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.

Mechanistic Hypotheses for Proprioceptive Sensing Within the Avian Lumbosacral Spinal Cord.

Animals need to accurately sense changes in their body position to perform complex movements. It is increasingly clear that the vertebrate central nervous system contains a variety of cells capable of detecting body motion, in addition to the comparatively well-understood mechanosensory cells of the vestibular system and the peripheral proprioceptors. One such intriguing system is the lower spinal cord and column in birds, also known as the avian lumbosacral organ (LSO), which is thought to act as a set of balance sensors that allow birds to detect body movements separately from head movements detected by the vestibular system. Here, we take what is known about proprioceptive, mechanosensory spinal neurons in other vertebrates to explore hypotheses for how the LSO might sense mechanical information related to movement. Although the LSO is found only in birds, recent immunohistochemical studies of the avian LSO have hinted at similarities between cells in the LSO and the known spinal proprioceptors in other vertebrates. In addition to describing possible connections between avian spinal anatomy and recent findings on spinal proprioception as well as sensory and sensorimotor spinal networks, we also present some new data that suggest a role for sensory afferent peptides in LSO function. Thus, this perspective articulates a set of testable ideas on mechanisms of LSO function grounded in the emerging spinal proprioception scientific literature.

Molecular Markers of Mechanosensation in Glycinergic Neurons in the Avian Lumbosacral Spinal Cord.

Birds are exceptionally adept at controlling their body position. For example, they can coordinate rapid movements of their body while stabilizing their head. Intriguingly, this ability may rely in part on a mechanosensory organ in the avian lower spinal cord called the lumbosacral organ (LSO). However, molecular mechanotransduction mechanisms have not been identified in the avian spinal cord. Here, we report the presence of glycinergic neurons in the LSO that exhibit immunoreactivity for myosin7a and espin, molecules essential for function and maintenance of hair cells in the inner ear. Specifically, we find glycinergic cell bodies near the central canal and processes that extend laterally to the accessory lobes and spinal ligaments. These LSO neurons are reminiscent of glycinergic neurons in a recently-described lateral spinal proprioceptive organ in zebrafish that detects spinal bending. The avian LSO, however, is located inside a series of fused vertebrae called the synsacrum, which constrains spinal bending. We suggest the LSO may be a modification and elaboration of a preexisting mechanosensory spinal network in vertebrates. A mechanistic understanding of its function may be an important clue to understanding the evolution and development of avian locomotion.

Differential development of myelin in zebra finch song nuclei.

Songbirds learn vocalizations by hearing and practicing songs. As song develops, the tempo becomes faster and more precise. In the songbird brain, discrete nuclei form interconnected myelinated circuits that control song acquisition and production. The myelin sheath increases the speed of action potential propagation by insulating the axons of neurons and by reducing membrane capacitance. As the brain develops, myelin increases in density, but the time course of myelin development across discrete song nuclei has not been systematically studied in a quantitative fashion. We tested the hypothesis that myelination develops differentially across time and song nuclei. We examined myelin development in the brains of the zebra finch (Taeniopygia guttata) from chick at posthatch day (d) 8 to adult (up to 147 d) in five major song nuclei: HVC (proper name), robust nucleus of the arcopallium (RA), Area X, lateral magnocellular nucleus of the anterior nidopallium, and medial portion of the dorsolateral thalamic nucleus (DLM). All of these nuclei showed an increase in the density of myelination during development but at different rates and to different final degrees. Exponential curve fits revealed that DLM showed earlier myelination than other nuclei, and HVC showed the slowest myelination of song nuclei. Together, these data show differential maturation of myelination in different portions of the song system. Such differential maturation would be well placed to play a role in regulating the development of learned song.

Distribution and co-expression of adrenergic receptor-encoding mRNA in the mouse inferior colliculus.

Adrenergic receptors are mediators of adrenergic and noradrenergic modulation throughout the brain. Previous studies have provided evidence for the expression of adrenergic receptors in the midbrain auditory nucleus, the inferior colliculus (IC), but have not examined the cellular patterns of expression in detail. Here, we utilize multichannel fluorescent in situ hybridization to detect the expression of adrenergic receptor-encoding mRNA in the inferior colliculus of male and female mice. We found expression of α1 , α2A , and ß2 receptor-encoding mRNA throughout all areas of the IC. While we observed similar levels of expression of α1 receptor-encoding mRNA across the subregions of the IC, α2A and ß2 receptor-encoding mRNA was expressed differentially. To account for developmental changes in noradrenergic receptor expression, we measured expression levels in mice aged P15, P20, and P60. We observed little change in levels of expression across these ages. To ascertain the modulatory potential of multiple adrenergic receptor subtypes in a single IC cell, we measured co-expression of α1 , α2A , and ß2 receptor-encoding mRNA. We found greater proportions of cells in the IC that expressed no adrenergic receptor-encoding mRNA, α1 and α2A adrenergic receptor-encoding mRNA, and α1, α2A, and ß2 receptor-encoding mRNA than would be predicted by independent expression of each receptor subtype. These data suggest a coordinated pattern of adrenergic receptor expression in the IC and provide the first evidence for adrenergic receptor expression and co-expression in the subregions of the mouse auditory midbrain.

