Effects of excess thyroid hormone on cell death,cell proliferation,and new neuron incorporation in the adult zebra finch telencephalon

Widespread telencephalic neuronal replacement occurs throughout life in birds. We explored the potential relationship between thyroxine (T₄) and cell turnover in the adult male zebra finch. We found that many cells in the zebra finch brain, including long‐projection neurons in the high vocal center (HVC), stained positively with an antibody to thyroid hormone receptors (TR). Labeling was generally weak in the ventricular zone (VZ) that gives rise to new neurons but some proliferative VZ cells and/or their progeny, identified by [³H]‐thymidine labeling, co‐labeled with anti‐TR antibody. Acute T₄ treatment dramatically increased the number of pyknotic and TUNEL‐positive cells in HVC and other telencephalic regions. In contrast, degenerating cells were never observed in the archistriatum or sub‐telencephalic regions, suggesting that excess T₄ augments cell death selectively in regions that show naturally occurring neuronal turnover. VZ mitotic activity was not altered shortly after acute T₄ treatment at a dosage that stimulated cell death, although [³H]‐labeling intensity per cell was slightly reduced. Moreover, the incorporation rates for neurons formed shortly before or after acute hormone treatment were no different from control values. Chronic T₄ treatment resulted in a reduction in the total number of HVC neurons. Thus, hyperthyroidism augmented neuronal death, which was not compensated for by neuronal replacement. Collectively, these results indicate that excess T₄ affects adult neuronal turnover in birds, and raises the possibility that thyroxine plays an important role in the postnatal development of the avian brain and vocal behavior. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 323–341, 2002     

Interspecies avian brain chimeras reveal that large brain size differences are influenced by cell-interdependent processes

Like humans, birds that exhibit vocal learning have relatively delayed telencephalon maturation, resulting in a disproportionately smaller brain prenatally but enlarged telencephalon in adulthood relative to vocal non-learning birds. To determine if this size difference results from evolutionary changes in cell-autonomous or cell-interdependent developmental processes, we transplanted telencephala from zebra finch donors (a vocal-learning species) into Japanese quail hosts (a vocal non-learning species) during the early neural tube stage (day 2 of incubation), and harvested the chimeras at later embryonic stages (between 9-12 days of incubation). The donor and host tissues fused well with each other, with known major fiber pathways connecting the zebra finch and quail parts of the brain. However, the overall sizes of chimeric finch telencephala were larger than non-transplanted finch telencephala at the same developmental stages, even though the proportional sizes of telencephalic subregions and fiber tracts were similar to normal finches. There were no significant changes in the size of chimeric quail host midbrains, even though they were innervated by the physically smaller zebra finch brain, including the smaller retinae of the finch eyes. Chimeric zebra finch telencephala had a decreased cell density relative to normal finches. However, cell nucleus size differences between each species were maintained as in normal birds. These results suggest that telencephalic size development is partially cell-interdependent, and that the mechanisms controlling the size of different brain regions may be functionally independent.     

Afferent influences on cell death and birth during development of a cortical nucleus necessary for learned vocal behavior in zebra finches

Forebrain nuclei that control learned vocal behavior in zebra finches are anatomically distinct and interconnected by a simple pattern of axonal pathways. In the present study, we examined afferent regulation of neuronal survival during development of the robust nucleus of the archistriatum (RA). RA projection neurons form the descending motor pathway of cortical vocal-control regions and are believed to be directly involved in vocal production.RA receives afferent inputs from two other cortical regions, the lateral magnocellular nucleus of the anterior neostriatum (lMAN) and the higher vocal center (HVC).However, because the ingrowth of HVC afferent input is delayed, lMAN projection neurons provide the majority of afferent input to RA during early vocal learning. lMAN afferent input to RA is of particular interest because lMAN is necessary for vocal learning only during a restricted period of development. By making lesions of lMAN in male zebra finches at various stages of vocal development (20-60 days of age) and in adults (>90-days old), we asked whether the survival of RA neurons depends on lMAN afferent input, and if so whether such dependence changes over the course of vocal learning. The results showed that removal of lMAN afferent input induced the loss of over 40% of RA neurons among birds in early stages of vocal development(20 days of age). However, lMAN lesions lost the ability to induce RA neuron death among birds in later stages of vocal development (40 days of age and older). These findings indicate that many RA neurons require lMAN afferent input for their survival during early vocal learning, whereas the inability of lMAN lesions to induce RA neuron death in older birds may indicate a reduced requirement for afferent input or perhaps the delayed ingrowth of HVC afferent input (at approx. 35 days of age)provides an alternate source of afferent support. Removal of lMAN afferent input also dramatically increased the incidence of mitotic figures in RA, but only among 20-day-old birds at 2 days post-lesion. The early, acute nature of the mitotic events raises the possibility that cell division in RA may be regulated by lMAN afferent input.     

