Different types of Drosophila dopaminergic neurons (DANs) reinforce memories of unique valence and provide state-dependent motivational control [1]. Prior studies suggest that the compartment architecture of the mushroom body (MB) is the relevant resolution for distinct DAN functions [2, 3]. Here we used a recent electron microscope volume of the fly brain [4] to reconstruct the fine anatomy of individual DANs within three MB compartments. We find the 20 DANs of the γ5 compartment, at least some of which provide reward teaching signals, can be clustered into 5 anatomical subtypes that innervate different regions within γ5. Reconstructing 821 upstream neurons reveals input selectivity, supporting the functional relevance of DAN sub-classification. Only one PAM-γ5 DAN subtype (γ5fb) receives direct recurrent input from γ5β’2a mushroom body output neurons (MBONs) and behavioral experiments distinguish a role for these DANs in memory revaluation from those reinforcing sugar memory. Other DAN subtypes receive major, and potentially reinforcing, inputs from putative gustatory interneurons or lateral horn neurons, which can also relay indirect feedback from the MB. We similarly reconstructed the single aversively reinforcing PPL1-γ1pedc DAN. The γ1pedc DAN inputs are mostly different to those of γ5 DANs and are clustered onto distinct branches of its dendritic tree, presumably separating its established roles in aversive reinforcement and appetitive motivation [5, 6]. Additional tracing identified neurons that provide broad input to γ5, β’2a and γ1pedc DANs suggesting that distributed DAN populations can be coordinately regulated. These connectomic and behavioral analyses therefore reveal additional complexity of dopaminergic reinforcement circuits between and within MB compartments.

The ability to discriminate sensory stimuli with overlapping features is thought to arise in brain structures called expansion layers, where neurons carrying information about sensory features make combinatorial connections onto a much larger set of cells. For 50 years, expansion coding has been a prime topic of theoretical neuroscience, which seeks to explain how quantitative parameters of the expansion circuit influence sensory sensitivity, discrimination, and generalization. Here, we investigate the developmental events that produce the quantitative parameters of the arthropod expansion layer, called the mushroom body. Using Drosophila melanogaster as a model, we employ genetic and chemical tools to engineer changes to circuit development. These allow us to produce living animals with hypothesis-driven variations on natural expansion layer wiring parameters. We then test the functional and behavioral consequences. By altering the number of expansion layer neurons (Kenyon cells) and their dendritic complexity, we find that input density, but not cell number, tunes neuronal odor selectivity. Simple odor discrimination behavior is maintained when the Kenyon cell number is reduced and augmented by Kenyon cell number expansion. Animals with increased input density to each Kenyon cell show increased overlap in Kenyon cell odor responses and become worse at odor discrimination tasks.

The ability to discriminate sensory stimuli with overlapping features is thought to arise in brain structures called expansion layers, where neurons carrying information about sensory features make combinatorial connections onto a much larger set of cells. For 50 years, expansion coding has been a prime topic of theoretical neuroscience, which seeks to explain how quantitative parameters of the expansion circuit influence sensory sensitivity, discrimination, and generalization. Here, we investigate the developmental events that produce the quantitative parameters of the arthropod expansion layer, called the mushroom body. Using Drosophila melanogaster as a model, we employ genetic and chemical tools to engineer changes to circuit development. These allow us to produce living animals with hypothesis-driven variations on natural expansion layer wiring parameters. We then test the functional and behavioral consequences. By altering the number of expansion layer neurons (Kenyon cells) and their dendritic complexity, we find that input density, but not cell number, tunes neuronal odor selectivity. Simple odor discrimination behavior is maintained when the Kenyon cell number is reduced and augmented by Kenyon cell number expansion. Animals with increased input density to each Kenyon cell show increased overlap in Kenyon cell odor responses and become worse at odor discrimination tasks.

The basal ganglia play a critical role in the regulation of voluntary action in vertebrates. Our understanding of the function of the basal ganglia relies heavily upon anatomical information, but continued progress will require an understanding of the specific functional roles played by diverse cell types and their connectivity. An increasing number of mouse lines allow extensive identification, characterization, and manipulation of specified cell types in the basal ganglia. Despite the promise of genetically modified mice for elucidating the functional roles of diverse cell types, there is relatively little anatomical data obtained directly in the mouse. Here we have characterized the retrograde labeling obtained from a series of tracer injections throughout the dorsal striatum of adult mice. We found systematic variations in input along both the medial-lateral and anterior-posterior neuraxes in close agreement with canonical features of basal ganglia anatomy in the rat. In addition to the canonical features we have provided experimental support for the importance of non-canonical inputs to the striatum from the raphe nuclei and the amygdala. To look for organization at a finer scale we have analyzed the correlation structure of labeling intensity across our entire dataset. Using this analysis we found substantial local heterogeneity within the large-scale order. From this analysis we conclude that individual striatal sites receive varied combinations of cortical and thalamic input from multiple functional areas, consistent with some earlier studies in the rat that have suggested the presence of a combinatorial map.