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.

Expression of the potassium-chloride co-transporter, KCC2, within the avian song system.

Songbirds learn to produce vocalizations early in life by listening to, then copying the songs of conspecific males. The anterior forebrain pathway, homologous to a basal ganglia-forebrain circuit, is essential for song learning. The projection between the striato-pallidal structure, Area X, and the medial portion of the dorsolateral thalamic nucleus (DLM) is strongly hyperpolarizing in adults, due to a very negative chloride reversal potential (Person & Perkel, Neuron 46:129-140, 2005). The chloride reversal potential is determined, in part, by the expression level of a neuron-specific potassium-chloride cotransporter, KCC2, which is developmentally upregulated in mammals. To determine whether a similar upregulation in KCC2 expression occurs at the Area X to DLM synapse during development, we examined the expression level of KCC2 in adult zebra finches across the song system as well as during development in the Area X - DLM synapse. We demonstrate that KCC2 is expressed in a subset of neurons throughout the song system, including HVC (used as a proper name), robust nucleus of the arcopallium (RA), lateral magnocellular nucleus of the anterior nidopallium (LMAN), Area X, and DLM. The majority of pallidal-like projection neurons in Area X showed KCC2 immunoreactivity. In adults, KCC2 expression was robust within DLM, and was upregulated between 14 and 24 days post hatching, before the onset of song learning. Light and electron microscopic analysis indicated that KCC2 immunoreactivity is strongly associated with the plasma membrane. Thus, in the song system as in the mammalian brain, KCC2 expression is well placed to modulate the GABAA reversal potential.

Dopaminergic modulation of basal ganglia output through coupled excitation-inhibition.

Learning and maintenance of skilled movements require exploration of motor space and selection of appropriate actions. Vocal learning and social context-dependent plasticity in songbirds depend on a basal ganglia circuit, which actively generates vocal variability. Dopamine in the basal ganglia reduces trial-to-trial neural variability when the bird engages in courtship song. Here, we present evidence for a unique, tonically active, excitatory interneuron in the songbird basal ganglia that makes strong synaptic connections onto output pallidal neurons, often linked in time with inhibitory events. Dopamine receptor activity modulates the coupling of these excitatory and inhibitory events in vitro, which results in a dynamic change in the synchrony of a modeled population of basal ganglia output neurons receiving excitatory and inhibitory inputs. The excitatory interneuron thus serves as one biophysical mechanism for the introduction or modulation of neural variability in this circuit.

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.

Extreme Postnatal Scaling in Bat Feeding Performance: A View of Ecomorphology from Ontogenetic and Macroevolutionary Perspectives.

Ecomorphology studies focus on understanding how anatomical and behavioral diversity result in differences in performance, ecology, and fitness. In mammals, the determinate growth of the skeleton entails that bite performance should change throughout ontogeny until the feeding apparatus attains its adult size and morphology. Then, interspecific differences in adult phenotypes are expected to drive food resource partitioning and patterns of lineage diversification. However, Formal tests of these predictions are lacking for the majority of mammal groups, and thus our understanding of mammalian ecomorphology remains incomplete. By focusing on a fundamental measure of feeding performance, bite force, and capitalizing on the extraordinary morphological and dietary diversity of bats, we discuss how the intersection of ontogenetic and macroevolutionary changes in feeding performance may impact ecological diversity in these mammals. We integrate data on cranial morphology and bite force gathered through longitudinal studies of captive animals and comparative studies of free-ranging individuals. We demonstrate that ontogenetic trajectories and evolutionary changes in bite force are highly dependent on changes in body and head size, and that bats exhibit dramatic, allometric increases in bite force during ontogeny. Interspecific variation in bite force is highly dependent on differences in cranial morphology and function, highlighting selection for ecological specialization. While more research is needed to determine how ontogenetic changes in size and bite force specifically impact food resource use and fitness in bats, interspecific diversity in cranial morphology and bite performance seem to closely match functional differences in diet. Altogether, these results suggest direct ecomorphological relationships at ontogenetic and macroevolutionary scales in bats.

Seasonal variations in auditory processing in the inferior colliculus of Eptesicus fuscus.