Acetylcholinesterase in central vocal control nuclei of the zebra finch (<Emphasis Type="Italic">Taeniopygia guttata</Emphasis>)

The distribution of acetylcholinesterase (AChE) in the central vocal control nuclei of the zebra finch was studied using enzyme histochemistry. AChE fibres and cells are intensely labelled in the forebrain nucleus area X, strongly labelled in high vocal centre (HVC) perikarya, and moderately to lightly labelled in the somata and neuropil of vocal control nuclei robust nucleus of arcopallium (RA), medial magnocellular nucleus of the anterior nidopallium (MMAN) and lateral magnocellular nucleus of the anterior nidopallium (LMAN). The identified sites of cholinergic and/or cholinoceptive neurons are similar to the cholinergic presence in vocal control regions of other songbirds such as the song sparrow, starling and another genus of the zebra finch (Poephila guttata), and to a certain extent in parallel vocal control regions in vocalizing birds such as the budgerigar. AChE presence in the vocal control system suggests innervation by either afferent projecting cholinergic systems and/or local circuit cholinergic neurons. Co-occurrence with choline acetyltransferase (ChAT) indicates efferent cholinergic projections. The cholinergic presence in parts of the zebra finch vocal control system, such as the area X, that is also intricately wired with parts of the basal ganglia, the descending fibre tracts and brain stem nuclei could underlie this circuitry’s involvement in sensory processing and motor control of song.     

Targeted neuronal death affects neuronal replacement and vocal behavior in adult songbirds

In the high vocal center (HVC) of adult songbirds, increases in spontaneous neuronal replacement correlate with song changes and with cell death. We experimentally induced death of specific HVC neuron types in adult male zebra finches using targeted photolysis. Induced death of a projection neuron type that normally turns over resulted in compensatory replacement of the same type. Induced death of the normally nonreplaced type did not stimulate their replacement. In juveniles, death of the latter type increased recruitment of the replaceable kind. We infer that neuronal death regulates the recruitment of replaceable neurons. Song deteriorated in some birds only after elimination of replaceable neurons. Behavioral deficits were transient and followed by variable degrees of recovery. This raises the possibility that induced neuronal replacement can restore a learned behavior.     

Early developmental stress negatively affects neuronal recruitment to avian song system nucleus HVC

Adverse environmental conditions can impact the life history trajectory of animals. Adaptive responses enable individuals to cope with unfavorable conditions, but altered metabolism and resource allocation can bear long‐term costs. In songbirds, early developmental stress can cause lifelong changes in learned song, a culturally transmitted trait, and nestlings experiencing developmental stress develop smaller song control nucleus HVCs. We investigated whether nutrition‐related developmental stress impacts neurogenesis in HVC, which may explain how poor nutrition leads to smaller HVC volume. We provided different quality diets (LOW and HIGH) by varying the husks‐to‐seeds ratio to zebra finch families for the first 35 days after the young hatched (PHD). At PHD14–18 and again at nutritional independence (PHD35), juveniles were injected with different cell division markers. To monitor growth, we took body measures at PHD10, 17, and 35. At PHD35 the number of newly recruited neurons in HVC and the rate of proliferation in the adjacent ventricular zone (VZ) were counted. Males raised on the LOW diet for their first weeks of life had significantly fewer new neurons in HVC than males raised on the HIGH diet. At the time when these new HVC neurons were born and labeled in the VZ (PHD17) the birds exposed to the LOW diet had significantly lower body mass. At PHD35 body mass or neuronal proliferation no longer differed. Our study shows that even transitory developmental stress can have negative consequences on the cellular processes underlying the development of neural circuits. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 107–118, 2016     

