A high-throughput screen in mESCs to identify the cross-talk between signaling, endocytosis, and pluripotency.

Embryonic stem cell fate is regulated by various cellular processes. Recently, the process of endocytosis has been implicated in playing a role in the maintenance of self-renewal and pluripotency of mouse embryonic stem cells. A previous siRNA-based screen interrogated the function of core components of the endocytic machinery in maintaining the pluripotency of embryonic stem cells, revealing a crucial role for clathrin mediated endocytosis. A number of other proteins involved in key signaling pathways have also been shown to both regulate and be regulated by endocytosis. We collated a list of 1141 genes in connection to the term "endocytosis" from Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO), excluding those previously interrogated, and examined the effect of their knockdown on the pluripotency of mouse embryonic stem cells (mESCs) using levels of green fluorescent protein driven by the Oct4 promoter. We used high-throughput screening followed by an automated MATrix LABoratory (MATLAB)-based analysis pipeline and assessed changes in GFP fluorescence as a readout for ESC pluripotency. Through this screen we identified a number of genes, many hitherto not associated with stem cell pluripotency, which upon knockdown either resulted in a significant increase or decrease of GFP fluorescence. We further present validation for some of these hits. We present a workflow aimed to identify genes involved in signaling pathways which can be regulated by endocytosis, and that affect the pluripotency of ESCs.

Cell-cell adhesions in embryonic stem cells regulate the stability and transcriptional activity of ß-catenin.

E-cadherin (CDH1) is involved in maintaining cell-cell adhesions in embryonic stem cells (ESCs). However, its function in the context of cell fate decisions is largely unknown. Using mouse ESCs (mESCs), we demonstrate that E-cadherin and ß-catenin interact at the membrane and continue to do so upon internalization within the cell. Cdh1-/- mESCs failed to form tight colonies, with altered differentiation, marker expression and retention of pluripotency factors during differentiation. Interestingly, Cdh1-/- mESCs showed dramatically reduced ß-catenin levels. Transcriptional profiling of Cdh1-/- mESCs displayed a significant alteration in the expression of a subset of ß-catenin targets in a cell state- and GSK3ß-dependent manner. Our findings hint at hitherto unknown roles played by E-cadherin in regulating the activity of ß-catenin in ESCs.

Intrinsic motivation for singing in songbirds is enhanced by temporary singing suppression and regulated by dopamine.

Behaviors driven by intrinsic motivation are critical for development and optimization of physical and brain functions, but their underlying mechanisms are not well studied due to the complexity and autonomy of the behavior. Songbirds, such as zebra finches, offer a unique opportunity to study neural substrates of intrinsic motivation because they spontaneously produce many renditions of songs with highly-quantifiable structure for vocal practice, even in the absence of apparent recipients ("undirected singing"). Neural substrates underlying intrinsic motivation for undirected singing are still poorly understood partly because singing motivation cannot be easily manipulated due to its autonomy. Also, undirected singing itself acts as an internal reward, which could increase singing motivation, leading to difficulty in measuring singing motivation independent of singing-associated reward. Here, we report a simple procedure to easily manipulate and quantify intrinsic motivation for undirected singing independent of singing-associated reward. We demonstrate that intrinsic motivation for undirected singing is dramatically enhanced by temporary suppression of singing behavior and the degree of enhancement depends on the duration of suppression. Moreover, by examining latencies to the first song following singing suppression as a measure of singing motivation independent of singing-associated reward, we demonstrate that intrinsic singing motivation is critically regulated by dopamine through D2 receptors. These results provide a simple experimental tool to manipulate and measure the intrinsic motivation for undirected singing and illustrate the importance of zebra finches as a model system to study the neural basis of intrinsically-motivated behaviors.

Introductory gestures before songbird vocal displays are shaped by learning and biological predispositions.

