Inflammation Induced by Natural Neuronal Death and LPS Regulates Neural Progenitor Cell Proliferation in the Healthy Adult Brain.

Inflammation is typically considered a negative response to injury or insult; however, recent advances demonstrate that inflammatory cells regulate development, plasticity, and homeostasis through anticytotoxic, progenerative responses. Here, we extend analyses of neuroinflammation to natural neurodegenerative and homeostatic states by exploiting seasonal plasticity in cytoarchitecture of the avian telencephalic song control nucleus, high vocal center [HVC (proper name)], in the songbird Gambel's white-crowned sparrow (Zonotrichia leucophrys gambelii). We report that local injection of the endotoxin lipopolysaccharide into HVC of birds in both breeding (high circulating testosterone level) and nonbreeding (low circulating testosterone level) conditions increased neural progenitor cell proliferation in the nearby but distinct ventricular zone. Additionally, we found that oral administration of the anti-inflammatory drug minocycline during seasonal regression of HVC reduced microglia activation in HVC and prevented the normal proliferative response in the ventricular zone to apoptosis in HVC. Our results suggest that local neuroinflammation positively regulates neural progenitor cell proliferation and, in turn, contributes to the previously described repatterning of HVC cytoarchitecture following seasonally induced neuronal loss.

Brain-Derived Neurotrophic Factor Has a Transsynaptic Trophic Effect on Neural Activity in an Adult Forebrain Circuit.

While hormone-driven plasticity in the adult brain is well studied, the underlying cellular and molecular mechanisms are less well understood. One example of this is seasonal plasticity in the avian brain, where song nuclei exhibit hormonally driven changes in response to changing photoperiod and circulating sex steroid hormones. Hormone receptor activation in song nucleus HVC (proper name) elicits a robust change in activity in target nucleus RA (robust nucleus of the arcopallium), but the molecular signal responsible for this is unknown. This study addressed whether brain-derived neurotrophic factor (BDNF) mediates a transsynaptic effect from HVC to RA in male Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii). In situ hybridization confirmed an increase in BDNF expression in HVC neurons of birds switched to a long-day (LD) photoperiod plus systemically elevated testosterone (T) levels, compared with short-day (SD) conditions. BDNF expression was virtually absent in RA neurons of SD birds, increasing to barely detectable levels in a small subset of cells in LD+T birds. Infusion of BDNF protein adjacent to the RA of SD birds caused an increase in the spontaneous neuron firing rate. Conversely, the infusion of ANA12, a specific antagonist of the tyrosine-related kinase B (TrkB) for BDNF, prevented the increase in RA neuron firing rate in LD+T birds. These results indicate that BDNF is sufficient, and TrkB receptor activation is necessary, for the transsynaptic trophic effect exerted by HVC on RA. The dramatic change in the activity of RA neurons during the breeding season provides a clear example of transsynaptic BDNF effects in the adult brain in a functionally relevant circuit.SIGNIFICANCE STATEMENT Sex steroid hormones drive changes in brain circuits in all vertebrates, both within specific neurons and on their synaptic targets. Such changes can lead to profound changes in behavior, but little is known about the precise molecular mechanisms that underlie this process. We addressed this question in a seasonally breeding songbird and found that the trophic effects of one forebrain song nucleus on its target are mediated transsynaptically by the neurotrophin BDNF. This suggests that, in addition to their role in development, neurotrophins have critical roles in adult brain plasticity.

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 ß-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.

Seasonal changes in neuronal turnover in a forebrain nucleus in adult songbirds.

Neuronal death and replacement, or neuronal turnover, in the adult brain are one of many fundamental processes of neural plasticity. The adult avian song control circuit provides an excellent model for exploring mature neuronal death and replacement by new neurons. In the song control nucleus, HVC of adult male Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelli) nearly 68,000 neurons are added each breeding season and die during the subsequent nonbreeding season. To accommodate large seasonal differences in HVC neuron number, the balance between neuronal addition and death in HVC must differ between seasons. To determine whether maintenance of new HVC neurons changes within and between breeding and nonbreeding conditions, we pulse-labeled two different cohorts of new HVC neurons under both conditions and quantified their maintenance. We show that the maintenance of new HVC neurons, as well as new nonneuronal cells, was higher at the onset of breeding conditions than at the onset of nonbreeding conditions. Once a steady-state HVC volume and neuronal number were attained in either breeding or nonbreeding conditions, neuronal and nonneuronal maintenance were similarly low. We found that new neuronal number correlated with a new nonneuronal number within each cohort of new neurons. Together, these data suggest that sex steroids promote the survival of an initial population of new neurons and nonneuronal cells entering HVC. However, once HVC is fully grown or regressed, neuronal and nonneuronal cell turnover is regulated by a common mechanism likely independent of direct sex steroid signaling.

