Genome size varies widely among organisms and is known to affect vertebrate development, morphology, and physiology. In amphibians, genome size is hypothesized to contribute to loss of lateforming structures, although this hypothesis has mainly been discussed in salamanders. Here we estimated genome size for 22 anuran species and combined this novel data set with existing genome size data for an additional 234 anuran species to determine whether larger genome size is associated with loss of a late-forming anuran sensory structure, the tympanic middle ear. We established that genome size is negatively correlated with development rate across 90 anuran species and found that genome size evolution is correlated with evolutionary loss of the middle ear bone (columella) among 241 species (224 eared and 17 earless). We further tested whether the development of the tympanic middle ear could be constrained by large cell sizes and small body sizes during key stages of tympanic middle ear development (metamorphosis). Together, our evidence suggests that larger genomes, slower development rate, and smaller body sizes at metamorphosis may contribute to the loss of the anuran tympanic middle ear. We conclude that increases in anuran genome size, although less drastic than those in salamanders, may affect development of late-forming traits.

Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARH) are a diverse group of neurons that project widely to different brain regions. It is unknown how this small population of neurons organizes its afferent projections. In this study, we hypothesized that individual ARH POMC neurons exclusively innervate select target regions. To investigate this hypothesis, we first verified that only a fraction of ARH POMC neurons innervate the lateral hypothalamus (LH), the paraventricular nucleus of the hypothalamus (PVN), the periaqueductal gray (PAG), or the ventral tegmental area (VTA) using the retrograde tracer cholera toxin B (CTB). Next, two versions of CTB conjugated to distinct fluorophores were injected bilaterally into two of the regions such that PVN and VTA, PAG and VTA, or LH and PVN received tracers simultaneously. These pairs of target sites were chosen based on function and location. Few individual ARH POMC neurons projected to two brain regions at once, suggesting that there are ARH POMC neuron subpopulations organized by their afferent projections. We also investigated whether increasing the activity of POMC neurons could increase the number of ARH POMC neurons labeled with CTB, implying an increase in new synaptic connections to downstream regions. However, chemogenetic enhancement of POMC neuron activity did not increase retrograde tracing of CTB back to ARH POMC neurons from either the LH, PVN, or VTA. Overall, subpopulations of ARH POMC neurons with distinct afferent projections may serve as a way for the POMC population to organize its many functions.

The notion that speech becomes less fluent during stressful speaking conditions has received little empirical test, and no research has tested this relationship in older adult participants. We analyzed speeches produced during the Trier Social Stress Test (TSST) or during a less stressful placebo (pTSST) version of the task. We measured young and older adults' speech fillers (e.g., um), unfilled pauses (at least 1 s in duration), and other disfluencies (e.g., repetitions, repairs). Neither young nor older adult participants rated themselves as having greater stress in the TSST than pTSST condition, but behavioral effects were obtained. Participants in the TSST condition produced more mid-phrase speech fillers and unfilled pauses than participants in the pTSST condition. Young adults produced more unfilled pauses than older adults overall, and older adults produced more mid-phrase fillers than young adults. Critically, age group interacted with experimental condition, such that older speakers produced disproportionately more mid-phrase fillers than young adults in the TSST compared to the pTSST condition. In sum, the negative effects of the TSST on fluency were generally similar across age, but this specific age-related increase in mid-phrase fillers indicates that older adults' word retrieval may have been particularly negatively affected. Findings are generally consistent with previous research and add to understanding of how factors internal to the speaker (i.e., demographic, personality, and cognitive variables) and factors external to the speaker (i.e., variables regarding the situation, context, or content of speech) combine to affect speech fluency.

Following nerve stimulation, there are two distinct phases of Ca 2+-dependent neurotransmitter release: a fast, synchronous release phase, and a prolonged, asynchronous release phase. Each of these phases is tightly regulated and mediated by distinct mechanisms. Synaptotagmin 1 is the major Ca 2+ sensor that triggers fast, synchronous neurotransmitter release upon Ca 2+ binding by its C 2 A and C 2 B domains. It has also been implicated in the inhibition of asynchronous neurotransmitter release, as blocking Ca 2+ binding by the C 2 A domain of synaptotagmin 1 results in increased asynchronous release. However, the mutation used to block Ca 2+ binding in the previous experiments (aspartate to asparagine mutations, syt D-N) had the unintended side effect of mimicking Ca 2+ binding, raising the possibility that the increase in asynchronous release was directly caused by ostensibly constitutive Ca 2+ binding. Thus, rather than modulating an asynchronous sensor, syt D-N may be mimicking one. To directly test the C 2 A inhibition hypothesis, we utilized an alternate C 2 A mutation that we designed to block Ca 2+ binding without mimicking it (an aspartate to glutamate mutation, syt D-E). Analysis of both the original syt D-N mutation and our alternate syt D-E mutation at the Drosophila neuromuscular junction showed differential effects on asynchronous release, as well as on synchronous release and the frequency of spontaneous release. Importantly, we found that asynchronous release is not increased in the syt D-E mutant. Thus, our work provides new mechanistic insight into synaptotagmin 1 function during Ca 2 +-evoked synaptic transmission and demonstrates that Ca 2+ binding by the C 2 A domain of synaptotagmin 1 does not inhibit asynchronous neurotransmitter release in vivo. Significance statement This study provides mechanistic insights into synaptotagmin function during asynchronous neurotransmitter release and supports a dramatically different hypothesis regarding the mechanisms triggering asynchronous vesicle fusion. Using two distinct C 2 A mutations that block Ca 2+ binding, we report opposing effects on synchronous, spontaneous, and asynchronous neurotransmitter release. Importantly, our data demonstrate that Ca 2+ binding by the C 2 A

Agonist binding to the mu opioid receptor (MOR) results in conformational changes that allow recruitment of G-proteins, activation of downstream effectors and eventual desensitization and internalization, all of which could affect receptor mobility. The present study employed single particle tracking (SPT) of quantum dot labeled FLAG-tagged MORs to examine shifts in MOR mobility after agonist binding. FLAG-MORs on the plasma membrane were in both mobile and immobile states under basal conditions. Activation of FLAG-MORs with DAMGO caused an acute increase in the fraction of mobile MORs, and free portions of mobile tracks were partially dependent on interactions with G-proteins. In contrast, 10-minute exposure to DAMGO or morphine increased the fraction of immobile FLAG-MORs. While the decrease in mobility with prolonged DAMGO exposure corresponded to an increase in colocalization with clathrin, the increase in colocalization was present in both mobile and immobile FLAG-MORs. Thus, no single mobility state of the receptor accounted for colocalization with clathrin. These findings demonstrate that SPT can be used to track agonist-dependent changes in MOR mobility over time, but that the mobility states observed likely arise from a diverse set of interactions and will be most informative when examined in concert with particular downstream effectors.