It is known that during lens differentiation a number of fibre cell specific membrane proteins change their expression profiles. In this study we have investigated how the profiles of the two most abundant fibre cell membrane proteins AQP0 (formerly known as Major Intrinsic Protein, MIP) and MP20 change as a function of fibre cell differentiation. While AQP0 was always found associated with fibre cell membranes, MP20 was initially found in the cytoplasm of peripheral fibre cells before becoming inserted into the membranes of deeper fibre cells. To determine at what stage in fibre cell differentiation MP20 becomes inserted into the membrane, sections were double-labelled with an antibody against MP20, and propidium iodide, a marker of cell nuclei. This showed that membrane insertion of MP20 occurs in a discrete transition zone that coincided with the degradation of cell nuclei. To test the significance of the membrane insertion of MP20 to overall lens function, whole lenses were incubated for varying times in a solution containing either Texas Red-dextran or Lucifer yellow as markers of extracellular space. Lenses were fixed and then processed for immunocytochemistry. Analysis of these sections showed that both tracer dyes were excluded from the extracellular space in an area that coincided with insertion of MP20 into the plasma membrane. Our results suggest that the insertion of MP20 into fibre cell membranes coincides with the creation of a barrier that restricts the diffusion of molecules into the lens core via the extracellular space.

Fruit flies (Drosophila melanogaster) are small insects, with correspondingly small power budgets. Despite this, they perform sophisticated neural computations in real time. Careful study of these insects is revealing how some of these circuits work. Insights from these systems might be helpful in designing other low power circuits.

AIM: Ethanol-induced locomotor sensitization is a behavioral manifestation of physiological responses to repeated ethanol exposures. While ethanol exerts direct effects on multiple neurotransmitter systems in the brain, ethanol-induced changes in metabolic state, including acute hyperglycemia and inhibition of insulin signaling, also have plausible roles in the expression of ethanol-related behaviors through direct and indirect effects on brain function. The current experiments examined whether insulin administration or the resultant hypoglycemia might attenuate the development of sensitization to the locomotor stimulant effect of ethanol.

MAIN METHODS: Male and female DBA/2J mice received daily injections of 5 or 10 IU/kg insulin before or after a stimulating dose of ethanol and subsequent testing in an automated activity monitor. Blood glucose levels were determined upon the completion of the experiments.

KEY FINDINGS: Insulin injected prior to ethanol blunted the acute stimulant response as well as the acquisition and expression of locomotor sensitization, while insulin given after ethanol did not affect the development of the sensitized response. In a separate experiment, mice given glucose concurrently with insulin developed ethanol-induced locomotor sensitization normally.

SIGNIFICANCE: These experiments suggest that insulin attenuates the development of ethanol-induced locomotor sensitization, and that blood glucose levels can largely account for this effect. Further studies of the role of ethanol-induced metabolic states should provide novel information on the expression of ethanol-related behaviors.

The insulin signaling pathway regulates multiple physiological processes, including energy metabolism, organismal growth, aging and reproduction. Here we show that genetic manipulations in Drosophila melanogaster that impair the function of insulin-producing cells or of the insulin-receptor signaling pathway in the nervous system lead to increased sensitivity to the intoxicating effects of ethanol. These findings suggest a previously unknown role for this highly conserved pathway in regulating the behavioral responses to an addictive drug.

Understanding how glucose transporter isoform 4 (GLUT4) redistributes to the plasma membrane during insulin stimulation is a major goal of glucose transporter research. GLUT4 molecules normally reside in numerous intracellular compartments, including specialized storage vesicles and early/recycling endosomes. It is unclear how these diverse compartments respond to insulin stimulation to deliver GLUT4 molecules to the plasma membrane. For example, do they fuse with each other first or remain as separate compartments with different trafficking characteristics? Our recent live cell imaging studies are helping to clarify these issues. Using Rab proteins as specific markers to distinguish between storage vesicles and endosomes containing GLUT4, we demonstrate that it is primarily internal GLUT4 storage vesicles (GSVs) marked by Rab10 that approach and fuse at the plasma membrane and GSVs don't interact with endosomes on their way to the plasma membrane. These new findings add strong support to the model that GSV release from intracellular retention plays a major role in supplying GLUT4 molecules onto the PM under insulin stimulation.

At the beginning of the final larval (fifth) instar of Manduca sexta, imaginal precursors including wing discs and eye primordia initiate metamorphic changes, such as pupal commitment, patterning and cell proliferation. Juvenile hormone (JH) prevents these changes in earlier instars and in starved final instar larvae, but nutrient intake overcomes this effect of JH in the latter. In this study, we show that a molecular marker of pupal commitment, broad, is up-regulated in the wing discs by feeding on sucrose or by bovine insulin or Manduca bombyxin in starved final instar larvae. This effect of insulin could not be prevented by JH. In vitro insulin had no effect on broad expression but relieved the suppression of broad expression by JH. This effect of insulin was directly on the disc as shown by its reduction in the presence of insulin receptor dsRNA. In starved penultimate fourth instar larvae, broad expression in the wing disc was not up-regulated by insulin. The discs became responsive to this action of insulin during the molt to the fifth instar together with the ability to become pupally committed in response to 20-hydroxyecdysone. Thus, the Manduca bombyxin acts as a metamorphosis-initiating factor in the imaginal precursors.