Number Of Neurons In HVC Research Articles

Adult neurogenesis is necessary for functional regeneration of a forebrain neural circuit

In adult songbirds, new neurons are born in large numbers in the proliferative ventricular zone in the telencephalon and migrate to the adjacent song control region HVC (acronym used as proper name) [A. Reiner et al., J. Comp. Neurol. 473, 377-414 (2004)]. Many of these new neurons send long axonal projections to the robust nucleus of the arcopallium (RA). The HVC-RA circuit is essential for producing stereotyped learned song. The function of adult neurogenesis in this circuit has not been clear. A previous study suggested that it is important for the production of well-structured songs [R. E. Cohen, M. Macedo-Lima, K. E. Miller, E. A. Brenowitz, J. Neurosci. 36, 8947-8956 (2016)]. We tested this hypothesis by infusing the neuroblast migration inhibitor cyclopamine into HVC of male Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii) to block seasonal regeneration of the HVC-RA circuit. Decreasing the number of new neurons in HVC prevented both the increase in spontaneous electrical activity of RA neurons and the improved structure of songs that would normally occur as sparrows enter breeding condition. These results show that the incorporation of new neurons into the adult HVC is necessary for the recovery of both electrical activity and song behavior in breeding birds and demonstrate the value of the bird song system as a model for investigating adult neurogenesis at the level of long projection neural circuits.

Development of a novel model: Using songbirds to elucidate the pro-neurogenic role of microglia in healthy and natural neurodegenerative states

Abstract Inflammatory cells like microglia exert beneficial effects on neuronal survival, synaptic maintenance, and circuit plasticity. Most research has focused on neural injury or insult; roles in normal development and homeostasis remain less clear. To characterize the behavior of microglia under non-pathological conditions, we exploit seasonal death and replacement of neurons in a sensorimotor brain region responsible for singing behavior in white-crowned sparrow, Zonotrichia leucophrys. The song control nucleus HVC incorporates 50,000 new projection neurons into an existing pool of 100,000 neurons within one week of transitioning to breeding conditions. Within four days of transitioning back to nonbreeding conditions, an equal number of HVC neurons dies. Seasonal neuronal death in HVC increases neural stem cell proliferation in the adjacent ventral ventricular zone. This growth and regression correlates to respective increases and decreases in song production rate and quality. We found that microglia activation mediates pro-neurogenic responses in each of two paradigms: (i) inflammation induced locally with lipopolysaccharide microinjection in HVC was sufficient to induce neural stem cell proliferation; (ii) inflammation, including activation of microglia, was necessary to induce neural stem cell proliferation following seasonal neuronal loss. Having established that activation of microglia drives pro-neurogenic responses under non-pathological conditions, we are enabling future mechanistic studies with essential genetic and molecular tools, including a high quality annotated genome and transcriptome, cell-type specific molecular markers, in vivo imaging, and CRISPR/Cas9 mutagenesis.

Testosterone-induced neuroendocrine changes in the medial preoptic area precede song activation and plasticity in song control nuclei of female canaries.

Testosterone plays a key role in the control of seasonal changes in singing behavior and its underlying neural circuitry. After administration of exogenous testosterone, song quality and song control nuclei volumes change over the course of weeks, but song rate increases within days. The medial preoptic nucleus (POM) controls sexual motivation and testosterone action in POM increases sexually motivated singing. In this study, we investigated the time course of testosterone action in the song control nuclei and POM, at the gross anatomical and cellular level. Photosensitive female canaries were injected with BrdU to label newborn neurons. One day later they were transferred to a long-day photoperiod and implanted with testosterone-filled or empty implants. Brains and blood were collected 1, 2, 9 or 21days later. Testosterone increased POM volume within 1day, whereas the volume of song control nuclei increased significantly only on day 21 even if a trend was already observed for HVC on day 9. The density of newborn neurons in HVC, labeled by Bromodeoxyuridine (BrdU) and doublecortin, was increased by testosterone on days 9 and 21 although a trend was already detectable on day 2. In POM, testosterone increased the number and size of aromatase-immunoreactive neurons already after 1day. This rapid action of testosterone in POM supports its proposed role in controlling singing motivation. Although testosterone increased the number of newborn neurons in HVC rapidly (9, possibly 2days), it is unlikely that these new neurons affect singing behavior before they mature and integrate into functional circuits.

