A juvenile locomotor program promotes vocal learning in zebra finches.

The evolution and development of complex, learned motor skills are thought to be closely associated with other locomotor movement and cognitive functions. However, it remains largely unknown how different neuromuscular programs may interconnect during the protracted developmental process. Here we use a songbird to examine the behavioral and neural substrates between the development of locomotor movement and vocal-motor learning. Juvenile songbirds escalate their locomotor activity during the sensitive period for vocal learning, followed by a surge of vocal practice. Individual variability of locomotor production is positively correlated with precision of tutor imitation and duration of multi-syllable sequences. Manipulation of juvenile locomotion significantly impacts the precision of vocal imitation and neural plasticity. The locomotor program developed during the sensitive period of vocal learning may enrich the neural substrates that promote the subsequent development of vocal learning.

Seasonal regulation of singing-driven gene expression associated with song plasticity in the canary, an open-ended vocal learner.

Songbirds are one of the few animal taxa that possess vocal learning abilities. Different species of songbirds exhibit species-specific learning programs during song acquisition. Songbirds with open-ended vocal learning capacity, such as the canary, modify their songs during adulthood. Nevertheless, the neural molecular mechanisms underlying open-ended vocal learning are not fully understood. We investigated the singing-driven expression of neural activity-dependent genes (Arc, Egr1, c-fos, Nr4a1, Sik1, Dusp6, and Gadd45ß) in the canary to examine a potential relationship between the gene expression level and the degree of seasonal vocal plasticity at different ages. The expression of these genes was differently regulated throughout the critical period of vocal learning in the zebra finch, a closed-ended song learner. In the canary, the neural activity-dependent genes were induced by singing in the song nuclei throughout the year. However, in the vocal motor nucleus, the robust nucleus of the arcopallium (RA), all genes were regulated with a higher induction rate by singing in the fall than in the spring. The singing-driven expression of these genes showed a similar induction rate in the fall between the first year juvenile and the second year adult canaries, suggesting a seasonal, not age-dependent, regulation of the neural activity-dependent genes. By measuring seasonal vocal plasticity and singing-driven gene expression, we found that in RA, the induction intensity of the neural activity-dependent genes was correlated with the state of vocal plasticity. These results demonstrate a correlation between vocal plasticity and the singing-driven expression of neural activity-dependent genes in RA through song development, regardless of whether a songbird species possesses an open- or closed-ended vocal learning capacity.

A memory-driven auditory program ensures selective and precise vocal imitation in zebra finches.

In the vocal learning model, the juvenile first memorizes a model sound, and the imprinted memory gradually converts into vocal-motor output during the sensorimotor integration. However, early acquired memory may not precisely represent the fine structures of a model sound. How do juveniles ensure precise model imitation? Here we show that juvenile songbirds develop an auditory learning program by actively and attentively engaging with tutor's singing during the sensorimotor phase. The listening/approaching behavior requires previously acquired model memory and the individual variability of approaching behavior correlates with the precision of tutor song imitation. Moreover, it is modulated by dopamine and associated with forebrain regions for sensory processing. Overall, precise vocal learning may involve two steps of auditory processing: a passive imprinting of model memory occurs during the early sensory period; the previously acquired memory then guides an active and selective engagement of the re-exposed model to fine tune model imitation.

Neural activation in response to conspecific songs in zebra finch (Taeniopygia guttata) embryos and nestlings.

Classic studies on the effects of auditory stimulation in embryonic birds have largely been limited to precocial taxa. In altricial taxa, physiological responses of embryos and, subsequently, the behavioral responses of nestlings have begun to receive increasing attention, yet it remains unclear whether and to what specificity neural responses are generated in ovo. Using in-situ hybridization for an immediate early gene, ZENK, we detected significant neural activation in both the embryos and nestlings of an altricial songbird, the zebra finch (Taeniopygia guttata) when exposed to conspecific song playbacks relative to silence. In turn, embryonic ZENK responses to heterospecific songs were intermediate in strength. These results are consistent with physiological evidence for conspecific song selectivity in embryos of other altricial songbird taxa.

Vocal practice regulates singing activity-dependent genes underlying age-independent vocal learning in songbirds.

