Recent advances in optogenetic techniques have generated new tools for controlling neuronal activity, with a wide range of neuroscience applications. The most commonly used approach has been the optical activation of the light-gated ion channel channelrhodopsin-2 (ChR2). However, targeted single-cell-level optogenetic activation with temporal precessions comparable to the spike timing remained challenging. Here we report fast (≤1 ms), selective, and targeted control of neuronal activity with single-cell resolution in hippocampal slices. Using temporally focused laser pulses (TEFO) for which the axial beam profile can be controlled independently of its lateral distribution, large numbers of channels on individual neurons can be excited simultaneously, leading to strong (up to 15 mV) and fast (≤1 ms) depolarizations. Furthermore, we demonstrated selective activation of cellular compartments, such as dendrites and large presynaptic terminals, at depths up to 150 μm. The demonstrated spatiotemporal resolution and the selectivity provided by TEFO allow manipulation of neuronal activity, with a large number of applications in studies of neuronal microcircuit function in vitro and in vivo.high-resolution neuronal stimulation | channelrhodopsin | temporal focusing | circuit mapping | electrophysiology A rtificial stimulation and inhibition of neuronal activity has applications ranging from fundamental neurobiology questions (1-4) to potential clinical treatment of neuropsychiatric disorders (5, 6). Historically, this task has been achieved mainly by electrical stimulation. Yet the recent development of genetic techniques for sensitizing neurons to optical stimulation (7-12) and silencing (13-16) have, for the first time, provided cell typespecific control of neuronal activity, which has been successfully used to address significant biological questions (1)(2)(3)(17)(18)(19). In the most widely used approach, genetically expressed light-gated ion channels, such as channelrhodopsin-2 (ChR2) (20, 21), or ion pumps, such as halorhodopsin or archaerhodopsin-3 (22, 23), and optical methods for triggering their function are combined to control neuronal activity. However, although optical activation of ChR2 has been suitable to induce population activity in a large number of neurons, directed, efficient, and fast stimulation of single cells has not been feasible. The main reason for that is the low channel conductance of ChR2 (21, 24). To achieve sufficient depolarizations, a large number of channels have to be activated nearly simultaneously, which typically extend over an area of tens of square micrometers. Although conventional one-photon excitation satisfies this requirement, light scattering and the extended axial beam parameter of the excitation area lead typically to inevitable activation of neurons in an untargeted fashion. Some success in addressing these issues has recently been reported (25, 26) using a two-photon scanning (27) approach. Although these studies (25, 26) could show high spatial resolution, the necessar...

The Schaffer collateral pathway provides hippocampal CA1 pyramidal cells with a fairly homogeneous excitatory synaptic input that is spread out across several hundred micrometers of their apical dendritic arborizations. A progressive increase in synaptic conductance, with distance from the soma, has been reported to reduce the location dependence that should result from this arrangement. The excitatory synaptic contacts within this pathway primarily use AMPA- and NMDA-type glutamate receptors. To investigate the underlying mechanism of the increased distal excitatory postsynaptic conductance, we used outside-out patches and a fast application system to characterize the properties and distribution of synaptic glutamate receptors across the range of apical dendrites receiving Schaffer collateral input. We observed an approximately twofold increase in AMPA-mediated current amplitude (0.3-0.6 nA) in the range of CA1 apical dendrites that receive a uniform density of Schaffer collateral input (approximately 100-250 micrometer from soma). NMDA-mediated current amplitude, however, remained unchanged. We analyzed the current kinetics, agonist affinity, single-channel conductance, maximum open probability, and reversal potential of AMPA receptors and did not find any differences. Instead, the number of AMPA receptors present in our patches increased approximately twofold. These data suggest that an increase in the number of AMPA receptors present at distal synapses may play an important role in the distance-dependent scaling of Schaffer collateral synapses.

Nonlinear interactions between coactive synapses enable neurons to discriminate between spatiotemporal patterns of inputs. Using patterned postsynaptic stimulation by two-photon glutamate uncaging, here we investigate the sensitivity of synaptic Ca2+ signalling and long-term plasticity in individual spines to coincident activity of nearby synapses. We find a proximodistally increasing gradient of nonlinear NMDA receptor (NMDAR)-mediated amplification of spine Ca2+ signals by a few neighbouring coactive synapses along individual perisomatic dendrites. This synaptic cooperativity does not require dendritic spikes, but is correlated with dendritic Na+ spike propagation strength. Furthermore, we show that repetitive synchronous subthreshold activation of small spine clusters produces input specific, NMDAR-dependent cooperative long-term potentiation at distal but not proximal dendritic locations. The sensitive synaptic cooperativity at distal dendritic compartments shown here may promote the formation of functional synaptic clusters, which in turn can facilitate active dendritic processing and storage of information encoded in spatiotemporal synaptic activity patterns.