HVC microlesions do not destabilize the vocal patterns of adult male zebra finches with prior ablation of LMAN

The songs of adult male zebra finches (Taeniopygia guttata) arise by an integration of activity from two neural pathways that emanate from the telencephalic nucleus HVC (proper name). One pathway descends directly from HVC to the vocal premotor nucleus RA (the robust nucleus of the arcopallium) whereas a second pathway descends from HVC into a basal ganglia circuit (the anterior forebrain pathway, AFP) that also terminates in RA. Although HVC neurons that project directly to RA outnumber those that contribute to the AFP, both populations are distributed throughout HVC. Thus, partial ablation (microlesion) of HVC should damage both pathways in a proportional manner. We report here that bilateral HVC microlesions in adult male zebra finches produce an immediate loss of song stereotypy from which birds recover, in some cases within 3 days. The contribution of the AFP to the onset of song destabilization was tested by ablating the output nucleus of this circuit (LMAN, the lateral magnocellular nucleus of the anterior nidopallium) prior to bilateral HVC microlesions. Song stereotypy was largely unaffected. Together, our findings suggest that adult vocal production involves nonproportional integration of two streams of neural activity with opposing effects on song--HVC's direct projection to RA underlies production of stereotyped song whereas the AFP seems to facilitate vocal variation. However, the rapid recovery of song in birds with HVC microlesions alone suggests the presence of dynamic corrective mechanisms that favor vocal stereotypy.     

Birth,migration, incorporation,and death of vocal control neurons in adult songbirds

Neurogenesis continues in the brain of adult birds. These cells are born in the ventricular zone of the lateral ventricles. Young neurons then migrate long distances guided, in part, by radial cell processes and become incorporated throughout most of the telencephalon. In songbirds, the high vocal center (HVC), which is important for the production of learned song, receives many of its neurons after hatching. HVC neurons which project to the robust nucleus of the archistriatum to form part of the efferent pathway for song production, and HVC interneurons continue to be added throughout life. In contrast, Area X-projecting HVC cells, thought to be part of a circuit necessary for song learning but not essential for adult song production, are only born in the embryo. New neurons in HVC of juvenile and adult birds replace older cells that die. There is a correlation between seasonal cell turnover rates (addition and loss) and testosterone levels in adult male canaries. Available evidence suggests that steroid hormones control the recruitment and/or survival of new HVC neurons, but not their production. The functions of neuronal replacement in adult birds remain unclear. However, rates of HVC neuron turnover are highest at times of year when canaries modify their songs. Replaceable HVC neurons may participate in the modification of perceptual memories or motor programs for song production. In contrast, permanent HVC neurons could hold long-lasting song-related information. The unexpected large-scale production of neurons in the adult brain holds important clues about brain function and, in particular, about the neural control of a learned behavior—birdsong. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 585–601, 1997     

Testosterone-induced changes in adult canary brain are reversible

Brain nuclei that control song are larger in male canaries, which sing, than in females, which sing rarely or not at all. Treatment of adult female canaries with testosterone (T) induces song production and causes song-control nuclei to grow, approaching the volumes observed in males. For example, the higher vocal center (HVC) of adult females approximately doubles in size by 1 month following the onset of T treatment. Male HVC projects to a second telencephalic nucleus, RA (the robust nucleus of the archistriatum), which projects in turn to the vocal motor neurons. Whether HVC makes a similar connection in female canaries is not known, although HVC and RA are not functionally connected in female zebra finches, a species in which testosterone does not induce neural or behavioral changes in the adult song system. This experiment investigated whether HVC makes an efferent projection to RA in normal adult female canaries, or if T is necessary to induce the growth of this connection. In addition, we examined whether T-induced changes in adult female canary brain are reversible. Adult female canaries received systemic T implants that were removed after 4 weeks; these birds were killed 4 weeks after T removal (Testosterone-Removal, T-R). Separate groups of control birds received either (a) T implants for 4 weeks which were not removed (Testosterone-Control, T-C) or (b) empty implants (Untreated Control, øO-C). Crystals of the fluorescent tracer DiI were placed in the song-control nucleus HVC in order to anterogradely label both efferent targets of HVC, RA and Area X. Projections from HVC to RA and Area X were present in all treatment groups including untreated controls, and did not appear to differ either qualitatively or quantitatively. Thus, formation of efferent connections from HVC may be prerequisite to hormone-induced expression of song behavior in adult songbirds. The volumes of RA and Area X were measured using the distribution of anterograde label as well as their appearance in Nissl-stained tissue. RA was larger in T-treated control birds than in untreated controls. Experimental birds in which T was given and then removed (T-R) had RA volumes closer in size to untreated controls (ø-C). Because the volume of RA in T-treated controls (T-C) was larger than that of birds that did not receive T (ø-C), we conclude that the volume of RA increased in both T-C and T-R birds but regressed upon removal of T in T-R birds. Surprisingly, the volume of Area X did not increase in T-treated birds. Birds in this study were maintained on short days, suggesting that T-induced growth of Area X reported previously may have resulted from an interaction between T and another seasonal or photoperiodic factor induced by exposure to long daylengths. © 1993 John Wiley & Sons, Inc.     