Early-life stress affects song complexity, song learning and volume of the brain nucleus RA in adult male song sparrows

The developmental stress hypothesis proposes that the honesty of birdsong is maintained by costs incurred during development, such that song in adulthood reflects exposure to early-life stressors. We determined the effects of early-life (7–60 days of age) food restriction or corticosterone (CORT) treatment on adult song production and neuroanatomy in male song sparrows, Melospiza melodia. When males were adults, we quantified song type repertoire size, syllable repertoire size, song-learning accuracy, trill deviation (the speed of frequency modulation in a trill) and song type stereotypy. We also analysed the volumes of the song control nuclei HVC, the robust nucleus of the arcopallium (RA), area X and the number of neurons in HVC. Song type and syllable repertoire sizes of food-restricted and CORT-treated birds were smaller than those of controls. Food restriction, but not CORT treatment, also reduced song-learning accuracy. We observed no effects of either treatment on trill deviation or song type stereotypy. However, trill deviation was significantly related to paternal repertoire size, suggesting a heritable component to some aspects of vocal learning. The volume of RA was smaller in food-restricted birds than in control or CORT-treated birds. Neither treatment affected the volumes of HVC or area X, or the number of neurons in HVC. Our results suggest that song complexity and song-learning accuracy may be honest indicators of a male song sparrow's early ontogeny and that early-life stress has long-lasting effects on the song control system in this species. However, measures of vocal performance (trill deviation, song type stereotypy) do not appear to be significantly affected by early-life stress in song sparrows.

A dose–response study of estradiol's effects on the developing zebra finch song system

To gauge the sensitivity of the female zebra finch song system to estradiol (E2), we used subcutaneous implants to administer various doses of E2 to hatchling female zebra finches. Four different doses of E2 were administered: 50, 15, 5 and 0-μg via subcutaneous silicon “ropes” at hatching, and the brains were examined in adulthood. Further, we examined whether masculinization was all-or-none once a threshold was reached or if the morphology of the song system would show a graded response to the various doses of E2. Finally, we asked if the various dependent measures – volume of song nuclei, neuron size, and neuron number – would show differential sensitivity to E2. Fifteen micrograms was sufficient to masculinize many aspects of the song system and was often as effective as 50-μg, causing a dramatic difference relative to the 0-μg group. Different aspects of the song system seemed differentially sensitive to the effects of E2: volumes of song control nuclei, the size of RA neurons, and the number of HVC neurons were significantly masculinized by 15-μg E2, but the number of RA neurons and HVC and lMAN soma sizes required 50-μg. The results suggest that several developmental processes are influenced by E2, possibly because of multiple sites of action or multiple processes that respond to E2.

Model of Birdsong Learning Based on Gradient Estimation by Dynamic Perturbation of Neural Conductances

We propose a model of songbird learning that focuses on avian brain areas HVC and RA, involved in song production, and area LMAN, important for generating song variability. Plasticity at HVC --> RA synapses is driven by hypothetical "rules" depending on three signals: activation of HVC --> RA synapses, activation of LMAN --> RA synapses, and reinforcement from an internal critic that compares the bird's own song with a memorized template of an adult tutor's song. Fluctuating glutamatergic input to RA from LMAN generates behavioral variability for trial-and-error learning. The plasticity rules perform gradient-based reinforcement learning in a spiking neural network model of song production. Although the reinforcement signal is delayed, temporally imprecise, and binarized, the model learns in a reasonable amount of time in numerical simulations. Varying the number of neurons in HVC and RA has little effect on learning time. The model makes specific predictions for the induction of bidirectional long-term plasticity at HVC --> RA synapses.