Adult Neurogenesis Leads to the Functional Reconstruction of a Telencephalic Neural Circuit.

UNLABELLED: Seasonally breeding songbirds exhibit pronounced annual changes in song behavior, and in the morphology and physiology of the telencephalic neural circuit underlying production of learned song. Each breeding season, new adult-born neurons are added to the pallial nucleus HVC in response to seasonal changes in steroid hormone levels, and send long axonal projections to their target nucleus, the robust nucleus of the arcopallium (RA). We investigated the role that adult neurogenesis plays in the seasonal reconstruction of this circuit. We labeled newborn HVC neurons with BrdU, and RA-projecting HVC neurons (HVCRA) with retrograde tracer injected in RA of adult male white-crowned sparrows (Zonotrichia leucophrys gambelii) in breeding or nonbreeding conditions. We found that there were many more HVCRA neurons in breeding than nonbreeding birds. Furthermore, we observed that more newborn HVC neurons were back-filled by the tracer in breeding animals. Behaviorally, song structure degraded as the HVC-RA circuit degenerated, and recovered as the circuit regenerated, in close correlation with the number of new HVCRA neurons. These results support the hypothesis that the HVC-RA circuit degenerates in nonbreeding birds, and that newborn neurons reconstruct the circuit in breeding birds, leading to functional recovery of song behavior. SIGNIFICANCE STATEMENT: We investigated the role that adult neurogenesis plays in the seasonal reconstruction of a telencephalic neural circuit that controls song behavior in white-crowned sparrows. We showed that nonbreeding birds had a 36%-49% reduction in the number of projection neurons compared with breeding birds, and the regeneration of the circuit in the breeding season is due to the integration of adult-born projection neurons. Additionally, song structure degraded as the circuit degenerated and recovered as the circuit regenerated, in close correlation with new projection neuron number. This study demonstrates that steroid hormones can help reestablish functional neuronal circuits following degeneration in the adult brain and shows non-injury-induced degeneration and reconstruction of a neural circuit critical for producing a learned behavior.

It takes a seasoned bird to be a good listener: communication between the sexes.

Birds commonly use sound for communication between the sexes. In many songbird species, only males sing and there are pronounced sex differences in the neural song control circuits. By contrast, the auditory circuitry is largely similar in males and females. Both sexes learn to recognize vocalizations heard as juveniles and this shapes auditory response selectivity. Mating vocalizations are restricted to the breeding season, when sex steroid levels are elevated. Auditory cells, from the ear to the cortex, are hormone sensitive. Estrogens are synthesized in the brain and can modulate the activity of auditory neurons. In species that breed seasonally, elevated levels of estradiol in females transiently enhance their auditory responses to conspecific vocalizations, resulting in sex differences in audition.

Network analysis of microRNA and mRNA seasonal dynamics in a highly plastic sensorimotor neural circuit.

BACKGROUND: Adult neurogenesis and the incorporation of adult-born neurons into functional circuits requires precise spatiotemporal coordination across molecular networks regulating a wide array of processes, including cell proliferation, apoptosis, neurotrophin signaling, and electrical activity. MicroRNAs (miRs) - short, non-coding RNA sequences that alter gene expression by post-transcriptional inhibition or degradation of mRNA sequences - may be involved in the global coordination of such diverse biological processes. To test the hypothesis that miRs related to adult neurogenesis and related cellular processes are functionally regulated in the nuclei of the avian song control circuit, we used microarray analyses to quantify changes in expression of miRs and predicted target mRNAs in the telencephalic nuclei HVC, the robust nucleus of arcopallium (RA), and the basal ganglia homologue Area X in breeding and nonbreeding Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelli). RESULTS: We identified 46 different miRs that were differentially expressed across seasons in the song nuclei. miR-132 and miR-210 showed the highest differential expression in HVC and Area X, respectively. Analyzing predicted mRNA targets of miR-132 identified 33 candidate target genes that regulate processes including cell cycle control, calcium signaling, and neuregulin signaling in HVC. Likewise, miR-210 was predicted to target 14 mRNAs differentially expressed across seasons that regulate serotonin, GABA, and dopamine receptor signaling and inflammation. CONCLUSIONS: Our results identify potential miR-mRNA regulatory networks related to adult neurogenesis and provide opportunities to discover novel genetic control of the diverse biological processes and factors related to the functional incorporation of new neurons to the adult brain.