Differential estrogen accumulation among populations of projection neurons in the higher vocal center of male canaries

The higher vocal center (HVC) of adult male canries undergoes a seasonal change in volume that corresponds to seasonal modifications of vocal behavior: HVC is large when birds produce stereotyped song (spring) and is small when birds produce plastic song and add new song syllables into their vocal repertoires (fall). We reported previously that systemic exposure to testosterone (T) produces an increase in the volume of HVC similar to that observed with long-day photoperiods. T-induced growth of HVC occured regardless of wheter the borders of HVC were defined by Nissl-staining, the distribution of androgen-concentrating cells, or the distribution of projection neurons [separate neuronal populations within HVC project to the robust nucleus of the archistriatum (RA) and to Area X of the avian striatum (X)]. In the present study we used steroid autoradiography to determine whether T can influence the distribution of HVC cells that bind estrogen, and we combined estrogen autoradiography with retrograde labeling to determine whether HVC neurons that project to RA versus X differ in their ability to accumulate estrogen. Results showed that T increased the volume of Nissl-defined HVC and although HVC contained a low density of estrogen-concentrating cells, T increased the spatial distribution of these cells to match the Nissl borders of HVC. We also identified a region containing a high density of estrogenconcentrating cells located medial to HVC [we call this region paraHVC (pHVC)], and T also increased the volume of pHVC. pHVC also contained numerous X-projecting neurons, but few if any RA-projecting neurons. Double-labeling analysis revealed the RA-projecting neurons did not accumulate estrogen, a small percentage of X-projecting neurons in HVC accumulated estrogen, and the majority of X-projecting neurons in pHVC showed heavy accumulation of estrogen. The data reported here and in our previous article suggest distinct roles for gonadal steroids within the HVC-pHVC complex: estrogens are concentrated by neurons that project to a striatal region that influences vocal production during song learning (X), whereas androgens are concentrated primarily by neurons that project to a motor region that is involved in vocal production during both song learning and the recitation of already-learned song (RA). © 1995 John Wiley & Sons, Inc.     

LMAN lesions prevent song degradation after deafening without reducing HVC neuron addition

In some songbirds perturbing auditory feedback can promote changes in song structure well beyond the end of song learning. One factor that may drive vocal change in such deafened birds is the ongoing addition of new vocal-motor neurons into the song system. Without auditory feedback to guide their incorporation, the addition of these new neurons could disrupt the established song pattern. To assess this hypothesis, the authors determined if neuronal recruitment into the vocal motor nucleus HVC is affected by neural signals that influence vocal change in adult deafened birds. Such signals appear to be conveyed via LMAN, a nucleus in the anterior forebrain that is necessary for vocal change after deafening. Here the authors tested whether LMAN lesions might restrict song degradation after deafening by reducing the addition or survival of new HVC neurons that would otherwise corrupt the ongoing song pattern. Using [3H]thymidine autoradiography to identify neurons generated in adult zebra finches, it was shown here that LMAN lesions do not reduce the number or percent of new HVC neurons surviving for either several weeks or months after [3H]thymidine labeling. However, the authors confirmed previous reports that LMAN lesions restrict vocal change after deafening. These data suggest that neurons incorporated into the adult HVC may form behaviorally adaptive connections without requiring auditory feedback, and that any role such neurons may play in promoting vocal change after adult deafening requires anterior forebrain pathway output.     

