The axon initial segment (AIS) is a specialized region in neurons where action potentials are initiated. It is commonly assumed that this process requires a high density of voltage-gated sodium (Na(+)) channels. Paradoxically, the results of patch-clamp studies suggest that the Na(+) channel density at the AIS is similar to that at the soma and proximal dendrites. Here we provide data obtained by antibody staining, whole-cell voltage-clamp and Na(+) imaging, together with modeling, which indicate that the Na(+) channel density at the AIS of cortical pyramidal neurons is approximately 50 times that in the proximal dendrites. Anchoring of Na(+) channels to the cytoskeleton can explain this discrepancy, as disruption of the actin cytoskeleton increased the Na(+) current measured in patches from the AIS. Computational models required a high Na(+) channel density (approximately 2,500 pS microm(-2)) at the AIS to account for observations on action potential generation and backpropagation. In conclusion, action potential generation requires a high Na(+) channel density at the AIS, which is maintained by tight anchoring to the actin cytoskeleton.

Two-photon calcium imaging of neuronal populations enables optical recording of spiking activity in living animals, but standard laser scanners are too slow to accurately determine spike times. Here we report in vivo imaging in mouse neocortex with greatly improved temporal resolution using random-access scanning with acousto-optic deflectors. We obtained fluorescence measurements from 34-91 layer 2/3 neurons at a 180-490 Hz sampling rate. We detected single action potential-evoked calcium transients with signal-to-noise ratios of 2-5 and determined spike times with near-millisecond precision and 5-15 ms confidence intervals. An automated 'peeling' algorithm enabled reconstruction of complex spike trains from fluorescence traces up to 20-30 Hz frequency, uncovering spatiotemporal trial-to-trial variability of sensory responses in barrel cortex and visual cortex. By revealing spike sequences in neuronal populations on a fast time scale, high-speed calcium imaging will facilitate optical studies of information processing in brain microcircuits.

Previous studies focusing on the temporal rules governing changes in synaptic strength during spike timing-dependent synaptic plasticity (STDP) have paid little attention to the fact that synaptic inputs are distributed across complex dendritic trees. During STDP, propagation of action potentials (APs) back to the site of synaptic input is thought to trigger plasticity. However, in pyramidal neurons, backpropagation of single APs is decremental, whereas high-frequency bursts lead to generation of distal dendritic calcium spikes. This raises the question whether STDP learning rules depend on synapse location and firing mode. Here, we investigate this issue at synapses between layer 2/3 and layer 5 pyramidal neurons in somatosensory cortex. We find that low-frequency pairing of single APs at positive times leads to a distance-dependent shift to long-term depression (LTD) at distal inputs. At proximal sites, this LTD could be converted to long-term potentiation (LTP) by dendritic depolarizations suprathreshold for BAC-firing or by high-frequency AP bursts. During AP bursts, we observed a progressive, distance-dependent shift in the timing requirements for induction of LTP and LTD, such that distal synapses display novel timing rules: they potentiate when inputs are activated after burst onset (negative timing) but depress when activated before burst onset (positive timing). These findings could be explained by distance-dependent differences in the underlying dendritic voltage waveforms driving NMDA receptor activation during STDP induction. Our results suggest that synapse location within the dendritic tree is a crucial determinant of STDP, and that synapses undergo plasticity according to local rather than global learning rules.

Spatiotemporal activity patterns in three-dimensionally organized cellular networks are fundamental to the function of the nervous system. Despite advances in functional imaging of cell populations, a method to resolve local network activity in three dimensions has been lacking. Here we introduce a three-dimensional (3D) line-scan technology for two-photon microscopy that permits fast fluorescence measurements from several hundred cells distributed in 3D space. We combined sinusoidal vibration of the microscope objective at 10 Hz with 'smart' movements of galvanometric x-y scanners to repeatedly scan the laser focus along a closed 3D trajectory. More than 90% of cell somata were sampled by the scan line within volumes of 250 microm side length. Using bulk-loading of calcium indicator, we applied this method to reveal spatiotemporal activity patterns in neuronal and astrocytic networks in the rat neocortex in vivo. Two-photon population imaging using 3D scanning opens the field for comprehensive studies of local network dynamics in intact tissue.