The development of highly complex vocal skill, like human language and bird songs, is underlain by learning. Vocal learning, even when occurring in adulthood, is thought to largely depend on a sensitive/critical period during postnatal development, and learned vocal patterns emerge gradually as the long-term consequence of vocal practice during this critical period. In this scenario, it is presumed that the effect of vocal practice is thus mainly limited by the intrinsic timing of age-dependent maturation factors that close the critical period and reduce neural plasticity. However, an alternative, as-yet untested hypothesis is that vocal practice itself, independently of age, regulates vocal learning plasticity. Here, we explicitly discriminate between the influences of age and vocal practice using a songbird model system. We prevented zebra finches from singing during the critical period of sensorimotor learning by reversible postural manipulation. This enabled to us to separate lifelong vocal experience from the effects of age. The singing-prevented birds produced juvenile-like immature song and retained sufficient ability to acquire a tutored song even at adulthood when allowed to sing freely. Genome-wide gene expression network analysis revealed that this adult vocal plasticity was accompanied by an intense induction of singing activity-dependent genes, similar to that observed in juvenile birds, rather than of age-dependent genes. The transcriptional changes of activity-dependent genes occurred in the vocal motor robust nucleus of the arcopallium (RA) projection neurons that play a critical role in the production of song phonology. These gene expression changes were accompanied by neuroanatomical changes: dendritic spine pruning in RA projection neurons. These results show that self-motivated practice itself changes the expression dynamics of activity-dependent genes associated with vocal learning plasticity and that this process is not tightly linked to age-dependent maturational factors.

Recurrent development of song idiosyncrasy without auditory inputs in the canary, an open-ended vocal learner.

Complex learned behaviors, like bird song and human speech, develop under the influence of both genetic and environmental factors. Accordingly, learned behaviors comprise species specificity and individual variability. Auditory information plays a critical role in vocal learning by songbirds, both to memorize tutor songs and to monitor own vocalizations. Nevertheless, audition-deprived songbirds develop structured, species-specific song patterns. It remains to be elucidated how the auditory input contributes to the development of individual variability of song characteristics. Here we show that an open-ended vocal learner, the canary, annually recapitulates individually unique songs without audition. Although the total number of syllable types was reduced by auditory deprivation, other vocal phenotypes examined in the syllable, phrase, and syntax of songs were conserved between the 1st and 2nd years, both in deafened and intact birds. In deafened canaries, approximately 60% of the syllables were yearly reproduced with consistent acoustic features, whereas the remaining syllables were replaced with new ones in an annual cycle of song development. These results indicate that the open-ended vocal learning of canaries involves an audition-independent mechanism for the development of recurrent song idiosyncrasy.

Lentiviral-Mediated Transgenesis in Songbirds.

Transgenesis involves the insertion of an exogenous gene into an animal's genome, which allows the identification of the expressed phenotypes in brain function or behavior. Lentiviral-mediated transgenesis offers unique transduction potency making it possible to deliver and stably integrate transgenes into a wide variety of dividing and nondividing cells. The ability to establish long-term expression of such transgenes allows their use for transgenesis which is especially useful in organisms lacking quality pluripotent stem cell lines and which is otherwise difficult to produce via traditional pronuclear microinjection, such as songbirds. Here we describe a protocol to generate the transgenic songbird, the zebra finch, by producing and inserting lentiviral-mediated transgene into the blastoderm of freshly laid eggs. This protocol includes procedures for production of lentiviral vectors, injection of a virus into zebra finch embryos, and postinjection care. The implementation of the songbird transgenic approach provides a leap toward basic and translational neuroscience that uses an animal model for speech and language and their pathologies. Additionally, the highly quantifiable song behavior, combined with a well-characterized song circuitry, offers an exciting opportunity to develop therapeutic strategies for neurological disorders.

Vocal matching and intensity of begging calls are associated with a forebrain song circuit in a generalist brood parasite.

