There is increased appreciation that dopamine (DA) neurons in the midbrain respond not only to reward 1 and reward-predicting cues 1,2 , but also to other variables such as distance to reward 3 , movements 4-9 , and behavioral choices 10,11. Based on these findings, a major open question is how the responses to these diverse variables are organized across the population of DA neurons. In other words, do individual DA neurons multiplex multiple variables, or are subsets of neurons specialized in encoding specific behavioral variables? The reason that this fundamental question has been difficult to resolve is that recordings from large populations of individual DA neurons have not been performed in a behavioral task with sufficient complexity to examine these diverse variables simultaneously. To address this gap, we used 2-photon calcium imaging through an implanted lens to record activity of >300 midbrain DA neurons in the ventral tegmental area (VTA) during a complex decision-making task. As mice navigated in a virtual reality (VR) environment, DA neurons encoded an array of sensory, motor, and cognitive variables. These responses were functionally clustered, such that subpopulations of neurons transmitted information about a subset of behavioral variables, in addition to encoding reward. These functional clusters were spatially organized, such that neighboring neurons were more likely to be part of the same cluster. Taken together with the topography between DA neurons and their projections, this specialization and anatomical organization may aid downstream circuits in correctly interpreting the wide range of signals transmitted by DA neurons. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

The precise nature of information flow through a biological network, which is governed by factors such as response sensitivities and noise propagation, greatly affects the operation of biological systems. Quantitative analysis of these properties is often difficult in naturally occurring systems but can be greatly facilitated by studying simple synthetic networks. Here, we report the construction of synthetic transcriptional cascades comprising one, two, and three repression stages. These model systems enable us to analyze sensitivity and noise propagation as a function of network complexity. We demonstrate experimentally steady-state switching behavior that becomes sharper with longer cascades. The regulatory mechanisms that confer this ultrasensitive response both attenuate and amplify phenotypical variations depending on the system's input conditions. Although noise attenuation allows the cascade to act as a low-pass filter by rejecting short-lived perturbations in input conditions, noise amplification results in loss of synchrony among a cell population. The experimental results demonstrating the above network properties correlate well with simulations of a simple mathematical model of the system. gene regulation ͉ gene network ͉ low-pass filter ͉ stochastic R egulatory cascades are ubiquitous in biological systems. For example, Escherichia coli and Saccharomyces cerevisiae regulatory networks contain transcriptional cascades with two or more stages (1-3). Many signal transduction programs and protein kinase pathways also take advantage of cascaded processes to regulate activities within living cells (4-6). In general, regulatory cascades exhibit several important features (7-8). Protein cascades provide an ultrasensitive ''all-or-none'' response to graded inputs where very small changes in input stimuli switch the output between low and high levels (9-10). Cascades direct temporal programs of successive gene expression as observed in the formation of flagella in E. coli (11), sporulation in budding yeast (12), or regulatory pathways that control bacterial cell cycle (13). In multicellular organisms, such as Drosophila and sea urchin, developmental programs require elaborate temporal ordering of events, often orchestrated by cascaded processes (14-15).Regulatory cascades are frequently found within more complex networks that incorporate additional control mechanisms [i.e., feed forward loops (3, 16), feedback (17), checkpoints (18), and single-input modules (3)]. A valuable approach to studying the properties of recurring network motifs is to decouple them as much as possible from other genetic regulatory elements (19-21). Examining network behavior in model systems can help discover the valuable properties and limitations of these motifs. Recent experimental studies of two-stage transcriptional cascades have examined quantitatively their steady-state sensitivity, § temporal programming (1), and noise properties (23). In addition, ultrasensitivity and attenuation of noise in longer cascades have been analyze...

One of the important challenges in the emerging field of synthetic biology is designing artificial networks that achieve coordinated behavior in cell communities. Here we present a synthetic multicellular bacterial system where receiver cells exhibit transient gene expression in response to a long-lasting signal from neighboring sender cells. The engineered sender cells synthesize an inducer, an acyl-homoserine lactone (AHL), which freely diffuses to spatially proximate receiver cells. The receiver cells contain a pulse-generator circuit that incorporates a feed-forward regulatory motif. The circuit responds to a long-lasting increase in the level of AHL by transiently activating, and then repressing, the expression of a GFP. Based on simulation models, we engineered variants of the pulse-generator circuit that exhibit different quantitative responses such as increased duration and intensity of the pulse. As shown by our models and experiments, the maximum amplitude and timing of the pulse depend not only on the final inducer concentration, but also on its rate of increase. The ability to differentiate between various rates of increase in inducer concentrations affords the system a unique spatiotemporal behavior for cells grown on solid media. Specifically, receiver cells can respond to communication from nearby sender cells while completely ignoring communication from senders cells further away, despite the fact that AHL concentrations eventually reach high levels everywhere. Because of the resemblance to naturally occurring feed-forward motifs, the pulse generator can serve as a model to improve our understanding of such systems.synthetic biology ͉ cell-cell signaling ͉ gene regulation ͉ feed-forward M any biochemical processes in cells exhibit transient responses to long-lasting changes in environmental, intercellular, and intracellular conditions. These transient responses can have very different time scales that range from milliseconds to several hours and can also occur across different spatial dimensions. Examples can be found both in single cells and multicellular organisms, such as the adaptation of tumbling probabilities to nutrient levels in bacterial chemotaxis (1), bacterial flagellar development (2), somitogenesis protein expression during embryo development (3), JAK͞STAT immune response pathways (4), circadian rhythms (5), and various feedforward regulatory motifs (6). Despite the prevalence and importance of pulse behavior in naturally occurring systems, their operating principles are not well understood quantitatively. Building and studying synthetic networks that exhibit similar behavior can be helpful for an improved understanding of the principles and kinetics behind such spatiotemporal patterns in gene expression, as well as for engineering cellular systems for synthetic biology (7-18).Recent studies have described small synthetic gene networks that can serve as model systems. These include the autorepressor (9), toggle switches (8, 14), the repressilator (10), the genetic clock (14), and di...

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A capability for scanning electron microscopy of wet biological specimens is presented. A membrane that is transparent to electrons protects the fully hydrated sample from the vacuum. The result is a hybrid technique combining the ease of use and ability to see into cells of optical microscopy with the higher resolution of electron microscopy. The resolution of low-contrast materials is Ϸ100 nm, whereas in high-contrast materials the resolution can reach 10 nm. Standard immunogold techniques and heavy-metal stains can be applied and viewed in the fluid to improve the contrast. Images present a striking combination of whole-cell morphology with a wealth of internal details. A possibility for direct inspection of tissue slices transpires, imaging only the external layer of cells. Simultaneous imaging with photons excited by the electrons incorporates data on material distribution, indicating a potential for multilabeling and specific scintillating markers.