Numerous animal displays begin with introductory gestures. For example, lizards start their head-bobbing displays with introductory push-ups, and many songbirds begin their vocal displays by repeating introductory notes (INs) before producing their learned song. Among songbirds, the acoustic structure and the number of INs produced before song vary considerably between individuals in a species. While similar variation in songs between individuals is a result of learning, whether variations in INs are also due to learning remains poorly understood. Here, using natural and experimental tutoring with male zebra finches, we show that mean IN number and IN acoustic structure are learned from a tutor. Interestingly, IN properties and how well INs were learned, were not correlated with the accuracy of song imitation and only weakly correlated with some features of songs that followed. Finally, birds artificially tutored with songs lacking INs still repeated vocalizations that resembled INs, before their songs, suggesting biological predispositions in IN production. These results demonstrate that INs, just like song elements, are shaped both by learning and biological predispositions. More generally, our results suggest mechanisms for generating variation in introductory gestures between individuals while still maintaining the species-specific structure of complex displays like birdsong.

Altering Opioid Neuromodulation in the Songbird Basal Ganglia Modulates Vocalizations.

Although the interplay between endogenous opioids and dopamine (DA) in the basal ganglia (BG) is known to underlie diverse motor functions, few studies exist on their role in modulating speech and vocalization. Vocal impairment is a common symptom of Parkinson's disease (PD), wherein DA depletion affects striosomes rich in µ-opioid receptors (µ-ORs). Symptoms of opioid addiction also include deficiencies in verbal functions and speech. To understand the interplay between the opioid system and BG in vocalization, we used adult male songbirds wherein high levels of µ-ORs are expressed in Area X, a BG region which is part of a circuit similar to the mammalian thalamocortical-basal ganglia loop. Changes in DA, glutamate and GABA levels were analyzed during the infusion of different doses of the µ-OR antagonist naloxone (50 and 100 ng/ml) specifically in Area X. Blocking µ-ORs in Area X with 100 ng/ml naloxone led to increased levels of DA in this region without altering the number of songs directed toward females (FD). Interestingly, this manipulation also led to changes in the spectro-temporal properties of FD songs, suggesting that altered opioid modulation in the thalamocortical-basal ganglia circuit can affect vocalization. Our study suggests that songbirds are excellent model systems to explore how the interplay between µ-ORs and DA modulation in the BG affects speech/vocalization.

Sensory feedback independent pre-song vocalizations correlate with time to song initiation.

The song of the adult male zebra finch is a well-studied example of a learned motor sequence. Song bouts begin with a variable number of introductory notes (INs) before actual song production. Previous studies have shown that INs progress from a variable initial state to a stereotyped final state before each song. This progression is thought to represent motor preparation, but the underlying mechanisms remain poorly understood. Here, we assessed the role of sensory feedback in the progression of INs to song. We found that the mean number of INs before song and the progression of INs to song were not affected by removal of two sensory feedback pathways (auditory or proprioceptive). In both feedback-intact and feedback-deprived birds, the presence of calls (other non-song vocalizations), just before the first IN, was correlated with fewer INs before song and an initial state closer to song. Finally, the initial IN state correlated with the time to song initiation. Overall, these results show that INs do not require real-time sensory feedback for progression to song. Rather, our results suggest that changes in IN features and their transition to song are controlled by internal neural processes, possibly involved in getting the brain ready to initiate a learned movement sequence.

Clathrin-Mediated Endocytosis Regulates a Balance between Opposing Signals to Maintain the Pluripotent State of Embryonic Stem Cells.

Endocytosis is implicated in the maintenance of embryonic stem cell (ESC) pluripotency, although its exact role and the identity of molecular players remain poorly understood. Here, we show that the clathrin heavy chain (CLTC), involved in clathrin-mediated endocytosis (CME), is vital for maintaining mouse ESC (mESC) pluripotency. Knockdown of Cltc resulted in a loss of pluripotency accompanied by reduced E-cadherin (E-CAD) levels and increased levels of transforming growth factor ß (TGF-ß) and extracellular signal-regulated kinase (ERK) signaling. We demonstrate that both E-CAD and TGF-ß receptor type 1 (TGF-ßR1) are internalized through CME in mESCs. While E-CAD is recycled, TGF-ßR1 is targeted for lysosomal degradation thus maintaining inverse levels of these molecules. Finally, we show that E-CAD interacts with ERK, and that the decreased pluripotency upon CME loss can be rescued by inhibiting TGF-ßR, MEK, and GSK3ß, or overexpressing E-CAD. Our results demonstrate that CME is critical for balancing signaling outputs to regulate ESC pluripotency, and possibly cell fate choices in early development.