Vocalizations produced by developing young early in life have simple acoustic features and are thought to be innate. Complex forms of early vocal learning are less likely to evolve in young altricial songbirds because the forebrain vocal-learning circuit is underdeveloped during the period when early vocalizations are produced. However, selective pressure experienced in early postnatal life may lead to early vocal learning that is likely controlled by a simpler brain circuit. We found the food begging calls produced by fledglings of the brown-headed cowbird (Molothrus ater), a generalist avian brood parasite, induced the expression of several immediate early genes and early circuit innervation in a forebrain vocal-motor pathway that is later used for vocal imitation. The forebrain neural activity was correlated with vocal intensity and variability of begging calls that appears to allow cowbirds to vocally match host nestmates. The begging-induced forebrain circuits we observed in fledgling cowbirds were not detected in nonparasitic passerines, including species that are close relatives to the cowbird. The involvement of forebrain vocal circuits during fledgling begging and its association with vocal learning plasticity may be an adaptation that provides young generalist brood parasites with a flexible signaling strategy to procure food from a wide range of heterospecific host parents.

Human mutant huntingtin disrupts vocal learning in transgenic songbirds.

Speech and vocal impairments characterize many neurological disorders. However, the neurogenetic mechanisms of these disorders are not well understood, and current animal models do not have the necessary circuitry to recapitulate vocal learning deficits. We developed germline transgenic songbirds, zebra finches (Taneiopygia guttata) expressing human mutant huntingtin (mHTT), a protein responsible for the progressive deterioration of motor and cognitive function in Huntington's disease (HD). Although generally healthy, the mutant songbirds had severe vocal disorders, including poor vocal imitation, stuttering, and progressive syntax and syllable degradation. Their song abnormalities were associated with HD-related neuropathology and dysfunction of the cortical-basal ganglia (CBG) song circuit. These transgenics are, to the best of our knowledge, the first experimentally created, functional mutant songbirds. Their progressive and quantifiable vocal disorder, combined with circuit dysfunction in the CBG song system, offers a model for genetic manipulation and the development of therapeutic strategies for CBG-related vocal and motor disorders.

Rudimentary substrates for vocal learning in a suboscine.

Vocal learning has evolved in only a few groups of mammals and birds. The key neuroanatomical and behavioural links bridging vocal learners and non-learners are still unknown. Here we show that a non-vocal-learning suboscine, the eastern phoebe, expresses neural and behavioural substrates that are associated with vocal learning in closely related oscine songbirds. In phoebes, a specialized forebrain region in the intermediate arcopallium seems homologous to the oscine song nucleus RA (robust nucleus of arcopallium) by its neural connections, expression of glutamate receptors and singing-dependent immediate-early gene expression. Lesion of this RA-like region induces subtle but consistent song changes. Moreover, the unlearned phoebe song unexpectedly develops through a protracted ontogeny. These features provide the first evidence of forebrain vocal-motor control in suboscines, which has not been encountered in other avian non-vocal-learners, and offer a potential configuration of brain and behaviour from which vocal learning might have evolved.

Differential androgen receptor expression and DNA methylation state in striatum song nucleus Area X between wild and domesticated songbird strains.

In songbirds, a specialized neural system, the song system, is responsible for acquisition and expression of species-specific vocal patterns. We report evidence for differential gene expression between wild and domesticated strains having different learned vocal phenotypes. A domesticated strain of the wild white-rumped munia, the Bengalese finch, has a distinct song pattern with a more complicated syntax than the wild strain. We identified differential androgen receptor (AR) expression in basal ganglia nucleus Area X GABAergic neurons between the two strains, and within different domesticated populations. Differences in AR expression were correlated with the mean coefficient of variation of the inter-syllable duration in the two strains. Differential AR expression in Area X was observed before the initiation of singing, suggesting that inherited and/or early developmental mechanisms may affect expression within and between strains. However, there were no distinct differences in regions upstream of the AR start codon among all the birds in the study. In contrast, an epigenetic modification, DNA methylation state in regions upstream of AR in Area X, was observed to differ between strains and within domesticated populations. These results provide insight into the molecular basis of behavioral evolution through the regulation of hormone-related genes and demonstrate the potential association between epigenetic modifications and behavioral phenotype regulation.

Decrease in ankle-brachial index over time and cardiovascular outcomes in patients with hemodialysis.

