Single cortical neurons in the mammalian brain receive signals arising from multiple sensory input channels. Dendritic integration of these afferent signals is critical in determining the amplitude and time course of the neurons' output signals. As of yet, little is known about the spatial and temporal organization of converging sensory inputs. Here, we combined in vivo two-photon imaging with whole-cell recordings in layer 2 neurons of the mouse vibrissal cortex as a means to analyze the spatial pattern of subthreshold dendritic calcium signals evoked by the stimulation of different whiskers. We show that the principle whisker and the surrounding whiskers can evoke dendritic calcium transients in the same neuron. Distance-dependent attenuation of dendritic calcium transients and the corresponding subthreshold depolarization suggest feed-forward activation. We found that stimulation of different whiskers produced multiple calcium hotspots on the same dendrite. Individual hotspots were activated with low probability in a stochastic manner. We show that these hotspots are generated by calcium signals arising in dendritic spines. Some spines were activated uniquely by single whiskers, but many spines were activated by multiple whiskers. These shared spines indicate the existence of presynaptic feeder neurons that integrate and transmit activity arising from multiple whiskers. Despite the dendritic overlap of whisker-specific and shared inputs, different whiskers are represented by a unique set of activation patterns within the dendritic field of each neuron.excitatory synapse in vivo | cortical circuits | mouse barrel cortex C ortical function relies on the network properties of connected neurons. In addition to conventional (1) and modern (2-4) structural reconstructions, electrophysiological (5, 6) and optical tools (7-10) are commonly applied to probe the functional connectivity among neurons. Recent advances in two-photon calcium imaging enabled an in vivo analysis of the dendritic organization of sensory inputs to layer 2/3 neurons in the mouse visual cortex (11). The sensory input sites were represented by local dendritic calcium influx through NMDA receptors called hotspots. The fine structure of the hotspots, whether they represented individual spines or small spinodendritic segments, was not determined. A major finding was that synaptic inputs sharing the same orientation preference are widely distributed throughout the dendritic field, and importantly, inputs with different orientation preference are interspersed. An important open question is whether the presence of such inputs with multiple orientations is related to the fact that, in the mouse visual cortex, neurons selective to different orientations are interspersed (12) rather than being organized into cortical columns. It has been suggested that, in the cat and in primates, the input connection scheme would be differently organized (13), because neurons with different orientation preferences are quite strictly clustered into orientation columns (14...

Neurons in the mammalian brain receive thousands of synaptic inputs on their dendrites. In many types of neurons, such as cortical pyramidal neurons, excitatory synapses are formed on fine dendritic protrusions called spines. Usually, an individual spine forms a single synaptic contact with an afferent axon. In this protocol, we describe a recently established experimental procedure for measuring intracellular calcium signals from dendritic spines in cortical neurons in vivo by using a combination of two-photon microscopy and whole-cell patch-clamp recordings. We have used mice as an experimental model system, but the protocol may be readily adapted to other species. This method involves data acquisition at high frame rates and low-excitation laser power, and is termed low-power temporal oversampling (LOTOS). Because of its high sensitivity of fluorescence detection and reduced phototoxicity, LOTOS allows for prolonged and stable calcium imaging in vivo. Key aspects of the protocol, which can be completed in 5-6 h, include the use of a variant of high-speed two-photon imaging, refined surgery procedures and optimized tissue stabilization.

Layer 5 pyramidal neurons process information from multiple cortical layers to provide a major output of cortex. Because of technical limitations it has remained unclear how these cells integrate widespread synaptic inputs located in distantly separated basal and tuft dendrites. Here, we obtained in vivo twophoton calcium imaging recordings from the entire dendritic field of layer 5 motor cortex neurons. We demonstrate that during subthreshold activity, basal and tuft dendrites exhibit spatially localized, small-amplitude calcium transients reflecting afferent synaptic inputs. During action potential firing, calcium signals in basal dendrites are linearly related to spike activity, whereas calcium signals in the tuft occur unreliably. However, in both dendritic compartments, spike-associated calcium signals were uniformly distributed throughout all branches. Thus, our data support a model of widespread, multibranch integration with a direct impact by basal dendrites and only a partial contribution on output signaling by the tuft.excitatory synapses | dendritic integration | mouse motor cortex P yramidal neurons feature extensive dendritic arborizations that receive and process widespread synaptic input. Much interest has been placed on the role of individual dendritic branches in determining neuronal output (1). The single branch has been hypothesized to function as a unit of plasticity (2), protein synthesis (3), and synaptic integration (4). Moreover, single dendritic branches have been shown to generate spatially restricted regenerative events, the so-called dendritic spikes, which can process and amplify local input (5). These features raise the question of whether the input-output relation of a cortical neuron is influenced primarily by single dendrites or by multibranch activity.Layer 5 (L5) pyramidal neurons serve as the major output cell type of neocortex. Their morphology is characterized by a set of basal dendrites as well as a set of distal tuft dendrites that extend into layer 1 and are separated from the soma by a long apical trunk (6). These compartments are known to be specialized in terms of the type of synaptic input that they receive (7). Furthermore, in vitro studies of dendritic physiology have revealed several active dendritic signals in these compartments such as back-propagation of action potentials (bAPs) (8, 9), NMDA spikes resulting from clustered synaptic activation (5), and calcium spikes initiated in the apical trunk (10, 11). Despite the presence of these dendritic specializations, little is known about the importance of these features for in vivo function. Previous in vivo studies were limited by the technical challenges of recording from deep basal structures in L5 (12) or of recording multiple individual dendrites simultaneously from the same neuron (13). A complete understanding of dendritic integration in L5 pyramidal neurons requires in vivo data from both the basal and tuft compartments with single dendritic branch resolution.Here we address the question of multibranch activi...

The sensory responses of cortical neuronal populations following training have been extensively studied. However, the spike firing properties of individual cortical neurons following training remain unknown. Here, we have combined two-photon Ca 2+ imaging and single-cell electrophysiology in awake behaving mice following auditory associative training. We find a sparse set (~5%) of layer 2/3 neurons in the primary auditory cortex, each of which reliably exhibits high-rate prolonged burst firing responses to the trained sound. Such bursts are largely absent in the auditory cortex of untrained mice. Strikingly, in mice trained with different multitone chords, we discover distinct subsets of neurons that exhibit bursting responses specifically to a chord but neither to any constituent tone nor to the other chord. Thus, our results demonstrate an integrated representation of learned complex sounds in a small subset of cortical neurons.