Greg J. Stuart

Patch-clamp recordings from the soma and dendrites of neurons in brain slices using infrared video microscopy.

A description is given of the implementation of infrared differential interference contrast (IR-DIC) video microscopy to an upright compound microscope. Using the improved resolution offered by IR-DIC a procedure is described for making patch-pipette recordings from visually identified neuronal somata and dendrites in brain slices. As an example of the application of this technique to electrophysiological recordings from small neuronal processes in brain slices we describe wholecell current-clamp and cell-attached and excised patchclamp recordings from the apical dendrites of layer V pyramidal neurons in slices of rat neocortex.

Action potential initiation and backpropagation in neurons of the mammalian CNS

Most neurons in the mammalian CNS encode and transmit information via action potentials. Knowledge of where these electrical events are initiated and how they propagate within neurons is therefore fundamental to an understanding of neuronal function. While work from the 1950s suggested that action potentials are initiated in the axon, many subsequent investigations have suggested that action potentials can also be initiated in the dendrites. Recently, experiments using simultaneous patch-pipette recordings from different locations on the same neuron have been used to address this issue directly. These studies show that the site of action potential initiation is in the axon, even when synaptic activation is powerful enough to elicit dendritic electrogenesis. Furthermore, these and other studies also show that following initiation, action potentials actively backpropagate into the dendrites of many neuronal types, providing a retrograde signal of neuronal output to the dendritic tree.

Action potential initiation and propagation in rat neocortical pyramidal neurons

1 Initiation and propagation of action potentials evoked by extracellular synaptic stimulation was studied using simultaneous dual and triple patch pipette recordings from different locations on neocortical layer 5 pyramidal neurons in brain slices from 4‐week‐old rats (P26–30) at physiological temperatures. 2 Simultaneous cell‐attached and whole‐cell voltage recordings from the apical trunk (up to 700 μm distal to the soma) and the soma indicated that proximal synaptic stimulation (layer 4) initiated action potentials first at the soma, whereas distal stimulation (upper layer 2/3) could initiate dendritic regenerative potentials prior to somatic action potentials following stimulation at higher intensity. 3 Somatic action potentials, once initiated, propagated back into the apical dendrites in a decremented manner which was frequency dependent. The half‐width of back‐propagating action potentials increased and their maximum rate of rise decreased with distance from the soma, with the peak of these action potentials propagating with a conduction velocity of approximately 0.5 m s−1. 4 Back‐propagation of action potentials into the dendritic tree was associated with dendritic calcium electrogenesis, which was particularly prominent during bursts of somatic action potentials. 5 When dendritic regenerative potentials were evoked prior to somatic action potentials, the more distal the dendritic recording was made from the soma the longer the time between the onset of the dendritic regenerative potential relative to somatic action potential. This suggested that dendritic regenerative potentials were initiated in the distal apical dendrites, possibly in the apical tuft. 6 At any one stimulus intensity, the initiation of dendritic regenerative potentials prior to somatic action potentials could fluctuate, and was modulated by depolarizing somatic or hyperpolarizing dendritic current injection. 7 Dendritic regenerative potentials could be initiated prior to somatic action potentials by dendritic current injections used to simulate the membrane voltage change that occurs during an EPSP. Initiation of these dendritic potentials was not affected by cadmium (200 μm), but was blocked by TTX (1 μm). 8 Dendritic regenerative potentials in some experiments were initiated in isolation from somatic action potentials. The voltage change at the soma in response to these dendritic regenerative events was small and subthreshold, showing that dendritic regenerative events are strongly attenuated as they spread to the soma. 9 Simultaneous whole‐cell recordings from the axon initial segment and the soma indicated that synaptic stimulation always initiated action potentials first in the axon. The further the axonal recording was made from the soma the greater the time delay between axonal and somatic action potentials, indicating a site of action potential initiation in the axon at least 30 μm distal to the soma. 10 Simultaneous whole‐cell recordings from the apical dendrite, soma and axon initial segment showed that action potentials were always initiated in the axon prior to the soma, and with the same latency difference, independent of whether dendritic regenerative potentials were initiated or not. 11 It is concluded that both the apical dendrites and the axon of neocortical layer 5 pyramidal neurons in P26–30 animals are capable of initiating regenerative potentials. Regenerative potentials initiated in dendrites, however, are significantly attenuated as they spread to the soma and axon. As a consequence, action potentials are always initiated in the axon before the soma, even when synaptic activation is intense enough to initiate dendritic regenerative potentials. Once initiated, the axonal action potentials are conducted orthogradely into the axonal arbor and retrogradely into the dendritic tree.

