Of Toasters and Molecular Ticker Tapes

Experiments in systems neuroscience can be seen as consisting of three steps: (1) selecting the signals we are interested in, (2) probing the system with carefully chosen stimuli, and (3) getting data out of the brain. Here I discuss how emerging techniques in molecular biology are starting to improve these three steps. To estimate its future impact on experimental neuroscience, I will stress the analogy of ongoing progress with that of microprocessor production techniques. These techniques have allowed computers to simplify countless problems; because they are easier to use than mechanical timers, they are even built into toasters. Molecular biology may advance even faster than computer speeds and has made immense progress in understanding and designing molecules. These advancements may in turn produce impressive improvements to each of the three steps, ultimately shifting the bottleneck from obtaining data to interpreting it

Optical Control of Metabotropic Glutamate Receptors

G-protein coupled receptors (GPCRs), the largest family of membrane signaling proteins, respond to neurotransmitters, hormones and small environmental molecules. The neuronal function of many GPCRs has been difficult to resolve because of an inability to gate them with subtype-specificity, spatial precision, speed and reversibility. To address this, we developed an approach for opto-chemical engineering native GPCRs. We applied this to the metabotropic glutamate receptors (mGluRs) to generate light-agonized and light-antagonized “LimGluRs”. The light-agonized “LimGluR2”, on which we focused, is fast, bistable, and supports multiple rounds of on/off switching. Light gates two of the primary neuronal functions of mGluR2: suppression of excitability and inhibition of neurotransmitter release. The light-antagonized “LimGluR2block” can be used to manipulate negative feedback of synaptically released glutamate on transmitter release. We generalize the optical control to two additional family members: mGluR3 and 6. The system works in rodent brain slice and in zebrafish in vivo, where we find that mGluR2 modulates the threshold for escape behavior. These light-gated mGluRs pave the way for determining the roles of mGluRs in synaptic plasticity, memory and disease

Genetic schemes and schemata in neurophysiology.

Information in nervous systems is often carried by neural ensembles--groups of neurons in transient functional linkage--and written in a code that involves the spatial locations of active neurons or synapses and the times at which activity occurs. Even in favorable neuroanatomical circumstances, studying neural ensemble function presents a serious experimental challenge. One recent strategy to overcome this challenge relies on protein-based sensors that provide direct optical images of neural activity, and on protein-based effectors that interfere with it. Because these molecules are encodable in DNA, they can be introduced into intact animals by genetic manipulation, and their expression pattern can be tailored to include--exclusively and at the same time comprehensively--the neurons of interest. Circumscribed populations of neurons can thus be studied in virtual isolation at defined stages of intact neural pathways

Selective photostimulation of genetically chARGed neurons.

To permit direct functional analyses of neural circuits, we have developed a method for stimulating groups of genetically designated neurons optically. Coexpression of the Drosophila photoreceptor genes encoding arrestin-2, rhodopsin (formed by liganding opsin with retinal), and the alpha subunit of the cognate heterotrimeric G protein--an explosive combination we term "chARGe"--sensitizes generalist vertebrate neurons to light. Illumination of a mixed population of neurons elicits action potentials selectively and cell-autonomously in its genetically chARGed members. In contrast to bath-applied photostimulants or caged neurotransmitters, which act indiscriminately throughout the illuminated volume, chARGe localizes the responsiveness to light. Distributed activity may thus be fed directly into a circumscribed population of neurons in intact tissue, irrespective of the spatial arrangement of its elements

The columnar and laminar organization of inhibitory connections to neocortical excitatory cells.

The cytoarchitectonic similarities of different neocortical regions have given rise to the idea of 'canonical' connectivity between excitatory neurons of different layers within a column. It is unclear whether similarly general organizational principles also exist for inhibitory neocortical circuits. Here we delineate and compare local inhibitory-to-excitatory wiring patterns in all principal layers of primary motor (M1), somatosensory (S1) and visual (V1) cortex, using genetically targeted photostimulation in a mouse knock-in line that conditionally expresses channelrhodopsin-2 in GABAergic neurons. Inhibitory inputs to excitatory neurons derived largely from the same cortical layer within a three-column diameter. However, subsets of pyramidal cells in layers 2/3 and 5B received extensive translaminar inhibition. These neurons were prominent in V1, where they might correspond to complex cells, less numerous in barrel cortex and absent in M1. Although inhibitory connection patterns were stereotypical, the abundance of individual motifs varied between regions and cells, potentially reflecting functional specializations

Transmission of olfactory information between three populations of neurons in the antennal lobe of the fly.

Three classes of neurons form synapses in the antennal lobe of Drosophila, the insect counterpart of the vertebrate olfactory bulb: olfactory receptor neurons, projection neurons, and inhibitory local interneurons. We have targeted a genetically encoded optical reporter of synaptic transmission to each of these classes of neurons and visualized population responses to natural odors. The activation of an odor-specific ensemble of olfactory receptor neurons leads to the activation of a symmetric ensemble of projection neurons across the glomerular synaptic relay. Virtually all excited glomeruli receive inhibitory input from local interneurons. The extent, odor specificity, and partly interglomerular origin of this input suggest that inhibitory circuits assemble combinatorially during odor presentations. These circuits may serve as dynamic templates that extract higher order features from afferent activity patterns