Hormonal and environmental control of song control region growth and new neuron addition in adult male house finches, Carpodacus mexicanus

In songbirds, testosterone (T) mediates seasonal changes in the sizes and neuroanatomical characteristics of brain regions that control singing (song control regions; SCRs). One model explaining the mechanisms of the growth of one SCR, the HVC, postulates that in the spring increasing photoperiod and circulating T concentrations enhance new neuron survival, thus increasing total neuron number. However, most research investigating the effects of T on new neuron survival has been done in autumn. The present study investigated the effects of photoperiod and T treatment on SCR growth and new neuron survival in the HVC in photosensitive adult male House Finches, Carpodacus mexicanus, under simulated spring-like conditions. Birds were castrated, given T-filled or empty Silastic capsules and maintained on short days (SD; 8L:16D) or long days (LD; 16L:8D). To mark new cells, birds received bromodeoxyuridine injections 11 days after experimental manipulations began and were sacrificed 28 days later. Testosterone treatment increased the sizes of two SCRs, the HVC and Robust nucleus of the arcopallium (RA). Exposure to LD did not affect HVC volume, but did increase RA volume. Testosterone treatment increased the total number of HVC neurons, but did not affect the number of new HVC neurons. Thus, T initiates SCR growth and increases neuron survival, but effects of T on new neuron incorporation may be limited in photosensitive birds under spring-like conditions. These results provide new insight into the effects of photoperiod and T treatment on vernal SCR growth and new neuron incorporation and support current models explaining this growth.

Antiandrogen blocks estrogen-induced masculinization of the song system in female zebra finches.

Song behavior and the neural song system that serves it are sexually dimorphic in zebra finches. In this species, males sing and females normally do not. The sex differences in the song system include sex differences in the proportion of neurons that express androgen receptors, which is higher in specific brain regions of males. Estradiol (E2) administered in early development profoundly masculinizes the song system of females, including the proportion of neurons expressing androgen receptors. We examined whether or not the expression of these androgen receptors was causally related to the E2-induced masculinization of this system by co-administering Flutamide, which blocks androgen action at the receptor, along with E2 at hatching. E2 alone had its usual masculinizing effect on the female song system, measured in adulthood: increasing the size of song nuclei, the size of neurons in HVC, RA, and 1MAN, and the number of neurons in HVC. E2's masculinizing action, however, was significantly diminished on all measures by co-administering Flutamide. Indeed, females receiving both E2 and Flutamide were never significantly more masculine than controls on any measure. Flutamide alone had no effect. Our results strongly suggest that the activation of androgen receptors is necessary for the E2-induced masculinization of the song system in females.

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.

A putative 5 α-reductase inhibitor demasculinizes portions of the zebra finch song system

One model of the sexual differentiation of the zebra finch song system holds that both major metabolites of testosterone, dihydrotestosterone (DHT) and estradiol (E 2), act together to masculinize the song system. To test this model, we administered a putative inhibitor of 5 α-reductase (MK-434) to decrease the synthesis of DHT from testosterone (T) in hatchling zebra finches. We tested MK-434's inhibition of 5 α-reductase, 5 β-reductase, and aromatase in vivo and in vitro. In vivo, MK-434 significantly inhibited 5 α-reductase activity but also reduced the activities of 5 β-reductase and aromatase. In vitro, MK-434 was extremely effective in inhibiting 5 α-reductase in the rat prostate but only slightly inhibited 5 α-reductase in the zebra finch telencephalon, where it also reduced aromatase and 5 β-reductase activities. These results suggest that MK-434 might differentially influence the availability of androgenic and estrogenic substrates, depending on the relative abundance of these enzymes in brain. MK-434 demasculinized (decreased) the number and decreased the density of RA neurons but did not significantly affect any other sexually dimorphic aspect of the song system, including the volumes of RA, HVC, and Area X; the size of neural somata in lMAN, HVC, and RA; and the number of neurons in HVC and lMAN. The differential influence of MK-434 on sexually dimorphic characteristics suggests that the various sexually dimorphic characteristics of the song system (1) are sensitive to different hormones, depending on the characteristic; or (2) have different sensitivities to hormone levels, some being easily affected by slightly reduced hormone levels whereas others are not; or (3) have markedly different critical periods depending on the characteristic. Regardless of the reason(s) for differential effects on the sexually dimorphic characteristics of the song system, the data clearly suggest that steroid hormones play a role in the normal masculine development of the song system.

