Mouse visual cortex is subdivided into multiple distinct, hierarchically organized areas that are interconnected through feedforward (FF) and feedback (FB) pathways. The principal synaptic targets of FF and FB axons that reciprocally interconnect primary visual cortex (V1) with the higher lateromedial extrastriate area (LM) are pyramidal cells (Pyr) and parvalbumin (PV)-expressing GABAergic interneurons. Recordings in slices of mouse visual cortex have shown that layer 2/3 Pyr cells receive excitatory monosynaptic FF and FB inputs, which are opposed by disynaptic inhibition. Most notably, inhibition is stronger in the FF than FB pathway, suggesting pathway-specific organization of feedforward inhibition (FFI). To explore the hypothesis that this difference is due to diverse pathway-specific strengths of the inputs to PV neurons we have performed subcellular Channelrhodopsin-2-assisted circuit mapping in slices of mouse visual cortex. Whole-cell patch-clamp recordings were obtained from retrobead-labeled FFV1→LM- and FBLM→V1-projecting Pyr cells, as well as from tdTomato-expressing PV neurons. The results show that the FFV1→LM pathway provides on average 3.7-fold stronger depolarizing input to layer 2/3 inhibitory PV neurons than to neighboring excitatory Pyr cells. In the FBLM→V1 pathway, depolarizing inputs to layer 2/3 PV neurons and Pyr cells were balanced. Balanced inputs were also found in the FFV1→LM pathway to layer 5 PV neurons and Pyr cells, whereas FBLM→V1 inputs to layer 5 were biased toward Pyr cells. The findings indicate that FFI in FFV1→LM and FBLM→V1 circuits are organized in a pathway- and lamina-specific fashion.

In most organisms, low ethanol doses induce increased activity, while high doses are sedating. To investigate the underlying mechanisms, we isolated Drosophila mutants with altered ethanol responsiveness. Mutations in white rabbit (whir), disrupting RhoGAP18B, are strongly resistant to the sedating effects of ethanol. This resistance can be suppressed by reducing the levels of Rho1 or Rac, implicating these GTPases in the behavioral response to ethanol. Indeed, expression of constitutively active forms of Rho1 or Rac1 in adult flies results in ethanol resistance similar to that observed in whir mutants. The whir locus produces several transcripts, RA-RD, which are predicted to encode three distinct RhoGAPs that share only the GAP domain. The RC transcript mediates the sedating effects of ethanol, while the RA transcript regulates its stimulant effects. Thus, distinct RhoGAPs, encoded by the same gene, regulate different manifestations of acute ethanol intoxication.

The superficial layers of the superior colliculus (sSC) receive retinal input and project to thalamic regions - the dorsal lateral geniculate (dLGN) and lateral posterior (LP; or pulvinar) nuclei -that convey visual information to cortex. A critical step towards understanding the functional impact of sSC neurons on these parallel thalamo-cortical pathways is determining whether different classes of sSC neurons, which are known to respond to different features of visual stimuli, innervate overlapping or distinct thalamic targets. Here, we identified a transgenic mouse line that labels sSC neurons that project to dLGN but not LP. We utilized selective expression of fluorophores and channelrhodopsin in this and previously characterized mouse lines to demonstrate that distinct cell types give rise to sSC projections to dLGN and LP. We further show that the glutamatergic sSC cell type that projects to dLGN also provides input to the sSC cell type that projects to LP. These results clarify the cellular origin of parallel sSC-thalamo-cortical pathways and reveal an interaction between these pathways via local connections within the sSC.

The human CRSP-Med coactivator complex is targeted by a diverse array of sequence-specific regulatory proteins. Using EM and single-particle reconstruction techniques, we recently completed a structural analysis of CRSP-Med bound to VP16 and SREBP-1a. Notably, these activators induced distinct conformational states upon binding the coactivator. Ostensibly, these different conformational states result from VP16 and SREBP-1a targeting distinct subunits in the CRSP-Med complex. To test this, we conducted a structural analysis of CRSP-Med bound to either thyroid hormone receptor (TR) or vitamin D receptor (VDR), both of which interact with the same subunit (Med220) of CRSP-Med. Structural comparison of TR- and VDR-bound complexes (at a resolution of 29 A) indeed reveals a shared conformational feature that is distinct from other known CRSP- Med structures. Importantly, this nuclear receptor-induced structural shift seems largely dependent on the movement of Med220 within the complex.

Activity in the motor cortex predicts movements, seconds before they are initiated. This preparatory activity has been observed across cortical layers, including in descending pyramidal tract neurons in layer 5. A key question is how preparatory activity is maintained without causing movement, and is ultimately converted to a motor command to trigger appropriate movements. Here, using single-cell transcriptional profiling and axonal reconstructions, we identify two types of pyramidal tract neuron. Both types project to several targets in the basal ganglia and brainstem. One type projects to thalamic regions that connect back to motor cortex; populations of these neurons produced early preparatory activity that persisted until the movement was initiated. The second type projects to motor centres in the medulla and mainly produced late preparatory activity and motor commands. These results indicate that two types of motor cortex output neurons have specialized roles in motor control.