Estrogens and non-estrogenic ovarian influences combine to promote the recruitment and decrease the turnover of new neurons in the adult female canary brain

The higher vocal center (HVC) of the songbird forebrain exhibits persistent neurogenesis in adulthood, particularly in a region of the mediocaudal neostriatum that is associated with a subventricular layer of estrogen receptive cells. We asked whether estrogens might influence adult neurogenesis, by assessing the effect of ovariectomy on HVC neuronal production in the adult female canary. Fifteen 1-year-old females were separated into groups of ovariectomized, estradiol-replaced ovariectomized, and gonadally intact birds. To label dividing cells and their progency, the birds were given [³H]thymidine for 8 days, killed 32 days later, and their brains autoradiographed. A significant rise was noted in the number of HVC neurons per section in estradiol-treated birds relative to the untreated control birds. The number of [³H]-thymidine-labeled HVC neurons was also higher in the estrogen-treated birds; however, the neuronal labeling index (LI) did not vary as a function of estradiol replacement, as the total number of HVC neurons rose in parallel with the added new neurons. In contrast, the neuronal LI did rise as a result of ovariectomy, and this ovariectomy-associated increase in the LI was not reversed by estradiol. Among non-neuronal cell types, the endothelial LI was higher in estrogen-treated birds than in their untreated counterparts, suggesting estrogen-associated angiogenesis. Radioimmunoassay confirmed that serum estradiol was reduced in the castrated birds. Since estrogen appeared to promote the survival of [³H]thymidine⁺ neurons, we next sought to determine whether estrogen acted directly on the newly generated neurons, or rather indirectly through an intermediary cell population. To this end, we asked whether the new neurons or their precursors expressed estrogen receptor immunoreactivity (ER-IR). Five adult male canaries were given [³H]thymidine for periods ranging from 2 to 28 days, killed at varying times up to 3 weeks therafter, then probed for ER-IR and autoradiographed. [³H]thymidine⁺ cells displayed no detectable ER-IR within their first 4 weeks of postmitotic life. Rather, during migration from the ventricular zone (VZ), the new neurons traversed a layer of mitotically quiescent, ER⁺ subventricular cells. Double labeling for ER-IR and cell-type selective antigens confirmed that these ER⁺ cells were neurons. These results indicate that the early survival of new neurons in the adult songbird HVC is promoted by estrogen, and may be mediated by the estrogen-stimulated paracrine release of neurotrophic agents by ER-IR subventricular neurons. Our data suggest that estrogen's promotion of neuronal survival may operate concurrently with an estrogen-independent ovarian suppression of neuronal mitogenesis.     

Sexually dimorphic neuron addition to an avian song-control region is not accounted for by sex differences in cell death

Only male zebra finches sing, and several brain regions implicated in song behavior exhibit marked sex differences in neuron number. In one region, the high vocal center (HVC), this dimorphism develops because the incorporation of new neurons is greater in males than in females during the first several weeks after hatching. Although estrogen (E₂) exposure stimulates neuron addition in females, it is not known where (E₂) acts, or to what extent sexual differentiation influences the production, specification, or survival of HVC neurons. In the present study we first reassessed sex and (E₂)-induced differences in cell degeneration within the HVC using the TUNEL technique to identify cells undergoing DNA fragmentation indicative of apoptosis. HVC neuron number, as well as the density and number of TUNEL-labeled and pyknotic cells within the HVC were measured in normal 20- and 30-day-old males and females, and in 30-day-old females implanted with E₂ on posthatch day 18. Although HVC neuron number was greater in males than in females, and was masculinized in E₂ females, no group differences were evident in the absolute number of dying cells. These results indicate that sex differences in cell survival within the HVC do not entirely account for sexually dimorphic neuron addition to this region. Rather, sexual differentiation acts on some HVC neurons before they complete their migration and/or early differentiation. Although the migratory route of HVC neurons is not known, a large number of E₂ receptor-containing cells (ER cells) reside just ventromedial to the HVC and adjacent to the proliferative ventricular zone. Next, we investigated whether these ER cells contribute to early-arising sex differences in HVC neuron addition. By combining [³H] thymidine autoradiography with immunocytochemistry for ERs, we first established that ER-expressing cells are not generated during posthatch sexually dimorphic HVC neuron addition, and thus are not young HVC neurons that transiently express ERs during their migration. Furthermore, in 25-day-old birds we found no sex difference in the density of pyknotic cells among this group of ER cells, suggesting that these cells do not promote the differential survival of HVC neuronal precursors migrating through this region. Rather, ER cells or other cell populations may establish sex differences in HVC neuron number by creating dimorphisms in cellular specification. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 61–71, 1997     