Testosterone Mediates Seasonal Growth of the Song Control Nuclei in a Tropical Bird.

In mid- to high-latitude songbirds, seasonal reproduction is stimulated by increasing day length accompanied by elevated plasma sex steroid levels, increased singing, and growth of the song control nuclei (SCN). Plasticity of the SCN and song behavior are primarily mediated by testosterone (T) and its metabolites in most species studied thus far. However, the majority of bird species are tropical and have less pronounced seasonal reproductive cycles. We have previously documented that equatorial rufous-collared sparrows (Zonotrichia capensis) exhibit seasonal neuroplasticity in the SCN. Manipulating T in these birds, however, did not alter singing behavior. In the current study, we investigated whether T mediates plasticity of the SCN in a similar manner to temperate songbirds. In the first experiment, we treated captive male birds with T or blank implants during the nonbreeding season. In a second experiment, we treated captive male birds with either blank implants, T-filled implants, T with flutamide (FLU; an androgen receptor antagonist) or T with FLU and 1,4,6-androstatriene-3,17-dione (ATD; an estrogen synthesis inhibitor) during the breeding season. In both experiments, the volumes of the brain areas high vocal center (HVC), Area X, and robust nucleus of the arcopallium (RA) were measured along with singing behavior. In summary, T stimulated growth of HVC and RA, and the combined effect of FLU and ATD reversed this effect in HVC. Area X was not affected by T treatment in either experiment. Neither T-treated birds nor controls sang in captivity during either experiment. Together, these data indicate that T mediates seasonal changes in the HVC and RA of both tropical and higher- latitude bird species even if the environmental signals differ. However, unlike most higher-latitude songbirds, we found no evidence that motivation to sing or growth of Area X are stimulated by T under captive conditions.

Neurogenesis in the adult avian song-control system.

New neurons are added throughout the forebrain of adult birds. The song-control system is a model to investigate the addition of new long-projection neurons to a cortical circuit that regulates song, a learned sensorimotor behavior. Neuroblasts destined for the song nucleus HVC arise in the walls of the lateral ventricle, and wander through the pallium to reach HVC. The survival of new HVC neurons is supported by gonadally secreted testosterone and its downstream effectors including neurotrophins, vascularization, and electrical activity of postsynaptic neurons in nucleus RA (robust nucleus of the arcopallium). In seasonal species, the HVC→RA circuit degenerates in nonbreeding birds, and is reconstructed by the incorporation of new projection neurons in breeding birds. There is a functional linkage between the death of mature HVC neurons and the birth of new neurons. Various hypotheses for the function of adult neurogenesis in the song system can be proposed, but this remains an open question.

Emerging from the bottleneck: benefits of the comparative approach to modern neuroscience.

Neuroscience has historically exploited a wide diversity of animal taxa. Recently, however, research has focused increasingly on a few model species. This trend has accelerated with the genetic revolution, as genomic sequences and genetic tools became available for a few species, which formed a bottleneck. This coalescence on a small set of model species comes with several costs that are often not considered, especially in the current drive to use mice explicitly as models for human diseases. Comparative studies of strategically chosen non-model species can complement model species research and yield more rigorous studies. As genetic sequences and tools become available for many more species, we are poised to emerge from the bottleneck and once again exploit the rich biological diversity offered by comparative studies.

Seasonal plasticity of precise spike timing in the avian auditory system.