Drosophila melanogaster can acquire a stable appetitive olfactory memory when the presentation of a sugar reward and an odor are paired. However, the neuronal mechanisms by which a single training induces long-term memory are poorly understood. Here we show that two distinct subsets of dopamine neurons in the fly brain signal reward for short-term (STM) and long-term memories (LTM). One subset induces memory that decays within several hours, whereas the other induces memory that gradually develops after training. They convey reward signals to spatially segregated synaptic domains of the mushroom body (MB), a potential site for convergence. Furthermore, we identified a single type of dopamine neuron that conveys the reward signal to restricted subdomains of the mushroom body lobes and induces long-term memory. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct dopamine neurons.

Ionotropic glutamate receptors principally mediate fast excitatory transmission in the brain. Among the three classes of ionotropic glutamate receptors, kainate receptors (KARs) have a unique brain distribution, which has been historically defined by (3)H-radiolabeled kainate binding. Compared with recombinant KARs expressed in heterologous cells, synaptic KARs exhibit characteristically slow rise-time and decay kinetics. However, the mechanisms responsible for these distinct KAR properties remain unclear. We found that both the high-affinity binding pattern in the mouse brain and the channel properties of native KARs are determined by the KAR auxiliary subunit Neto1. Through modulation of agonist binding affinity and off-kinetics of KARs, but not trafficking of KARs, Neto1 determined both the KAR high-affinity binding pattern and the distinctively slow kinetics of postsynaptic KARs. By regulating KAR excitatory postsynaptic current kinetics, Neto1 can control synaptic temporal summation, spike generation and fidelity.

Pigmentation divergence between Drosophila species has emerged as a model trait for studying the genetic basis of phenotypic evolution, with genetic changes contributing to pigmentation differences often mapping to genes in the pigment synthesis pathway and their regulators. These studies of Drosophila pigmentation have tended to focus on pigmentation changes in one body part for a particular pair of species, but changes in pigmentation are often observed in multiple body parts between the same pair of species. The similarities and differences of genetic changes responsible for divergent pigmentation in different body parts of the same species thus remain largely unknown. Here we compare the genetic basis of pigmentation divergence between Drosophila elegans and D. gunungcola in the wing, legs, and thorax. Prior work has shown that regions of the genome containing the pigmentation genes yellow and ebony influence the size of divergent male-specific wing spots between these two species. We find that these same two regions of the genome underlie differences in leg and thorax pigmentation; however, divergent alleles in these regions show differences in allelic dominance and epistasis among the three body parts. These complex patterns of inheritance can be explained by a model of evolution involving tissue-specific changes in the expression of Yellow and Ebony between D. elegans and D. gunungcola.

BRCA2 is an essential tumor suppressor protein involved in promoting faithful repair of DNA lesions. The activity of BRCA2 needs to be tuned precisely to be active when and where it is needed. Here, we quantified the spatio-temporal dynamics of BRCA2 in living cells using aberration-corrected multifocal microscopy (acMFM). Using multicolor imaging to identify DNA damage sites, we were able to quantify its dynamic motion patterns in the nucleus and at DNA damage sites. While a large fraction of BRCA2 molecules localized near DNA damage sites appear immobile, an additional fraction of molecules exhibits subdiffusive motion, providing a potential mechanism to retain an increased number of molecules at DNA lesions. Super-resolution microscopy revealed inhomogeneous localization of BRCA2 relative to other DNA repair factors at sites of DNA damage. This suggests the presence of multiple nanoscale compartments in the chromatin surrounding the DNA lesion, which could play an important role in the contribution of BRCA2 to the regulation of the repair process.

The microtubule cytoskeleton has proven to be an effective target for cancer therapeutics. One class of drugs, known as microtubule stabilizing agents (MSAs), binds to microtubule polymers and stabilizes them against depolymerization. The prototype of this group of drugs, Taxol, is an effective chemotherapeutic agent used extensively in the treatment of human ovarian, breast, and lung carcinomas. Although electron crystallography and photoaffinity labeling experiments determined that the binding site for Taxol is in a hydrophobic pocket in beta-tubulin, little was known about the effects of this drug on the conformation of the entire microtubule. A recent study from our laboratory utilizing hydrogen-deuterium exchange (HDX) in concert with various mass spectrometry (MS) techniques has provided new information on the structure of microtubules upon Taxol binding. In the current study we apply this technique to determine the binding mode and the conformational effects on chicken erythrocyte tubulin (CET) of another MSA, discodermolide, whose synthetic analogues may have potential use in the clinic. We confirmed that, like Taxol, discodermolide binds to the taxane binding pocket in beta-tubulin. However, as opposed to Taxol, which has major interactions with the M-loop, discodermolide orients itself away from this loop and toward the N-terminal H1-S2 loop. Additionally, discodermolide stabilizes microtubules mainly via its effects on interdimer contacts, specifically on the alpha-tubulin side, and to a lesser extent on interprotofilament contacts between adjacent beta-tubulin subunits. Also, our results indicate complementary stabilizing effects of Taxol and discodermolide on the microtubules, which may explain the synergy observed between the two drugs in vivo.