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Are you impulsive, or do you tend to be more deliberate? Have you ever felt the need to be cautious only to be dragged into something by a reckless accomplice? In this issue Hu et. al provide compelling evidence for molecular peer-pressure: two sodium channel (NaCh) isoforms with different demeanors located in the axon initial segment (AIS) – one a bit cautious and the other more impetuous. Neurons are continuously barraged by synaptic input which opens neurotransmitter receptors and induces changes in membrane potential (Vm). Once Vm becomes sufficiently elevated, voltage-gated NaChs open and initiate an action potential (AP), or spike, fulfilling the neuron’s role as an information integrator. Previous studies have shown that the AIS, a structure at the juncture between the soma and the axon, is rich in NaChs1 and initiates APs2. Once initiated, spikes propagate in two directions, forward down the axon to cause neurotransmitter release by depolarizing presynaptic terminals3, and backwards through the soma and then on to the dendrites. While the forward-propagating AP transmits information to downstream post-synaptic neurons, the back-propagating AP enables forms of synaptic plasticity4,5. The unique characteristics of the AIS that allow it to both initiate spikes with relative ease and then guarantee subsequent back-propagation have remained elusive.
Here, Hu and colleagues beautifully show, using quantitative immunostaining, electrophysiology (including the novel method of axonal bleb recording developed by the senior author) and computer modeling, that two NaCh sub-types, the high-threshold Nav1.2 and the low-threshold Nav1.6, are asymmetrically distributed in the AIS - precisely localizing these NaChs within the complex topography of the neuron. Nav1.2 is found mainly in the 25 μm of the AIS closest to the soma, and requires substantial depolarization for activation. Nav1.6, on the other hand, is found in more distal portions of the AIS, 25-50 μm from the soma, and is activated by relatively little depolarization6. This polarized configuration, low-threshold NaChs in the distal AIS flanked by high-threshold NaChs closer to the soma creates a new blueprint of AIS function which explains many of the unique properties of the AIS, including the faithful generation of back-propagating APs.
In this new model, APs are detonated by NaV1.6 channels because of their low threshold for activation and high channel density7. NaV1.6 channels sit in the perfect location to allow their easy initiation of APs – distal to the incoming dendritic excitation and insulated from it by somatic inhibitory neurotransmission and a reserve pool of timid NaV1.2 channels in the proximal AIS. If synaptic depolarization makes it as far as the distal AIS, the trigger-happy NaV1.6 figures the neuron deserves to spike. Once NaV1.6 channels are activated they rapidly depolarize the nearby area, coercing the hesitant NaV1.2 channels in the proximal AIS to open and generate a back-propagating AP. Having a reserve of high-threshold NaV1.2 channels proximal to the soma, the majority of which fail to open in response to the initial synaptic depolarization, provides a source of non-inactivated NaChs ready and waiting to initiate a back-propagating AP. Furthermore because NaV1.6 channels in the distal AIS have entered an inactive state by the time NaV1.2 channels open, a second forward-propagating AP is prevented. Although elements of this scheme are not perfectly clear, this mechanism of spike initiation followed by faithful generation of a back-propagating AP is both alluring and exciting.
The initiation of APs in the AIS is not a new concept. In fact the mechanism proposed above draws on years of work from groups dedicated to understanding the specific mechanism of spike generation. Eccles and colleagues first reported over 50 years ago that the AP appears first in the AIS of motoneurons and is followed by a back-propagating somatodendritic AP2. As electrophysiological and imaging techniques advanced so did our understanding of spike initiation. Pioneering studies by Colbert & Johnston8 used simultaneous recording from the soma and AIS of subicular neurons to demonstrate that during an AP, Vm rises more rapidly in the AIS, which occurs presumably due to the NaV1.6 localization found by Hu et al. In the soma, Colbert and Johnston showed that the onset of a spike occurs more slowly initially, due to what we now think is NaV1.6-mediated depolarization in the distal AIS, and is then followed by a rapid increase in Vm which now appears to be driven by NaV1.2 activation in the proximal AIS. Colbert & Johnston also show that somatic AP threshold is established by sodium channels ≈50 μm from the soma, where Hu et. al have localized NaV1.6. Although these authors did not know the identity of the NaChs subtypes driving APs, they proposed the idea of a ‘heminode’ beyond the AIS where APs originate – an idea that is conceptually validated by Hu et. al’s finding of a high concentration of NaV1.6 channels in the distal AIS.