Vertebrate audition is a dynamic process, capable of exhibiting both short- and long-term adaptations to varying listening conditions. Precise spike timing has long been known to play an important role in auditory encoding, but its role in sensory plasticity remains largely unexplored. We addressed this issue in Gambel's white-crowned sparrow (Zonotrichia leucophrys gambelii), a songbird that shows pronounced seasonal fluctuations in circulating levels of sex-steroid hormones, which are known to be potent neuromodulators of auditory function. We recorded extracellular single-unit activity in the auditory forebrain of males and females under different breeding conditions and used a computational approach to explore two potential strategies for the neural discrimination of sound level: one based on spike counts and one based on spike timing reliability. We report that breeding condition has robust sex-specific effects on spike timing. Specifically, in females, breeding condition increases the proportion of cells that rely solely on spike timing information and increases the temporal resolution required for optimal intensity encoding. Furthermore, in a functionally distinct subset of cells that are particularly well suited for amplitude encoding, female breeding condition enhances spike timing-based discrimination accuracy. No effects of breeding condition were observed in males. Our results suggest that high-resolution temporal discharge patterns may provide a plastic neural substrate for sensory coding.

Transsynaptic trophic effects of steroid hormones in an avian model of adult brain plasticity.

The avian song control system provides an excellent model for studying transsynaptic trophic effects of steroid sex hormones. Seasonal changes in systemic testosterone (T) and its metabolites regulate plasticity of this system. Steroids interact with the neurotrophin brain-derived neurotrophic factor (BDNF) to influence cellular processes of plasticity in nucleus HVC of adult birds, including the addition of newborn neurons. This interaction may also occur transsynpatically; T increases the synthesis of BDNF in HVC, and BDNF protein is then released by HVC neurons on to postsynaptic cells in nucleus RA where it has trophic effects on activity and morphology. Androgen action on RA neurons increases their activity and this has a retrograde trophic effect on the addition of new neurons to HVC. The functional linkage of sex steroids to BDNF may be of adaptive value in regulating the trophic effects of the neurotrophin and coordinating circuit function in reproductively relevant contexts.

Reactive neurogenesis in response to naturally occurring apoptosis in an adult brain.

Neuronal birth and death are tightly coordinated to establish and maintain properly functioning neural circuits. Disruption of the equilibrium between neuronal birth and death following brain injury or pharmacological insult often induces reactive, and in some cases regenerative, neurogenesis. Many neurodegenerative disorders are not injury-induced, however, so it is critical to determine if and how reactive neurogenesis occurs under noninjury-induced neurodegenerative conditions. Here, we used a model of naturally occurring neural degradation in a neural circuit that controls song behavior in Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii) and examined the temporal dynamics between neuronal birth and death. We found that during seasonal-like regression of the song, control nucleus HVC (proper name), caspase-mediated apoptosis increased within 2 d following transition from breeding to nonbreeding conditions and neural stem-cell proliferation in the nearby ventricular zone (VZ) increased shortly thereafter. We show that inhibiting caspase-mediated apoptosis in HVC decreased neural stem-cell proliferation in the VZ. In baseline conditions the extent of neural stem-cell proliferation correlated positively with the number of dying cells in HVC. We demonstrate that as apoptosis increased and the number of both recently born and pre-existing neurons in HVC decreased, the structure of song, a learned sensorimotor behavior, degraded. Our data illustrate that reactive neurogenesis is not limited to injury-induced neuronal death, but also can result from normally occurring degradation of a telencephalic neural circuit.

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 GABAA 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.

Estradiol selectively enhances auditory function in avian forebrain neurons.

Sex steroids modulate vertebrate sensory processing, but the impact of circulating hormone levels on forebrain function remains unclear. We tested the hypothesis that circulating sex steroids modulate single-unit responses in the avian telencephalic auditory nucleus, field L. We mimicked breeding or nonbreeding conditions by manipulating plasma 17ß-estradiol levels in wild-caught female Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii). Extracellular responses of single neurons to tones and conspecific songs presented over a range of intensities revealed that estradiol selectively enhanced auditory function in cells that exhibited monotonic rate level functions to pure tones. In these cells, estradiol treatment increased spontaneous and maximum evoked firing rates, increased pure tone response strengths and sensitivity, and expanded the range of intensities over which conspecific song stimuli elicited significant responses. Estradiol did not significantly alter the sensitivity or dynamic ranges of cells that exhibited non-monotonic rate level functions. Notably, there was a robust correlation between plasma estradiol concentrations in individual birds and physiological response properties in monotonic, but not non-monotonic neurons. These findings demonstrate that functionally distinct classes of anatomically overlapping forebrain neurons are differentially regulated by sex steroid hormones in a dose-dependent manner.

