Laser microscopy has generally poor temporal resolution, caused by the serial scanning of each pixel. This is a signifi cant problem for imaging or optically manipulating neural circuits, since neuronal activity is fast. To help surmount this limitation, we have developed a "scanless" microscope that does not contain mechanically moving parts. This microscope uses a diffractive spatial light modulator (SLM) to shape an incoming two-photon laser beam into any arbitrary light pattern. This allows the simultaneous imaging or photostimulation of different regions of a sample with three-dimensional precision. To demonstrate the usefulness of this microscope, we perform two-photon uncaging of glutamate to activate dendritic spines and cortical neurons in brain slices. We also use it to carry out fast (60 Hz) two-photon calcium imaging of action potentials in neuronal populations. Thus, SLM microscopy appears to be a powerful tool for imaging and optically manipulating neurons and neuronal circuits. Moreover, the use of SLMs expands the fl exibility of laser microscopy, as it can substitute traditional simple fi xed lenses with any calculated lens function.

We introduce an optical method to stimulate individual neurons in brain slices in any arbitrary spatiotemporal pattern, using two-photon uncaging of MNI-glutamate with beam multiplexing. This method has single-cell and three-dimensional precision. By sequentially stimulating up to a thousand potential presynaptic neurons, we generated detailed functional maps of inputs to a cell. We combined this approach with two-photon calcium imaging in an all-optical method to image and manipulate circuit activity.

Deciphering the circuitry of the neocortex requires knowledge of its components, making a systematic classification of neocortical neurons necessary. GABAergic interneurons contribute most of the morphological, electrophysiological and molecular diversity of the cortex, yet interneuron subtypes are still not well defined. To quantitatively identify classes of interneurons, 59 GFP-positive interneurons from a somatostatin-positive mouse line were characterized by whole-cell recordings and anatomical reconstructions. For each neuron, we measured a series of physiological and morphological variables and analyzed these data using unsupervised classification methods. PCA and cluster analysis of morphological variables revealed three groups of cells: one comprised of Martinotti cells, and two other groups of interneurons with short asymmetric axons targeting layers 2/3 and bending medially. PCA and cluster analysis of electrophysiological variables also revealed the existence of these three groups of neurons, particularly with respect to action potential time course. These different morphological and electrophysiological characteristics could make each of these three interneuron subtypes particularly suited for a different function within the cortical circuit.

We pulsed the activation of neurons using a femtosecond laser. Pyramidal neurons are depolarized and fire action potentials when high intensity mode-locked infrared light irradiates somatic membranes and axon initial segments. This depolarization is reversible, does not occur with CW laser light, and appears to be due to multiphoton excitation. We describe two regimes of multiphoton optical stimulation. Low intensity, long duration laser irradiation produces a sustained depolarization, insensitive to sodium channel blockers yet sensitive to antioxidants. On the other hand, high intensity, short duration irradiation can induce fast depolarizations, which appear due to different mechanism. The combination of multiphoton stimulation and optical probing could enable systematic analysis of circuits.

Dendritic spines mediate most excitatory synapses in the brain. Past theoretical work and recent experimental evidence have suggested that spines could contain sodium channels. We tested this by measuring the effect of the sodium channel blocker tetrodotoxin (TTX) on depolarizations generated by two-photon uncaging of glutamate on spines from mouse neocortical pyramidal neurons. In practically all spines examined, uncaging potentials were significantly reduced by TTX. This effect was postsynaptic and spatially localized to the spine and occurred with uncaging potentials of different amplitudes and in spines of different neck lengths. Our data confirm that spines from neocortical pyramidal neurons are electrically isolated from the dendrite and indicate that they have sodium channels and are therefore excitable structures. Spine sodium channels could boost synaptic potentials and facilitate action potential backpropagation.dendritic spines ͉ membrane potential ͉ neurons A s predicted by Ramón y Cajal (1), in the mammalian cortex, most synaptic contacts in pyramidal cells are made on dendritic spines (2). Thus, it is natural to wonder whether spines influence synaptic transmission. Indeed, theoretical work over the last six decades has explored the possibility that spines have an electrical function, filtering and/or perhaps amplifying synaptic potentials (3-10). At the same time, other calculations have argued that spines cannot have an electrical function, serving merely as biochemical compartments (11)(12)(13). This debate has been reopened by two-photon calcium imaging data that demonstrated that spines have voltage-sensitive calcium channels (14, 15). Therefore, it becomes possible that spines could also have other types of voltage-gated channels, including sodium and potassium channels, and, if so, that spines could be excitable structures (5, 16).In our recent studies, we have encountered evidence consistent with the existence of sodium channels in the spine. First, numerical simulations indicated that the measured densities of sodium channels in dendritic shafts are too low to sustain action potential (AP) back propagation in pyramidal neurons, and that additional sodium channels are likely to be present in spines to ensure effective back propagation (17). Also, optical measurements of membrane potential using second harmonic generation have shown that backpropagating APs invade spines without a significant decrement in amplitude (18). At the same time, we have found that the spine neck provides a barrier to the propagation of membrane potentials (19), so the full-blown backpropagating AP, rather than invading passively, could be locally generated at the spine. Together, these data suggest that sodium channels might exist in spines, and that they could help promote AP backpropagation.We have tested this hypothesis by using two-photon uncaging of glutamate to activate spines individually and examine whether their responses are affected by tetrodotoxin (TTX), a specific sodium channel blocker. We find that T...

Neuroactive compounds can be photoreleased by means of two-photon excitation using a new kind of transition metal-based caged compound.

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The use of spatial light modulators (SLMs) for two-photon laser microscopy is described. SLM phase modulation can be used to generate nearly any spatiotemporal pattern of light, enabling simultaneous illumination of any number of selected regions of interest. We take advantage of this flexibility to perform fast two-photon imaging or uncaging experiments on dendritic spines and neocortical neurons. By operating in the spatial Fourier plane, an SLM can effectively mimic any arbitrary optical transfer function and thus replace, in software, many of the functions provided by hardware in standard microscopes, such as focusing, magnification, and aberration correction.