INTRODUCTION: Abnormal ankle-brachial index (ABI) is associated with increased morbidity and mortality in hemodialysis patients. However, whether the decrease in ABI over time carries the prognostic value is unknown. The aim of this study was to assess whether the decrease in ABI over time was a good predictor of poor cardiovascular (CV) prognosis in hemodialysis patients. METHODS: This study enrolled 234 routine hemodialysis patients and 173 patients completed the follow-up. The ABI was measured by an ABI-form device at baseline and at the first year follow-up. The ΔABI was defined as ABI measured at the first year follow-up minus ABI measured at baseline. Progressors of ABI were defined as patients with ΔABI < -0.3. CV events were defined as CV death, hospitalization for unstable angina, nonfatal myocardial infarction, hospitalization for arrhythmia, hospitalization for congestive heart failure and stroke. RESULTS: The follow-up period was 37.8 ± 11.1 months. In the multivariate analysis, progressors of ABI (hazard ratio, 2.71; 95% confidence interval, 1.10-6.68, P = 0.03), decreased albumin and increased high-sensitivity C-reactive protein were associated with increased CV events. CONCLUSIONS: This longitudinal study showed ΔABI < -0.3 was independently associated with an increase in CV events. Hence, a great decrease in ABI over time might be a useful indicator of poor CV prognosis in hemodialysis patients.

Specialized motor-driven dusp1 expression in the song systems of multiple lineages of vocal learning birds.

Mechanisms for the evolution of convergent behavioral traits are largely unknown. Vocal learning is one such trait that evolved multiple times and is necessary in humans for the acquisition of spoken language. Among birds, vocal learning is evolved in songbirds, parrots, and hummingbirds. Each time similar forebrain song nuclei specialized for vocal learning and production have evolved. This finding led to the hypothesis that the behavioral and neuroanatomical convergences for vocal learning could be associated with molecular convergence. We previously found that the neural activity-induced gene dual specificity phosphatase 1 (dusp1) was up-regulated in non-vocal circuits, specifically in sensory-input neurons of the thalamus and telencephalon; however, dusp1 was not up-regulated in higher order sensory neurons or motor circuits. Here we show that song motor nuclei are an exception to this pattern. The song nuclei of species from all known vocal learning avian lineages showed motor-driven up-regulation of dusp1 expression induced by singing. There was no detectable motor-driven dusp1 expression throughout the rest of the forebrain after non-vocal motor performance. This pattern contrasts with expression of the commonly studied activity-induced gene egr1, which shows motor-driven expression in song nuclei induced by singing, but also motor-driven expression in adjacent brain regions after non-vocal motor behaviors. In the vocal non-learning avian species, we found no detectable vocalizing-driven dusp1 expression in the forebrain. These findings suggest that independent evolutions of neural systems for vocal learning were accompanied by selection for specialized motor-driven expression of the dusp1 gene in those circuits. This specialized expression of dusp1 could potentially lead to differential regulation of dusp1-modulated molecular cascades in vocal learning circuits.

The ratio of observed to predicted left ventricular mass is independently associated with increased cardiovascular events in patients with chronic kidney disease.

A condition involving the growth of the myocardium that exceeds hemodynamic needs has been reported and called as inappropriate left ventricular mass (LVM). The appropriateness of LVM can be estimated by the ratio of observed LVM to predicted LVM. The excessive growth of LVM is frequently noted in patients with chronic kidney disease (CKD). This study is designed to assess whether the ratio of observed to predicted LVM is a useful prognostic indicator of cardiovascular events in patients with moderate to advanced CKD. We consecutively enrolled 485 patients with CKD stages 3-5 from our Outpatient Department of Internal Medicine. Inappropriate LVM was defined as observed LVM more than 28% greater than the predicted value. The relative risk of cardiovascular events was analyzed by Cox-regression methods. There was a significant trend for a stepwise increase in the observed/predicted LVM ratio (P<0.001) and the prevalence of inappropriate LVM (P=0.003) corresponding to advances in CKD stages. In the multivariate analysis, old age, a history of coronary artery disease, congestive heart failure, atrial fibrillation, wide pulse pressure, decreased serum albumin and hemoglobin levels, left atrial diameter >4.7 cm and increased observed/predicted LVM were independently associated with increased cardiovascular events. Our findings show that increased observed/predicted LVM is independently associated with adverse cardiovascular outcomes in patients with CKD stages 3-5.

Association of hyperuricemia with renal outcomes, cardiovascular disease, and mortality.