Pre-Bout Neural Activity Changes in Premotor Nucleus HVC Correlate with Successful Initiation of Learned Song Sequence.

Preparatory activity, characterized by gradual, longer timescale changes in neural activity, is present in a number of different brain areas before the onset of simple movements and is believed to be important for movement initiation. However, relatively little is known about such activity before initiation of naturally learned movement sequences. The song of an adult male zebra finch is a well studied example of a naturally learned movement sequence and previous studies have shown robust premotor activity immediately before song. Here, I characterize longer timescale changes in neural activity in adult male zebra finch premotor nucleus HVC before onset of song bouts. I show that interneurons and a subset of basal-ganglia-projecting neurons change their activity several hundred milliseconds before song bout onset. Interneurons increased their activity, whereas basal-ganglia-projecting neurons either increased or decreased their activity. Such changes in neural activity were larger, started earlier, and were more common specifically before song bouts that began with the short, repetitive, introductory notes (INs) characteristic of zebra finch song bouts. Further, stronger and earlier changes were also correlated with successful song sequence initiation. Finally, a small fraction of basal-ganglia-projecting neurons that increased their activity before song bout onset did not have song or IN-related activity, suggesting a specialized preparatory role for such neurons. Overall, these data suggest that pre-bout activity in HVC represents preparatory activity important for initiation of a naturally learned movement sequence.SIGNIFICANCE STATEMENT Changes in neuronal activity well before the onset of simple movements are thought to be important for movement initiation. However, a number of animal movements consist of sequences of simple movements and relatively little is known about neuronal activity before such movement sequences. Using adult zebra finch song, a well studied example of a movement sequence, I show here that neurons in premotor nucleus HVC change their activity hundreds of milliseconds before song bout onset. In most neurons, the presence of such changes correlated with successful song sequence initiation. My results show the presence of preparatory neural activity in HVC and suggest a role for HVC in sequence initiation in addition to its established role in song sequence timing.

Emergence of context-dependent variability across a basal ganglia network.

Context dependence is a key feature of cortical-basal ganglia circuit activity, and in songbirds the cortical outflow of a basal ganglia circuit specialized for song, LMAN, shows striking increases in trial-by-trial variability and bursting when birds sing alone rather than to females. To reveal where this variability and its social regulation emerge, we recorded stepwise from corticostriatal (HVC) neurons and their target spiny and pallidal neurons in Area X. We find that corticostriatal and spiny neurons both show precise singing-related firing across both social settings. Pallidal neurons, in contrast, exhibit markedly increased trial-by-trial variation when birds sing alone, created by highly variable pauses in firing. This variability persists even when recurrent inputs from LMAN are ablated. These data indicate that variability and its context sensitivity emerge within the basal ganglia network, suggest a network mechanism for this emergence, and highlight variability generation and regulation as basal ganglia functions.

Behavioral and neural signatures of readiness to initiate a learned motor sequence.

All movements are thought to be "prepared" in the brain before initiation, and preparation can be impaired in motor diseases. However, little is known about what sort of preparation precedes self-initiated, naturally learned sequences of movements. Here we took advantage of a canonical example of a precisely timed learned motor sequence, adult zebra finch song, to examine motor preparation. We found that the sequences of short vocalizations, or introductory notes (INs), preceding song gradually increased in speed and converged on an acoustic endpoint highly similar across renditions, just before song initiation. The more the initial IN differed acoustically from the final IN, the greater the number of INs produced presong. Moreover, the song premotor nucleus HVC exhibited IN-related neural activity that progressed to a distinctive endpoint immediately before song. Together, our behavioral and neural data suggest that INs reflect a variable period of preparation during which the brain attains a common "ready" state each time sequence generation is about to begin.

Rats smell in stereo.

It has been hypothesized that rats and other mammals can use stereo cues to localize odor sources, but there is limited behavioral evidence to support this hypothesis. We found that rats trained on an odor-localization task can localize odors accurately in one or two sniffs. Bilateral sampling was essential for accurate odor localization, with internasal intensity and timing differences as directional cues. If the stimulus arrived at the correct point of the respiration cycle, internasal timing differences as short as 50 milliseconds sufficed. Neuronal recordings show that bulbar neurons responded differentially to stimuli from the left and stimuli from the right.