The time course of Mg(2+) block and unblock of NMDA receptors (NMDARs) determines the extent they are activated by depolarization. Here, we directly measure the rate of NMDAR channel opening in response to depolarizations at different times after brief (1 ms) and sustained (4.6 s) applications of glutamate to nucleated patches from neocortical pyramidal neurons. The kinetics of Mg(2+) unblock were found to be non-instantaneous and complex, consisting of a prominent fast component (time constant approximately 100 micros) and slower components (time constants 4 and approximately 300 ms), the relative amplitudes of which depended on the timing of the depolarizing pulse. Fitting a kinetic model to these data indicated that Mg(2+) not only blocks the NMDAR channel, but reduces both the open probability and affinity for glutamate, while enhancing desensitization. These effects slow the rate of NMDAR channel opening in response to depolarization in a time-dependent manner such that the slower components of Mg(2+) unblock are enhanced during depolarizations at later times after glutamate application. One physiological consequence of this is that brief depolarizations occurring earlier in time after glutamate application are better able to open NMDAR channels. This finding has important implications for spike-timing-dependent synaptic plasticity (STDP), where the precise (millisecond) timing of action potentials relative to synaptic inputs determines the magnitude and sign of changes in synaptic strength. Indeed, we find that STDP timing curves of NMDAR channel activation elicited by realistic dendritic action potential waveforms are narrower than expected assuming instantaneous Mg(2+) unblock, indicating that slow Mg(2+) unblock of NMDAR channels makes the STDP timing window more precise.

Spike-timing-dependent synaptic plasticity (STDP) by definition requires the temporal association of pre- and postsynaptic action potentials (APs). Yet, in cortical pyramidal neurons pairing unitary EPSPs with single APs at low frequencies is ineffective at generating plasticity. Using recordings from synaptically coupled layer 5 pyramidal neurons, we show here that high-frequency (200 Hz) postsynaptic AP bursts, rather than single APs, are required for both long-term potentiation (LTP) induction and NMDA channel activation during EPSP-AP pairing at low frequencies. Furthermore, we find that AP bursts can lead to LTP induction and NMDA channel activation during EPSP-AP pairing at both positive and negative times. High-frequency AP bursts generated supralinear calcium signals in basal dendrites suggesting the generation of dendritic calcium spikes, as has been observed previously in apical dendrites during AP burst firing at frequencies greater than 100 Hz. Consistent with a role of these dendritic calcium spikes in LTP induction, pairing EPSPs with low frequency (50 Hz) AP bursts was ineffective in generating LTP. Furthermore, supralinear calcium signals in basal dendrites during AP bursts were blocked by low concentrations of the T- and R-type calcium channel antagonist nickel, which also blocked LTP and NMDA channel activation. These data suggest an important role of dendritic calcium spikes during AP bursts in determining both the efficacy and time window for STDP induction.

Different streams of sensory information are transmitted to the cortex where they are merged into a percept in a process often termed 'binding.' Using recordings from triplets of rat cortical layer 2/3 and layer 5 pyramidal neurons, we show that specific subnetworks within layer 5 receive input from different layer 2/3 subnetworks. This cortical microarchitecture may represent a mechanism that enables the main output of the cortex (layer 5) to bind different features of a sensory stimulus.

Patch-clamp recording from dendrites has lead to a significant increase in our understanding of the mechanisms underlying signal integration and propagation in neurons. The majority of synaptic input to neurons, however, is made onto small-diameter dendrites, currently beyond the scope of patch-clamp recording techniques. Here we use both calcium and voltage imaging to investigate propagation of action potentials (APs) in fine basal dendrites of cortical layer 5 pyramidal neurons. High-frequency (200 Hz) AP bursts caused supralinear increases in dendritic calcium at distal, but not proximal, basal locations. Supralinear increases in dendritic calcium were also observed at distal basal locations during AP trains above a critical frequency (ϳ100 Hz). Using voltage imaging, we show that single APs undergo significant attenuation as they propagate into basal dendrites, whereas AP bursts lead to generation of dendritic calcium spikes. Focal and bath application of 4-AP increased the amplitude of calcium transients evoked by APs at distal, but not proximal, locations, suggesting that A-type potassium channels regulate AP backpropagation into basal dendrites. Finally, we show that pairing EPSPs with AP bursts is an effective means of activating synaptic NMDA receptors in basal dendrites. The experimental observations on the role of A-type potassium channels in regulation of AP backpropagation in basal dendrites, as well as the generation of dendritic calcium spikes during AP bursts, were reproduced in a morphologically realistic neuronal model with uniform distributions of dendritic sodium, calcium, and potassium channels. Together, these findings have important implications for understanding dendritic integration and synaptic plasticity in cortical basal dendrites.