Testosterone and the incidence of hormone target cells in song‐control nuclei of adult canaries

Adult male canaries learn to produce high-amplitude complex courtship songs each breeding season, whereas females do not, and brain nuclei involved with the production of song behavior are much larger in breeding males than in nonbreeding males or females (Nottebohm, 1980, 1981). However, treatment of adult females with testosterone (T) causes them to produce male-like song and stimulates pronounced growth of some song-control brain nuclei such as the caudal nucleus of the ventral hyperstriatum (HVc). We reexamined the effects of T on song-control nuclei in deafened birds. In order to examine whether the pattern of hormone accumulation varies as a function of circulating testosterone levels we described the distribution of testosterone-concentrating cells in HVc and the magnocellular nucleus of the anterior neostriatum (MAN) in hearing adult male, female, and T-treated female canaries, as well as in deaf T-treated and untreated females. In contrast to our previous findings (Bottjer, Schoonmaker, and Arnold, 1986a), we observed no tendency in this study for testosterone-induced growth of HVc to be attenuated in deafened birds. There was no difference between deaf and hearing birds in the incidence of labeled cells within HVc. We also observed no sex or hormone-induced differences in the percentage of hormone-concentrating cells in HVc: normal females have approximately the same proportion of hormone target cells as do males and T-treated females. However, males normally have many more neurons in HVc than do control females, and systemic exposure to testosterone induces a pronounced increase in the number of HVc neurons of adult females. Therefore, the absolute number of hormone target cells in HVc is likely to be much greater in males and T-treated females than in normal females. As in HVc, there were no differences among groups in the proportion of labeled cells within lateral MAN (IMAN), a nucleus that has been implicated in song learning (Bottjer, Miesner and Arnold, 1984). In contrast, the incidence of hormone target cells in medial MAN (mMAN) did vary as a function of hormonal condition: although mMAN of normal females is rarely visible in Nissl-stained sections and cells in this region are not hormone labeled, mMAN is clearly visible in Nissl-stained sections of males and T-treated females and contains many hormone-labeled cells. This testosterone-induced change in the phenotype of mMAN cells suggests a possible role for mMAN in learned song behavior.

Joint hormonal and sensory stimulation modulate neuronal number in adult canary brains.

Treatment of adult female canaries with testosterone (T) causes them to produce male-typical vocalizations and results in striking growth of brain nuclei that control song behavior (Nottebohm, 1980). The song-control nucleus HVc (caudal nucleus of the ventral hyperstriatum) contains cells that concentrate testosterone or its metabolites, suggesting that steroid hormones may induce the growth of HVc directly by regulating the expression of specific genes in those HVc neurons that have steroid receptors. However, we have previously provided evidence that is inconsistent with the idea that steroids promote growth of HVc solely via a direct action on hormone receptors: testosterone treatment of deafened adult females results in very little growth of HVc, relative to T-treated hearing birds (Bottjer et al., 1986b). Thus, birds in the former group undergo very little overall growth of HVc despite high circulating levels of hormone. We show here that the slightly increased size of HVc in T-treated deaf birds is attributable to an increase in neuronal spacing; the greatly increased size of HVc in T-treated hearing birds is due to an increase in neuronal number as well as spacing. There was virtually no increase in number of HVc neurons in T-treated deafened birds relative to control groups, whereas T-treated hearing birds showed a marked increase in neuron number. The song-control nucleus RA (robust nucleus of the archistriatum), which receives direct afferent input from HVc, also increases in size in response to testosterone treatment. However, the volume of RA increases in both hearing and deafened birds; this increase is primarily due to an increase in neuronal spacing as well as a small increase in neuron number. These results demonstrate that the number of neurons in a specific vocal-control nucleus (HVc) can change dramatically in adult canaries and suggest that some synergistic action of hormonal and sensory stimulation is necessary to induce such a change.