Developmental experience alters information coding in auditory midbrain and forebrain neurons

In songbirds, species identity and developmental experience shape vocal behavior and behavioral responses to vocalizations. The interaction of species identity and developmental experience may also shape the coding properties of sensory neurons. We tested whether responses of auditory midbrain and forebrain neurons to songs differed between species and between groups of conspecific birds with different developmental exposure to song. We also compared responses of individual neurons to conspecific and heterospecific songs. Zebra and Bengalese finches that were raised and tutored by conspecific birds, and zebra finches that were cross‐tutored by Bengalese finches were studied. Single‐unit responses to zebra and Bengalese finch songs were recorded and analyzed by calculating mutual information (MI), response reliability, mean spike rate, fluctuations in time‐varying spike rate, distributions of time‐varying spike rates, and neural discrimination of individual songs. MI quantifies a response's capacity to encode information about a stimulus. In midbrain and forebrain neurons, MI was significantly higher in normal zebra finch neurons than in Bengalese finch and cross‐tutored zebra finch neurons, but not between Bengalese finch and cross‐tutored zebra finch neurons. Information rate differences were largely due to spike rate differences. MI did not differ between responses to conspecific and heterospecific songs. Therefore, neurons from normal zebra finches encoded more information about songs than did neurons from other birds, but conspecific and heterospecific songs were encoded equally. Neural discrimination of songs and MI were highly correlated. Results demonstrate that developmental exposure to vocalizations shapes the information coding properties of songbird auditory neurons. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 70: 235–252, 2010.     

Rapamycin blocks the neuroprotective effects of sex steroids in the adult birdsong system

In adult songbirds, the telencephalic song nucleus HVC and its efferent target RA undergo pronounced seasonal changes in morphology. In breeding birds, there are increases in HVC volume and total neuron number, and RA neuronal soma area compared to nonbreeding birds. At the end of breeding, HVC neurons die through caspase‐dependent apoptosis and thus, RA neuron size decreases. Changes in HVC and RA are driven by seasonal changes in circulating testosterone (T) levels. Infusing T, or its metabolites 5α‐dihydrotestosterone (DHT) and 17 β‐estradiol (E2), intracerebrally into HVC (but not RA) protects HVC neurons from death, and RA neuron size, in nonbreeding birds. The phosphoinositide 3‐kinase (PI3K)‐Akt (a serine/threonine kinase)‐mechanistic target of rapamycin (mTOR) signaling pathway is a point of convergence for neuroprotective effects of sex steroids and other trophic factors. We asked if mTOR activation is necessary for the protective effect of hormones in HVC and RA of adult male Gambel's white‐crowned sparrows (Zonotrichia leucophrys gambelii). We transferred sparrows from breeding to nonbreeding hormonal and photoperiod conditions to induce regression of HVC neurons by cell death and decrease of RA neuron size. We infused either DHT + E2, DHT + E2 plus the mTOR inhibitor rapamycin, or vehicle alone in HVC. Infusion of DHT + E2 protected both HVC and RA neurons. Coinfusion of rapamycin with DHT + E2, however, blocked the protective effect of hormones on HVC volume and neuron number, and RA neuron size. These results suggest that activation of mTOR is an essential downstream step in the neuroprotective cascade initiated by sex steroid hormones in the forebrain.     

