. Arrows show BrdU-positive neurons. The arrowhead indicates a BrdU-positive cell that does not colabel with Hu. (D) The number of BrdU-positive neurons in HVC at time of death. The number of Hu- and BrdU-colabeled neurons in HVC is significantly lower in the hemisphere ipsilateral to muscimol infusion than in the contralateral, uninfused hemisphere and is lower than either hemisphere in vehicle-infused controls. *P ≤ 0.05. Contralateral hemispheres are shown in light gray, and ipsilateral are in black. All data are presented as mean ± SEM.HVC contains three types of neurons: HVC→RA and HVC→area X projection neurons and interneurons. During seasonal growth, most, if not all, neurons incorporated into HVC project to RA (ref. 8, but see ref. 9). Neural progenitor cells are born at the dorsal and ventral portion of the lateral ventricle and migrate from the ventricular zone (VZ) to HVC within 1 wk after birth (10). Over the next 2–3 wk, new neurons send axonal projections to RA (11). These new HVC→RA projection neurons are functional; they can fire action potentials in response to sound stimuli (12).Environment and experience play important roles in both brain development and adult neurogenesis. For example, during embryonic development, neural activity in the visual cortex is required for target selection by axons from the lateral geniculate nucleus (13). In adult rodents, voluntary exercise and hippocampal-dependent learning enhance neurogenesis in the dentate gyrus (reviewed in ref. 1). In adult songbirds, auditory experience and song production influence neuronal turnover in HVC (14–16). This literature suggests that adult neurogenesis in the vertebrate brain is activity-dependent. In vitro studies show that excitatory stimuli act directly on hippocampal neural progenitor cells and promote survival of new neurons (reviewed in ref. 17). Thus, activity-dependent mechanisms likely influence neuronal recruitment in vivo in both developing and adult brains.One factor that may influence the recruitment to and survival of new neurons in HVC is the electrical activity of their postsynaptic targets in RA (5). We hypothesized that inhibiting the electrical activity in RA neurons in vivo would reduce neuronal addition to adult HVC. We show that decreasing RA electrical activity does indeed reduce neuronal addition to HVC, indicating that target activity is essential for appropriate neuronal addition to HVC.     

Melatonin receptor density in Area X of European starlings is correlated with reproductive state and is unaffected by plasma melatonin concentration

Several of the song control nuclei of songbirds, including HVc (higher vocal center) and Area X, contain melatonin receptor (MelR). In laboratory-housed male starlings, the densities of MelR in Area X change markedly according to reproductive state. MelR are down-regulated when starlings are photostimulated (in full breeding condition) and are subsequently up-regulated when starlings become photorefractory (reproductively quiescent). However, seasonal regulation of MelR densities in Area X has only been investigated during the light phase of the light:dark cycle. Variation in MelR densities are physiologically relevant only if they also occur during the dark phase, when melatonin is present in the circulation. Brains from male starlings that were in different reproductive states but exposed to the same 18L:6D photoperiod were collected during either the mid-point of the light phase or the dark phase. Melatonin receptor distribution was assessed in vitro by 125Iodomelatonin (IMEL) receptor autoradiography. All photostimulated birds exhibited down-regulation of MelR in Area X, and all photorefractory birds exhibited high MelR density in Area X, regardless of time of sampling or plasma melatonin concentration. Thus, within each reproductive state, MelR density in Area X did not differ over the course of a circadian cycle. The functional significance of seasonal regulation of MelR in this song control nucleus remains unclear, but it is likely to involve a release of cellular inhibition by melatonin during photostimulation, with possible consequences for song learning, memory consolidation or regulation of the context of song production.     

Seasonal growth of song control nuclei precedes seasonal reproductive development in wild adult song sparrows

In seasonally breeding adult songbirds, the brain regions that control song undergo dramatic seasonal morphological changes. During late winter and early spring, increasing day length triggers an increase in circulating testosterone that ultimately causes several song nuclei to grow in volume. The timing of this growth relative to the seasonal development of the reproductive system is not known. This question was investigated in two populations of wild song sparrows (Melospiza melodia morphna). Both populations live at the same latitude (46 degrees N), but breed at different altitudes. One population resides on the Pacific coast in Washington, and the other resides in the foothills of the Cascade Mountains. Both populations experienced the same photoperiodic conditions, but the timing of seasonal reproductive development differed between populations. Coastal birds initiated gonadal recrudescence approximately 2 weeks earlier than montane birds. Despite this temporal difference in reproductive development, there were no differences between these groups in the seasonal growth of two song control nuclei, HVc and RA. During late February, both groups had low circulatory levels of testosterone (mean for coastal birds was 1.01 +/- 0.37 ng/ml; mean for montane birds was 1.41 +/- 0.26 ng/ml) and fully recrudesced song nuclei (for example, mean HVc volume in coastal birds was 1.77 +/- 0.08 mm(3); mean HVc volume in montane birds was 1.76 +/- 0.09). Also at this time, both populations were in the earliest stages of seasonal reproductive development as judged by the degree of gonadal recrudescence (mean gonad volume was less than 10% of typical breeding size in both populations). It is concluded that seasonal song system growth is completed before seasonal reproductive development in response to submaximal levels of circulating testosterone.