Recently, Kole & Stuart 9 unraveled a long standing mystery – if AIS NaCh density explains spike initiation, then why don’t recordings reveal a higher density of NaChs in the AIS than elsewhere in the neuron? Answering this question required a literal deconstruction of the AIS, a structure notorious for its dense cytoskeleton10. The AIS is rich in the adapter protein ankyrin G which helps cluster both NaChs11 and potassium channels12. Kole & Stuart demonstrated that disruption of the actin cytoskeleton, and presumably its ability to stabilize ankyrin G, caused a three-fold increase in the sodium current that could be recorded in the AIS of layer V pyramidal neurons9. This leads one to the hypothesis that the AIS is designed to be a hospitable home to NaChs, not to patch pipettes. The rigid cytoskeletal scaffolding which keeps NaChs entrenched at the AIS causes the membrane the channels exists in to be so inflexible that they cannot be pulled into the pipette tip for patch recording. These results thus confirmed immunohistological and sodium-imaging findings and reconciled previous electrophysiological findings. Overall these results highlight the high value neurons place on bidirectional spike propagation. They have evolved an anatomical distribution of NaChs at a location distinct from that of incoming synaptic input and developed an extensive cytoskeletal system to ensure its stability.
Hu and colleagues also address a recent controversy in the spike generation field - the possibility that NaCh activation is a cooperative process 13,14. When APs are recorded from the soma of layer V cortical neurons, their onset is so rapid that some believe they cannot be described using classic Hodgkin-Huxley models, but can be recreated if NaCh gating is cooperative. According to the cooperative gating model, the statistical probability of any given channel opening in an environment rich with NaChs, such as the AIS, would not only be determined by Vm but also by the open-state of nearby NaChs. However, Hu et. al report that neither partial blockade of voltage-gated NaChs with tetrodotoxin nor decreasing NaCh currents with a low sodium buffer alters the voltage dependence of channel activation. If NaCh activation was cooperative, one would expect that removing a subset of NaChs from the active pool of channels with tetrodotoxin would alter channel activation, while reducing the sodium driving force would not. This result should lay to rest the notion that unique, cooperative, NaCh gating occurs in the AIS to initiate APs and supports the idea that the rapid onset of APs in the soma results from recording distally from the site of AP initiation.
Is there a new integrated view of spike initiation in pyramidal neurons? Hu and colleagues combined their electrophysiological and immunohistochemical findings with elegant modeling experiments to confirm the roles of NaV1.6 and NaV1.2. By altering the relative amounts of NaV1.2 and NaV1.6 in their model the authors show that the forward propagating AP threshold is almost completely dependent on the impulsive NaV1.6, while the threshold for generating a back-propagating somatodendritic AP is controlled by the hesitant NaV1.2. It will be exciting to see which other unique biophysical parameters of NaV1.2 and NaV1.6 will be relevant to additional aspects of spike generation and neuronal excitability. Will the faster recovery from inactivation seen in NaV1.2 mean that they are more responsible for AP generation during high-frequency firing? Or will the ability of NaV1.6 to maintain high current amplitude during repeated activation put it in the driver’s seat during high-frequency spiking6? Will the differential effects of drugs modulating NaCh properties, i.e. phenytoin, carbamazepine, lamotrigine, etc, be better understood now that we know more about the specific ion channels mediating AP generation? With this detailed picture of the spike generation machinery we are much better equipped to answer these and other pressing questions.
As a final note, our understanding of spike generation has truly paralleled our technical advances in electrophysiological and imaging techniques. From Eccles and colleagues intracellular recordings from motoneurons, to our ability to make simultaneous patch clamp recordings from a single neuron at multiple locations, to in vivo recording of AP threshold, our knowledge of spike initiation continues to grow. Now techniques like voltage and sodium imaging and bleb recording are rapidly advancing our ability to characterize excitability in specific neuronal sub-structures. The most intriguing question that Hu and colleagues leave unanswered is how is NaCh distribution built and maintained? Which cytoskeletal components, signaling molecules, and NaCh domains are responsible? Does inappropriate trafficking or anchoring of NaChs underlie pathological states? The ion channel trafficking and cytoskeletal interaction which have been so elegantly studied in the synapse now must be understood in the AIS.