Calcium action potentials restricted to distal apical dendrites of rat neocortical pyramidal neurons

1 Simultaneous whole‐cell voltage and Ca2+ fluorescence measurements were made from the distal apical dendrites and the soma of thick tufted pyramidal neurons in layer 5 of 4‐week‐old (P28–32) rat neocortex slices to investigate whether activation of distal synaptic inputs can initiate regenerative responses in dendrites. 2 Dual whole‐cell voltage recordings from the distal apical trunk and primary tuft branches (540–940 μm distal to the soma) showed that distal synaptic stimulation (upper layer 2) evoking a subthreshold depolarization at the soma could initiate regenerative potentials in distal branches of the apical tuft which were either graded or all‐or‐none. These regenerative potentials did not propagate actively to the soma and axon. 3 Calcium fluorescence measurements along the apical dendrites indicated that the regenerative potentials were associated with a transient increase in the concentration of intracellular free calcium ([Ca2+]i) restricted to distal dendrites. 4 Cadmium added to the bath solution blocked both the all‐or‐none dendritic regenerative potentials and local dendritic [Ca2+]i transients evoked by distal dendritic current injection. Thus, the regenerative potentials in distal dendrites represent local Ca2+ action potentials. 5 Initiation of distal Ca2+ action potentials by a synaptic stimulus required coactivation of AMPA‐ and NMDA‐type glutamate receptor channels. 6 It is concluded that in neocortical layer 5 pyramidal neurons of P28–32 animals glutamatergic synaptic inputs to the distal apical dendrites can be amplified via local Ca2+ action potentials which do not reach threshold for axonal AP initiation. As amplification of distal excitatory synaptic input is associated with a localized increase in [Ca2+]i these Ca2+ action potentials could control the synaptic efficacy of the distal cortico‐cortical inputs to layer 5 pyramidal neurons.

Amplification of EPSPs by axosomatic sodium channels in neocortical pyramidal neurons

Simultaneous somatic and dendritic recordings were made from the same neocortical layer V pyramidal neuron, and current injection via the dendritic recording pipette was used to simulate the voltage change that occurs during an EPSP. At the soma, these simulated EPSPs increased nonlinearly with the amplitude of the dendritic current injection and with depolarization of the membrane potential. Bath application of the sodium channel blocker TTX decreased large (> 5 mV) EPSPs and also blocked amplification of EPSPs at depolarized membrane potentials, whereas calcium channel blockers had little effect. Local application of TTX to the soma and axon blocked EPSP amplification, whereas dendritic application had little effect. Simultaneous somatic and axonal recordings demonstrated that EPSP amplification was largest in the axon. These results show that EPSPs are amplified by voltage-activated sodium channels located close to the soma and in the axon.

Action potential generation requires a high sodium channel density in the axon initial segment

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 ∼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 (∼2,500 pS μm−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.

Initiation and spread of sodium action potentials in cerebellar purkinje cells

Simultaneous whole-cell recordings were made from the soma and dendrites of cerebellar Purkinje cells in rat brain slices. Sodium action potentials, evoked by either depolarizing current pulses or synaptic stimulation of parallel or climbing fibers, always occurred first at the soma and decreased in amplitude with increasing distance into the dendrites. Simultaneous somatic and axonal recordings showed that these action potentials were initiated in the axon. Outside-out patches excised from the soma and dendrites revealed that the sodium channel current density decreased with distance from the soma. Consistent with this finding, comparable attenuation was observed for evoked action potentials and simulated action potential waveforms when sodium channels were blocked. These results show that sodium action potentials in cerebellar Purkinje cells are initiated in the axon and then spread passively into the dendritic tree.

Axon Initial Segment Kv1 Channels Control Axonal Action Potential Waveform and Synaptic Efficacy

Action potentials are binary signals that transmit information via their rate and temporal pattern. In this context, the axon is thought of as a transmission line, devoid of a role in neuronal computation. Here, we show a highly localized role of axonal Kv1 potassium channels in shaping the action potential waveform in the axon initial segment (AIS) of layer 5 pyramidal neurons independent of the soma. Cell-attached recordings revealed a 10-fold increase in Kv1 channel density over the first 50 microm of the AIS. Inactivation of AIS and proximal axonal Kv1 channels, as occurs during slow subthreshold somatodendritic depolarizations, led to a distance-dependent broadening of axonal action potentials, as well as an increase in synaptic strength at proximal axonal terminals. Thus, Kv1 channels are strategically positioned to integrate slow subthreshold signals, providing control of the presynaptic action potential waveform and synaptic coupling in local cortical circuits.

Axonal initiation and active dendritic propagation of action potentials in substantia nigra neurons

The site of action potential initiation in substantia nigra neurons was investigated by using simultaneous somatic and dendritic whole-cell recording in brain slices. In many dopamine neurons, action potentials were observed first at the dendritic recording site. Anatomical reconstruction showed that in these neurons, the axon emerged from the dendrite from which the recording had been made. Action potentials showed little attention in the dendritic tree, which in dopamine neurons was shown to be due to recruitment of dendritic sodium channels and may be related to the dendritic release of dopamine. We conclude that in substantia nigra neurons, the site of action potential initiation, and thus the final site of synaptic integration, is in the axon. As the axon can originate from a dendrite, up to 240 microns away from the soma, synaptic input to the axon-bearing dendrite may be privileged with respect to its ability to influence action potential initiation.

Learning rules for spike timing-dependent plasticity depend on dendritic synapse location

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.