BACKGROUND AND OBJECTIVES: Hyperuricemia is an independent risk factor for mortality, cardiovascular disease, and renal disease in general population. However, the relationship between hyperuricemia with clinical outcomes in CKD remains controversial. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: The study investigated the association between uric acid with all-cause mortality, cardiovascular events, renal replacement therapy, and rapid renal progression (the slope of estimated GFR was less than -6 ml/min per 1.73 m(2)/y) in 3303 stages 3-5 CKD patients that were in the integrated CKD care system in one medical center and one regional hospital in southern Taiwan. RESULTS: In all subjects, the mean uric acid level was 7.9 ± 2.0 mg/dl. During a median 2.8-year follow-up, there were 471 (14.3%) deaths, 545 (16.5%) cardiovascular events, 1080 (32.3%) participants commencing renal replacement therapy, and 841 (25.5%) participants with rapid renal progression. Hyperuricemia increased risks for all-cause mortality and cardiovascular events (the adjusted hazard ratios for quartile four versus quartile one of uric acid [95% confidence interval] were 1.85 [1.40-2.44] and 1.42 [1.08-1.86], respectively) but not risks for renal replacement therapy (0.96 [0.79-1.16]) and rapid renal progression (1.30 [0.98-1.73]). CONCLUSIONS: In stages 3-5 CKD, hyperuricemia is a risk factor for all-cause mortality and cardiovascular events but not renal replacement therapy and rapid renal progression.

Increasing prevalence of peripheral artery occlusive disease in hemodialysis patients: a 2-year follow-up.

INTRODUCTION: Peripheral artery occlusive disease (PAOD) has been reported to be prevalent in hemodialysis patients and influence their mortality. Ankle-brachial index (ABI) <0.9 is a reliable marker for PAOD. The aims of the 2-year longitudinal study were to assess whether there was a progression in PAOD and to find out the determinants of ABI progression in hemodialysis patients. METHODS: This study enrolled 237 routine hemodialysis patients and 154 patients completed the 2-year follow-up. The ABI was measured by an ABI-form device at baseline and at the first and second year follow-up. The change in ABI (ΔABI) was defined as ABI measured at the second year follow-up minus ABI measured at baseline. RESULTS: The prevalence of ABI <0.9 increased yearly (10.4%, 22.7% and 27.9%, respectively; P < 0.001) and the values of ABI decreased yearly (1.11 ± 0.16, 0.97 ± 0.17 and 0.96 ± 0.19, respectively; P < 0.001) in the 154 follow-up patients. Multiple stepwise analysis identified fasting glucose level, calcium-phosphorous product, high-sensitivity C-reactive protein and homocysteine level as independent determinants of ΔABI. CONCLUSIONS: Our results demonstrated the prevalence of PAOD increased and the values of ABI decreased yearly in hemodialysis patients. The ABI progression was associated with high fasting glucose level, high calcium-phosphorous product, high-sensitivity C-reactive protein and low homocysteine levels.

Arterial stiffness in patients with chronic kidney disease.

INTRODUCTION: Brachial-ankle pulse wave velocity (baPWV) was a marker of arterial stiffness, and increased baPWV was associated with renal function progression in patients with chronic kidney disease (CKD). However, there was no study to evaluate the longitudinal change of baPWV in patients with CKD. The aims of this study were to assess whether there was a longitudinal change in baPWV and to find out the determinants of this change in patients with CKD. METHODS: This study enrolled 52 patients with CKD stages 3 to 5 from outpatient Department of Internal Medicine. The values of baPWV were measured by an ankle-brachial index-form device at baseline and at 2-year follow-up. Changes of parameters (Δ) were used to evaluate the relationship between biological markers, blood pressures, medications and baPWV. RESULTS: The values of baPWV decreased during the 2-year follow-up (2108.4 ± 893.9 and 1897.1 ± 472.4; P = 0.016). There were also significant reductions in systolic and diastolic blood pressures during the 2-year follow-up, and Δsystolic blood pressure and baseline baPWV were the major determinants of ΔbaPWV. CONCLUSIONS: The present longitudinal study showed that the values of baPWV decreased during the 2-year follow-up, and the major determinants of ΔbaPWV were Δsystolic blood pressure and baseline baPWV in patients with CKD. Good control of systolic blood pressure may be associated with the regression of baPWV in patients with CKD.

