The mammalian cerebral cortex is composed of neurons whose properties vary in a continuous fashion rather than falling into discrete cell types. In the mouse visual cortex, excitatory neurons in layer 2 and 3 (L2/3) form such a continuum along cortical depth, patterned by the graded expression of hundreds of genes. Here we sought to understand how this continuum develops and contributes to cortical wiring. Using single-nucleus multiomics (RNA- and ATAC-Seq) and spatial transcriptomics, we show that the L2/3 continuum is established in two phases. During the first postnatal week, a genetically hardwired program establishes a primitive continuum of cell identities spanning the depth of L2/3. The second program, promoted by visual experience, is later superimposed upon the preexisting continuum. This second phase is driven by activity-regulated transcription factors that drive the L2/3 depth-dependent expression of genes linked to synaptic function and plasticity. We show that neurons at different positions along the L2/3 continuum project preferentially to distinct higher visual areas and that visual deprivation disrupts targeting to some higher visual areas while sparing others. Thus, cortical continua emerge through a stepwise process in which genetic programs and sensory experience specify neuronal identity and sculpt intracortical wiring specificity.

The amphipod crustacean Parhyale hawaiensis has been put forward as an attractive organism for evolutionary developmental comparisons, and considerable effort is being invested in isolating developmental genes and studying their expression patterns in this species. The scope of these studies could be significantly expanded by establishing means for genetic manipulation that would enable direct studies of gene functions to be carried out in this species. Here, we report the use of the Minos transposable element for the genetic transformation of P. hawaiensis. Transformed amphipods can be obtained from approximately 30% of surviving individuals injected with both a Minos element carrying the 3xP3-DsRed fluorescent marker and with mRNA encoding the Minos transposase. Integral copies of the transposon are inserted into the host genome and are stably inherited through successive generations. We have used reporter constructs to identify a muscle-specific regulatory element from Parhyale, demonstrating that this transformation vector can be used to test the activity of cis-regulatory elements in this species. The relatively high efficiency of this transgenic methodology opens new opportunities for the direct study of cis-regulatory elements and gene functions in Parhyale, allowing functional studies to be carried out beyond previously established model systems in insects.

A recent study challenges the oft-held notion that ester bonds in prodrug molecules are cleaved rapidly and completely inside cells by endogenous, nonspecific esterases. Structure-activity relationship studies on acylated sugars reveal that regioisomeric compounds display disparate biological activity, suggesting that ester bonds can persist in a cellular context.

Pairwise sequence covariations are a signal of conserved RNA secondary structure. We describe a method for distinguishing when lack of covariation signal can be taken as evidence against a conserved RNA structure, as opposed to when a sequence alignment merely has insufficient variation to detect covariations. We find that alignments for several long noncoding RNAs previously shown to lack covariation support do have adequate covariation detection power, providing additional evidence against their proposed conserved structures.

One approach to super-resolution fluorescence microscopy, termed stochastic localization microscopy, relies on the nanometer scale spatial localization of individual fluorescent emitters that stochastically label specific features of the specimen. The precision of emitter localization is an important determinant of the resulting image resolution but is insufficient to specify how well the derived images capture the structure of the specimen. We address this deficiency by considering the inference of specimen structure based on the estimated emitter locations. By using estimation theory, we develop a measure of spatial resolution that jointly depends on the density of the emitter labels, the precision of emitter localization, and prior information regarding the spatial frequency content of the labeled object. The Nyquist criterion does not set the scaling of this measure with emitter number. Given prior information and a fixed emitter labeling density, our resolution measure asymptotes to a finite value as the precision of emitter localization improves. By considering the present experimental capabilities, this asymptotic behavior implies that further resolution improvements require increases in labeling density above typical current values. Our treatment also yields algorithms to enhance reliable image features. Overall, our formalism facilitates the rigorous statistical interpretation of the data produced by stochastic localization imaging techniques.

Upon exposure to ethanol, Drosophila display behaviors that are similar to ethanol intoxication in rodents and humans. Using an inebriometer to measure ethanol-induced loss of postural control, we identified cheapdate, a mutant with enhanced sensitivity to ethanol. Genetic and molecular analyses revealed that cheapdate is an allele of the memory mutant amnesiac. amnesiac has been postulated to encode a neuropeptide that activates the cAMP pathway. Consistent with this, we find that enhanced ethanol sensitivity of cheapdate can be reversed by treatment with agents that increase cAMP levels or PKA activity. Conversely, genetic or pharmacological reduction in PKA activity results in increased sensitivity to ethanol. Taken together, our results provide functional evidence for the involvement of the cAMP signal transduction pathway in the behavioral response to intoxicating levels of ethanol.

