1. Circuit mechanisms of cortical plasticity and dysfunction

Ingrid Bureau, Leopoldo Petreanu, Alla Karpova

The wiring diagram is fundamental to understanding cortical function and plasticity. However, little quantitative information about functional circuits is available. What are the sources of input to a neuronal subtype in a particular layer and column and what are their relative strengths? Which connections change with novel sensory experience? 

We developed laser scanning photostimulation (LSPS) into a quantitative and rapid tool for circuit analysis. In brain slices we mapped the excitatory circuits impinging onto L2/3 and L5B neurons. To determine if morphology can predict functional circuits between excitatory neurons, we directly compared functional LSPS maps with ‘geometric circuits’ computed from quantitative reconstructions of axons and dendrites. Functional connections were accurately predicted by geometry within a particular projection (with interesting exceptions), but the ratio of functional to geometric connectivity differed greatly (> 20 fold) between projections.  This finding implies new forms of specificity in cortical circuits.

We performed an unbiased search for experience-dependent synaptic pathways impinging onto two types of L5B pyramidal cells, regular-spiking and intrinsically- bursting. Experience-dependent changes in excitatory L2/3 ®  L5B synapses were cell-type specific. While regular-spiking neurons lost input from their home column in an experience-dependent manner, intrinsically-bursting cells gained input from surround columns.

We have used LSPS to explore the circuit and defects in animal models of fragile X mental retardation. We found specific defects in the function and plasticity of excitatory L 4 ®  L 2/3 synapses.

2. Microorganization of the sensory cortex

Takashi Sato with Noah Gray

We are using 2-photon microscopy combined with Ca²⁺ imaging to measure the activity in neuronal populations in the intact neocortex. Neurons are labeled with membrane permeable synthetic Ca²⁺ indicator. Ca²⁺ transients evoked by action potentials can be detected as sawtooth-shaped fluorescence changes. We have developed algorithms to detect and count individual spikes based on Ca²⁺ imaging. Our techniques allow us to study the firing of populations of neurons with well-defined spatial relationships. We are using these tools to learn about the micoorganization of cortical maps in the somatosensory cortex and how this organization is shaped by changes in sensory experience.

3. New approaches to circuit analysis: photostimulation

Leopoldo Petreanu, Daniel Huber, Volker Scheuss

Investigation of functional circuits in the neocortex is confounded by the intertwined nature of dendrites and axons. LSPS by UV glutamate uncaging is a tool for selectively stimulating neurons in brain slices close to their cell bodies, avoiding axons of passage, while recording from postsynaptic neurons.  Somata are preferentially excited because the size of the UV beam and the soma are roughly matched. Somewhere between 10-20 neurons are excited at every laser position, producing good signal levels. Since a laser beam can be moved quickly, LSPS can rapidly accumulate spatial maps of monosynaptic (excitatory or inhibitory) input for individual neurons with sub-laminar and sub-columnar resolution (< 100 mm). We make our LSPS mapping software and hardware available to several laboratories.

LSPS mapping by UV glutamate uncaging has significant limitations for circuit analysis. The averaging inherent to the technique obscures information about pair-wise connectivity. What fraction of cells are connected? What is the distribution of the sizes of unitary currents? Addressing these important questions requires single-cell stimulation. The conventional method for recording from pairs of neurons using microelectrodes is slow and inefficient, allowing only a tiny subset of projections, characterized by unusually high connectivity, to be probed.

We are developing optical mapping with single-cell resolution. We have two approaches to stimulating single neurons. First, 2-photon uncaging of glutamate can be spatially localized to a small (1 mm³) focal volume. Localization of excitation favors driving single cells. However, 2-photon uncaging preferentially excites dendrites because gluRs are more concentrated there. This is a disadvantage for a mapping technique, because dendrites are highly entangled, while somata are not. We are testing rapid stimulus patterns designed to selectively excite somata by 2-photon uncaging. So far this has met with only limited success.

Second, we are bypassing gluRs and express exogenous receptors  (e.g. P2X2, ATP receptors; TRPV1, capsaicin receptors, CHr2) in cortical neurons. We find that expression of Chr2 is a powerful tool for circuit analysis.

4. New approaches to circuit analysis: imaging

Tianyi Mao, Dan O'Connor, Vijay Iyer

Single unit studies employing tungsten microelectrodes have revolutionized our understanding of the functional organization of the brain. However, extracellular recordings have major drawbacks. Only one (or a few) neuron is interrogated at a time and the cell type and location are not well-defined. In addition, only neurons that respond vigorously to the stimulus of interest are usually studied, biasing the sample. 2P microscopy, in combination with fluorescent probes of neuronal function, has the potential to overcome these problems.

Direct measurement of membrane depolarization can be achieved with voltage-sensitive dyes .  However, imaging with single cell resolution has remained challenging except in the most favorable circumstances .  Alternatively, action potentials open voltage gated calcium channels (VGCCs) and cause Ca²⁺ influx into the cytoplasm which can be readily detected using [Ca²⁺] imaging . Populations of neurons can be loaded with membrane permeable Ca²⁺ indicators in vitro and in vivo . Using these techniques it has been possible to track the dynamics of populations of individual neurons in vitro and in vivo .

A drawback of [Ca²⁺] indicators is that it is not straightforward to relate fluorescence changes to number of spikes or spike timing. First, the coupling between activity and Ca²⁺ accumulations depend on a number of factors that are difficult to control: Ca²⁺ influx per action potential varies from cell to cell; the [Ca²⁺] change depends on the amount of [Ca²⁺] indicator trapped in the cell and the size of the compartment imaged; Ca²⁺ can enter the cell not only through VGCCs but also through synaptic receptors and through calcium-induced calcium release. Second, the relationship between fractional fluorescence changes and Ca²⁺ accumulations by itself is complex: resting Ca²⁺ levels vary from cell to cell and compartment to compartment; membrane permeable forms of [Ca²⁺] indicators are often trapped in intracellular compartments, producing variable static baseline fluorescence; Ca²⁺ indicators respond sublinearly to [Ca²⁺] increases. It is sometimes possible to detect saw-tooth shaped fluorescence transients corresponding to single spikes , but in our view the properties of the noise sources in the intact brain may not allow single spike detection reliably in vivo .

Genetically encoded [Ca²⁺] indicators (GECI) may offer an alternative to synthetic indicators. GECIs have been used in combination with 2P microscopy in Drosophila and mice . With current GECIs their slow response kinetics, small dynamic range, and nonlinear behavior only add to the problems discussed for synthetic indicators above (Pologruto et al., 2004) . However, progress in indicator development and data analysis tools are rapid and substantial advances can be expected over the next few years.

We are investing significant effort in testing and developing new GECIs. We are also building a 2P microscope that allows rapid random access imaging to track the dynamics of neural populations over times of ms.