Lack of a synergistic effect between estradiol and dihydrotestosterone in the masculinization of the zebra finch song system

Previous studies have suggested that both major active metabolites of testosterone, estradiol (E₂) and dihydrotestosterone (DHT), are needed for complete masculinization of the brain regions that control song in passerine birds. However, DHT treatment of hatchling female zebra finches has only small masculinizing effects on the song system. To assess whether E₂ and DHT have a synergistic effect on the masculinization of the zebra finch song system, female zebra finches were given Silastic implants of E₂ on the day of hatching (day 1) either without any additional hormone treatment or in combination with DHT on days 1, 14, or 70. At 105 to 110 days of age, we measured the volumes of Area X, higher vocal center (HVC), robust nucleus of the archistriatum (RA), soma sizes in HVC, RA, and the lateral magnocellular nucleus of the neostriatum (lMAN), and neuron density and number in RA. E₂ masculinized all of the measures in the song system with the exception of the number of neurons in RA. DHT did not synergize with E₂ to produce any additional masculinization of the attributes measured. These data demonstrate that the combination of E₂ and DHT did not result in the complete masculinization of the song control nuclei and argue against the importance of androgen in sexual differentiation of the song system. © 1995 John Wiley & Sons, Inc.     

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

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

Auditory-vocal cholinergic pathway in the songbird brain

Male zebra finches learn to imitate a tutor's song through auditory and motor learning. The two main song control nuclei in the zebra finch forebrain, the higher vocal center (HVC) and the robust nucleus of the archistriatum (RA), receive cholinergic innervation from the ventral paleostriatum (VP) of the basal forebrain which may play a key role in song learning. By injecting neuroanatomical tracers, we found a topographically segregated pathway from nucleus ovoidalis (Ov) to VP that in turn projects in a topographic fashion to HVC and RA. Ov is a major relay in the main ascending auditory pathway. The results suggest that the cholinergic neurons in the VP responsible for song learning are regulated by auditory information from the Ov.     

Cell death during development of a forebrain nucleus involved with vocal learning in zebra finches

Lateral MAN (magnocellular nucleus of the anterior neostriatum) is a forebrain nucleus that is known to be importantly involved with vocal learning in juvenile male zebra finches only during a restricted period of the learning process: lesions of lMAN completely disrupt song behavior in zebra finches prior to 50 days of age but have little or no effect in older juvenile or adult birds. The development of lMAN, as of other song-control regions, is delayed until the time that song behavior is being learned. Lateral MAN undergoes a substantial loss of neurons between 25 and 55 days of age, a time that encompasses initial stages of vocal production as well as the interval during which lMAN lesions become ineffective. In this study, we measured both the time course of neuronal loss and the incidence of pyknotic cells within lMAN during the period of cell loss. There is a pronounced loss of neurons from lMAN between 20 and 35 days, after which the adult number of neurons is established. The incidence of pyknosis is greatest at 20 days, around the time when the loss of live cells is also most pronounced, suggesting that the loss of neurons from lMAN is attributable to cell death. The loss of neurons occurs well before lesions of lMAN become ineffective in disrupting vocal behavior. Thus the neurons remaining in lMAN after the period of cell loss apparently undergo a substantial change in function at the time lesions lose effectiveness (about 55-60 days).     

The selectivity of canary HVC neurons for the Bird's Own Song: rate coding, temporal coding, or both?

The neuronal selectivity observed in the avian song system for the Bird's Own Song progressively emerged as an extraordinary fruitful model to investigate the neural code underlying the recognition of complex stimuli and the occurrence of learned behaviors. In adult zebra finch, neurons from the HVC (used as a proper name) show very selective auditory responses, firing more to presentation of the Bird's Own Song (BOS) than to reverse BOS or other conspecific songs. However, as adult zebra finches always produce the same stereotyped song, the presence of such highly selective neurons in birds with larger repertoire still remains an open question. Data presented here show that neurons selective for the BOS can be found in adult canary, a seasonal breeding bird which display a large repertoire. More precisely, we found that a large proportion of neurons (29/36) exhibits higher responses to presentation of the forward than to the reverse BOS, and that 22% of the cells were identified as selective on the basis of the d' value. For a cell that was extensively studied, we evaluated to what extent temporal stimulus-related structure predicts the acoustic stimulus using linear or non-linear artificial neural networks (ANN). These analyses indicated that the temporal structure contained in spike trains characterizes more accurately the stimulus than the firing rate. The limitations of applying ANN analyses to electrophysiological data are discussed and potential solutions to increase the confidence in these analysis are proposed.