BACKGROUND: It has become increasingly clear that molecular and neural mechanisms underlying learning and memory and drug addiction are largely shared. To confirm and extend these findings, we analyzed ethanol-responsive behaviors of a collection of Drosophila long-term memory mutants.

METHODS: For each mutant, sensitivity to the acute uncoordinating effects of ethanol was quantified using the inebriometer. Additionally, 2 distinct forms of ethanol tolerance were measured: rapid tolerance, which develops in response to a single brief exposure to a high concentration of ethanol vapor; and chronic tolerance, which develops following a sustained low-level exposure.

RESULTS: Several mutants were identified with altered sensitivity, rapid or chronic tolerance, while a number of mutants exhibited multiple defects.

CONCLUSIONS: The corresponding genes in these mutants represent areas of potential overlap between learning and memory and behavioral responses to alcohol. These genes also define components shared between different ethanol behavioral responses.

BACKGROUND: Increased ethanol intake, a major predictor for the development of alcohol use disorders, is facilitated by the development of tolerance to both the aversive and pleasurable effects of the drug. The molecular mechanisms underlying ethanol tolerance development are complex and are not yet well understood.

METHODS: To identify genetic mechanisms that contribute to ethanol tolerance, we examined the time course of gene expression changes elicited by a single sedating dose of ethanol in Drosophila, and completed a behavioral survey of strains harboring mutations in ethanol-regulated genes.

RESULTS: Enrichment for genes in metabolism, nucleic acid binding, olfaction, regulation of signal transduction, and stress suggests that these biological processes are coordinately affected by ethanol exposure. We also detected a coordinate up-regulation of genes in the Toll and Imd innate immunity signal transduction pathways. A multi-study comparison revealed a small set of genes showing similar regulation, including increased expression of 3 genes for serine biosynthesis. A survey of Drosophila strains harboring mutations in ethanol-regulated genes for ethanol sensitivity and tolerance phenotypes revealed roles for serine biosynthesis, olfaction, transcriptional regulation, immunity, and metabolism. Flies harboring deletions of the genes encoding the olfactory co-receptor Or83b or the sirtuin Sir2 showed marked changes in the development of ethanol tolerance.

CONCLUSIONS: Our findings implicate novel roles for these genes in regulating ethanol behavioral responses.

Transcriptional regulation is achieved through combinatorial interactions between regulatory elements in the human genome and a vast range of factors that modulate the recruitment and activity of RNA polymerase. Experimental approaches for studying transcription in vivo now extend from single-molecule techniques to genome-wide measurements. Parallel to these developments is the need for testable quantitative and predictive models for understanding gene regulation. These conceptual models must also provide insight into the dynamics of transcription and the variability that is observed at the single-cell level. In this Review, we discuss recent results on transcriptional regulation and also the models those results engender. We show how a non-equilibrium description informs our view of transcription by explicitly considering time- and energy-dependence at the molecular level.

Genetically encoded optical indicators hold the promise of enabling non-invasive monitoring of activity in identified neurons in behaving organisms. However, the interpretation of images of brain activity produced using such sensors is not straightforward. Several recent studies of sensory coding used G-CaMP 1.3-a calcium sensor-as an indicator of neural activity; some of these studies characterized the imaged neurons as having narrow tuning curves, a conclusion not always supported by parallel electrophysiological studies. To better understand the possible cause of these conflicting results, we performed simultaneous in vivo 2-photon imaging and electrophysiological recording of G-CaMP 1.3 expressing neurons in the antennal lobe (AL) of intact fruitflies. We find that G-CaMP has a relatively high threshold, that its signal often fails to capture spiking response kinetics, and that it can miss even high instantaneous rates of activity if those are not sustained. While G-CaMP can be misleading, it is clearly useful for the identification of promising neural targets: when electrical activity is well above the sensor’s detection threshold, its signal is fairly well correlated with mean firing rate and G-CaMP does not appear to alter significantly the responses of neurons that express it. The methods we present should enable any genetically encoded sensor, activator, or silencer to be evaluated in an intact neural circuit in vivo in Drosophila.