Impact of experience-dependent and -independent factors on gene expression in songbird brain.

Songbirds provide rich natural models for studying the relationships between brain anatomy, behavior, environmental signals, and gene expression. Under the Songbird Neurogenomics Initiative, investigators from 11 laboratories collected brain samples from six species of songbird under a range of experimental conditions, and 488 of these samples were analyzed systematically for gene expression by microarray. ANOVA was used to test 32 planned contrasts in the data, revealing the relative impact of different factors. The brain region from which tissue was taken had the greatest influence on gene expression profile, affecting the majority of signals measured by 18,848 cDNA spots on the microarray. Social and environmental manipulations had a highly variable impact, interpreted here as a manifestation of paradoxical "constitutive plasticity" (fewer inducible genes) during periods of enhanced behavioral responsiveness. Several specific genes were identified that may be important in the evolution of linkages between environmental signals and behavior. The data were also analyzed using weighted gene coexpression network analysis, followed by gene ontology analysis. This revealed modules of coexpressed genes that are also enriched for specific functional annotations, such as "ribosome" (expressed more highly in juvenile brain) and "dopamine metabolic process" (expressed more highly in striatal song control nucleus area X). These results underscore the complexity of influences on neural gene expression and provide a resource for studying how these influences are integrated during natural experience.

Seasonal changes in patterns of gene expression in avian song control brain regions.

Photoperiod and hormonal cues drive dramatic seasonal changes in structure and function of the avian song control system. Little is known, however, about the patterns of gene expression associated with seasonal changes. Here we address this issue by altering the hormonal and photoperiodic conditions in seasonally-breeding Gambel's white-crowned sparrows and extracting RNA from the telencephalic song control nuclei HVC and RA across multiple time points that capture different stages of growth and regression. We chose HVC and RA because while both nuclei change in volume across seasons, the cellular mechanisms underlying these changes differ. We thus hypothesized that different genes would be expressed between HVC and RA. We tested this by using the extracted RNA to perform a cDNA microarray hybridization developed by the SoNG initiative. We then validated these results using qRT-PCR. We found that 363 genes varied by more than 1.5 fold (>log(2) 0.585) in expression in HVC and/or RA. Supporting our hypothesis, only 59 of these 363 genes were found to vary in both nuclei, while 132 gene expression changes were HVC specific and 172 were RA specific. We then assigned many of these genes to functional categories relevant to the different mechanisms underlying seasonal change in HVC and RA, including neurogenesis, apoptosis, cell growth, dendrite arborization and axonal growth, angiogenesis, endocrinology, growth factors, and electrophysiology. This revealed categorical differences in the kinds of genes regulated in HVC and RA. These results show that different molecular programs underlie seasonal changes in HVC and RA, and that gene expression is time specific across different reproductive conditions. Our results provide insights into the complex molecular pathways that underlie adult neural plasticity.

Immediate and long-term effects of testosterone on song plasticity and learning in juvenile song sparrows.

Steroid sex hormones play critical roles in the development of brain regions used for vocal learning. It has been suggested that puberty-induced increases in circulating testosterone (T) levels crystallize a bird's repertoire and inhibit future song learning. Previous studies show that early administration of T crystallizes song repertoires but have not addressed whether new songs can be learned after this premature crystallization. We brought 8 juvenile song sparrows (Melospiza melodia) into the laboratory in the late summer and implanted half of them with subcutaneous T pellets for a two week period in October. Birds treated with T tripled their singing rates and crystallized normal songs in 2 weeks. After T removal, subjects were tutored by 4 new adults. Birds previously treated with T tended toward learning fewer new songs post T, consistent with the hypothesis that T helps to close the song learning phase. However, one T-treated bird proceeded to learn several new songs in the spring, despite singing perfectly crystallized songs in the fall. His small crystallized fall repertoire and initial lag behind other subjects in song development suggest that this individual may have had limited early song learning experience. We conclude that an exposure to testosterone sufficient for crystallization of a normal song repertoire does not necessarily prevent future song learning and suggest that early social experiences might override the effects of hormones in closing song learning.