Stepwise increases in left ventricular mass index and decreases in left ventricular ejection fraction correspond with the stages of chronic kidney disease in diabetes patients.

AIMS: Patients with diabetic nephropathy are reported to have a high prevalence of left ventricular structural and functional abnormalities. This study was designed to assess the determinants of left ventricular mass index (LVMI) and left ventricular ejection fraction (LVEF) in diabetic patients at various stages of chronic kidney disease (CKD). METHODS: This cross-sectional study enrolled 285 diabetic patients with CKD stages 3 to 5 from our outpatient department of internal medicine. Clinical and echocardiographic parameters were compared and analyzed. RESULTS: We found a significant stepwise increase in LVMI (P < 0.001), LVH (P < 0.001), and LVEF <55% (P = 0.013) and a stepwise decrease in LVEF (P = 0.038) corresponding to advance in CKD stages. CONCLUSIONS: Our findings suggest that increases in LVMI and decreases in LVEF coincide with advances in CKD stages in patients with diabetes.

Echocardiographic parameters are independently associated with increased cardiovascular events in patients with chronic kidney disease.

BACKGROUND: Patients with chronic kidney disease (CKD) are associated with increased cardiovascular (CV) morbidity and mortality. Echocardiographic measures of heart structure and function have been reported to predict adverse CV outcomes in various pathologic conditions. The aim of this study is to assess whether echocardiographic parameters are independently associated with increased CV events in patients with CKD Stages 3-5. METHODS: We consecutively enrolled 505 CKD patients from our outpatient department of internal medicine. CV events were defined as CV death, hospitalization for unstable angina, non-fatal myocardial infarction, sustained ventricular arrhythmia, hospitalization for congestive heart failure, transient ischemia attack and stroke. The relative CV events' risk was analyzed by Cox regression methods. RESULTS: In the multivariate analysis, old age, the presence of diabetes, coronary artery disease and atrial fibrillation; decreased serum albumin and hematocrit levels; left atrial diameter (LAD) >4.7 cm [hazard ratio (HR), 2.141; 95% confidence interval (CI), 1.155-3.971, P = 0.016]; increased left ventricular mass index (LVMI) (HR, 1.006; 95% CI, 1.002 to 1.010, P = 0.003) and left ventricular ejection fraction (LVEF) <55% (HR, 2.007; 95% CI, 1.007-3.743, P = 0.028) were independently associated with increased CV events. CONCLUSIONS: Our findings show that LAD >4.7 cm, increased LVMI and LVEF <55% are independently associated with adverse CV outcomes in CKD patients. Screening CKD patients by means of echocardiography may help identify a high-risk group of poor CV prognosis.

Echocardiographic parameters are independently associated with rate of renal function decline and progression to dialysis in patients with chronic kidney disease.

BACKGROUND AND OBJECTIVES: Cardiac abnormalities were frequently noted in patients with chronic kidney disease (CKD). This study is designed to assess whether echocardiographic parameters are associated with rate of renal function decline and progression to dialysis in CKD stage 3 to 5 patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This longitudinal study enrolled 415 patients. The renal end point was defined as commencement of dialysis. The change in renal function was measured by estimated GFR (eGFR) slope. RESULTS: Progression to dialysis was predicted by wide pulse pressure, low albumin, low hemoglobin, high calcium-phosphorous product, proteinuria, diuretics use, and concentric left ventricular hypertrophy (LVH) (hazard ratio, 2.03; 95% confidence interval [CI], 1.00 to 4.10; P = 0.05). The eGFR slope was negatively associated with total cholesterol, uric acid, proteinuria, diuretics use, and left atrial (LA) diameter (change in slope, -0.50; 95% CI, -0.89 to -0.11; P = 0.01) and positively associated with albumin and left ventricular ejection fraction (LVEF) (change in slope, 0.06; 95% CI, 0.03 to 0.08; P < 0.001). CONCLUSIONS: Our study in patients of CKD stage 3 to 5 demonstrated that concentric LVH was associated with progression to dialysis, and that increased LA diameter and decreased LVEF were associated with faster renal function decline. Echocardiography may help identify high-risk groups with progressive decline in renal function to dialysis and rapid progression of renal dysfunction in CKD stage 3 to 5 patients.