Site of Action Potential Initiation in Layer 5 Pyramidal Neurons

Abstract

Fundamental to an understanding of how neurons integrate synaptic input is the knowledge of where within a neuron this information is converted into an output signal, the action potential. Although it has been known for some time that action potential initiation occurs within the axon of neurons, the precise location has remained elusive. Here, we provide direct evidence using voltage-sensitive dyes that the site of action potential initiation in cortical layer 5 pyramidal neurons is ϳ35 m from the axon hillock. This was the case during action potential generation under a variety of conditions, after axonal inhibition, and at different stages of development. Once initiated action potentials propagated down the axon in a saltatory manner. Experiments using local application of low-sodium solution and TTX, as well as an investigation of the influence of axonal length on action potential properties, provided evidence that the initial 40 m of the axon is essential for action potential generation. To morphologically identify the relationship between the site of action potential initiation and axonal myelination, we labeled oligodendrocytes supplying processes to the proximal region of the axon. These experiments indicated that the axon initial segment was ϳ40 m in length, and the first node of Ranvier was ϳ90 m from the axon hillock. Experiments targeting the first node of Ranvier suggested it was not involved in action potential initiation. In conclusion, these results indicate that, in layer 5 pyramidal neurons, action potentials are generated in the distal region of the axon initial segment.

Axonal bleb recording

“…Considering this velocity difference, we speculated that the initiation of action potentials occurs at a site less than 48 μm from the soma. This finding is consistent with the previous electrophysiological [20,21] and recent voltage-sensitive dye imaging results [39,40] in neocortical layer 5 pyramidal neurons. Using dual somatic and axonal bleb recordings in hippocampal CA3 pyramidal neurons, a recent study also revealed that, in response to somatic stimulation, action potentials are initiated in the proximal region of the axon and subsequently back-propagated into the dendrites [41] .…”

Ankyrin-Dependent and -Independent Mechanisms Orchestrate Axonal Compartmentalization of L1 Family Members Neurofascin and L1/Neuron–Glia Cell Adhesion Molecule

“…The axonal initial segment (IS) is a highly specialized subdomain of the axon in which membrane proteins [such as voltage-gated sodium channels (VGNCs), potassium channels, and cell adhesion molecules] as well as cytoskeletal components (such as specialized ankyrins and spectrins) accumulate at high density (Kordeli et al, 1995;Berghs et al, 2000;Pan et al, 2006) (for review, see Salzer, 2003;Hedstrom and Rasband, 2006). As a result, the IS is a site of action potential initiation, in which neuronal firing is regulated (Coombs et al, 1957;Frankenhaeuser and Huxley, 1964;Khaliq and Raman, 2006;Palmer and Stuart, 2006;Van Wart and Matthews, 2006). Additionally, cell adhesion molecules (CAMs) clustered at the IS can serve to guide synaptogenesis during development (Ango et al, 2004).…”

Imaging membrane potential in dendrites and axons of single neurons

“…Average of four and seven recordings, with each recording the result of averaging ∼40 action potentials. c to f from [41] applied voltage-sensitive dyes to map the site of action potential initiation in cortical pyramidal neurons [43], finding, in contrast to observations in cerebellar Purkinje neurons [10, but see 27], that action potential initiation occurs at the distal end of the axon initial segment. The same study also showed that once initiated, action potentials propagate down the axon in a saltatory manner.…”