Eptesicus fuscus is typical of temperate zone bats in that both sexes undergo marked seasonal changes in behavior, endocrine status, and reproductive status. Acoustic communication plays a key role in many seasonal behaviors. For example, males emit specialized vocalizations during mating in the fall, and females use different specialized vocalizations to communicate with infants in late spring. Bats of both sexes use echolocation for foraging during times of activity, but engage in little sound-directed behavior during torpor and hibernation in winter. Auditory processing might be expected to reflect these marked seasonal changes. To explore the possibility that seasonal changes in hormonal status could drive functional plasticity in the central auditory system, we examined responses of single neurons in the inferior colliculus throughout the year. The average first spike latency in females varied seasonally, almost doubling in spring compared to other times of year. First spike latencies in males remained relatively stable throughout the year. Latency jitter for both sexes was higher in winter and spring than in summer or fall. Females had more burst responders than other discharge patterns throughout the year whereas males had more transient responders at all times of year except fall, when burst responses were the predominant type. The percentage of simple discharge patterns (sustained and transient) was higher in males than females in the spring and higher in females than males in the fall. In females, the percentage of shortpass duration-tuned neurons doubled in summer and remained elevated through fall and early winter. In males, the percentage of shortpass duration-tuned cells increased in spring and the percentage of bandpass duration-tuned cells doubled in the fall. These findings suggest that there are clear seasonal changes in basic response characteristics of midbrain auditory neurons in Eptesicus, especially in temporal response properties and duration sensitivity. Moreover, the pattern of changes is different in males and females, suggesting that hormone-driven plasticity adjusts central auditory processing to fit the characteristics of vocalizations specific to seasonal behavioral patterns.

Hepatic Lipidosis in a Research Colony of Big Brown Bats (Eptesicus fuscus).

During a nearby construction project, a sudden decrease in food intake and guano production occurred in an outdoor colony of big brown bats (Eptesicus fuscus), and one animal was found dead. Investigation revealed that the project was generating a large amount of noise and vibration, which disturbed the bats' feeding. Consequently the bats were moved into an indoor enclosure away from the construction noises, and the colony resumed eating. Over the next 3 wk, additional animals presented with clinical signs of lethargy, weight loss, ecchymoses, and icterus and were necropsied. Gross necropsy of the affected bats revealed large, pale yellow to tan, friable livers with rounded edges that floated when placed in 10% neutral-buffered formalin. Some bats had ecchymoses on the webbing and skin and gross perirenal hemorrhage. Histologic examination showed hepatic and renal tubular lipidosis. The clinical and pathologic signs of hemorrhage and icterus were suggestive of hepatic failure. Hepatic lipidosis was attributed to stress and inappetence associated with environmental perturbations. Once the environmental stressor was removed, the colony morbidity and mortality decreased. However, 2 y later, a series of new environmental stressors triggered additional deaths associated with hepatic lipidosis. Over a 9-y period, 21 cases of hepatic lipidosis were diagnosed in this bat colony.

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.

Stimulus-specific adaptation in specialized neurons in the inferior colliculus of the big brown bat, Eptesicus fuscus.

The inferior colliculus (IC) of the big brown bat (Eptesicus fuscus) contains specialized neurons that respond exclusively to highly specific spectrotemporal patterns such as sinusoidally frequency modulated (SFM) signals or directional frequency modulated sweeps (FM). Other specialized cells with I-shaped frequency response areas (FRAs) are tuned to very narrow frequency bands (1-2 kHz) in an amplitude-tolerant manner. In contrast, non-specialized neurons respond to any stimulus with energy in their frequency response area. IC neurons in several mammalian species, including bats, demonstrate stimulus-specific adaptation (SSA), a reduction in response to a high-probability stimulus. To evaluate the relation between stimulus selectivity and SSA, we presented sounds using an oddball stimulus paradigm and recorded extracellular responses of IC neurons. SFM-selective cells (n = 10), FM-selective cells (n = 7), and cells with I-shaped FRAs (n = 13) did not show SSA under any of the conditions tested (NSSI = 0.009, 0.033, 0.020 respectively). However, non-specialized neurons (n = 52) exhibited various levels of SSA (NSSI = 0.163), with a subset of these cells displaying strong adaptation. These findings suggest that SSA is not a ubiquitous characteristic of all neurons in the bat IC, but is present only in a subset of non-specialized neurons.

Development of echolocation and communication vocalizations in the big brown bat, Eptesicus fuscus.

Big brown bats form large maternity colonies of up to 200 mothers and their pups. If pups are separated from their mothers, they can locate each other using vocalizations. The goal of this study was to systematically characterize the development of echolocation and communication calls from birth through adulthood to determine whether they develop from a common precursor at the same or different rates, or whether both types are present initially. Three females and their six pups were isolated from our captive breeding colony. We recorded vocal activity from postnatal day 1 to 35, both when the pups were isolated and when they were reunited with their mothers. At birth, pups exclusively emitted isolation calls, with a fundamental frequency range <20 kHz, and duration >30 ms. By the middle of week 1, different types of vocalizations began to emerge. Starting in week 2, pups in the presence of their mothers emitted sounds that resembled adult communication vocalizations, with a lower frequency range and longer durations than isolation calls or echolocation signals. During weeks 2 and 3, these vocalizations were extremely heterogeneous, suggesting that the pups went through a babbling stage before establishing a repertoire of stereotyped adult vocalizations around week 4. By week 4, vocalizations emitted when pups were alone were identical to adult echolocation signals. Echolocation and communication signals both appear to develop from the isolation call, diverging during week 2 and continuing to develop at different rates for several weeks until the adult vocal repertoire is established.