Beyond a Transmission Cable-New Technologies to Reveal the Richness in Axonal Electrophysiology.

The axon is a neuronal structure capable of processing, encoding, and transmitting information. This assessment contrasts with a limiting, but deeply rooted, perspective where the axon functions solely as a transmission cable of somatodendritic activity, sending signals in the form of stereotypical action potentials. This perspective arose, at least partially, because of the technical difficulties in probing axons: their extreme length-to-diameter ratio and intricate growth paths preclude the study of their dynamics through traditional techniques. Recent findings are challenging this view and revealing a much larger repertoire of axonal computations. Axons display complex signaling processes and structure-function relationships, which can be modulated via diverse activity-dependent mechanisms. Additionally, axons can exhibit patterns of activity that are dramatically different from those of their corresponding soma. Not surprisingly, many of these recent discoveries have been driven by novel technology developments, which allow for in vitro axon electrophysiology with unprecedented spatiotemporal resolution and signal-to-noise ratio. In this review, we outline the state-of-the-art in vitro toolset for axonal electrophysiology and summarize the recent discoveries in axon function it has enabled. We also review the increasing repertoire of microtechnologies for controlling axon guidance which, in combination with the available cutting-edge electrophysiology and imaging approaches, have the potential for more controlled and high-throughput in vitro studies. We anticipate that a larger adoption of these new technologies by the neuroscience community will drive a new era of experimental opportunities in the study of axon physiology and consequently, neuronal function.

High-frequency limit of neural stimulation with ChR2.

Optogenetic technology based on light activation of genetically targeted single component opsins such as Channelrhodopsin-2 (ChR2) has been changing the way neuroscience research is conducted. This technology is becoming increasingly important for neural engineering as well. The efficiency of neural stimulation with ChR2 drops at high frequencies, often before the natural limit of the neuron is reached. This study aims to investigate the underlying mechanisms that limit the efficiency of the stimulation at high frequencies. The study analyzes the dynamics of the spikes induced by ChR2 in comparison to control stimulations using patch clamp current injection. It shows that the stimulation dynamics is limited by two mechanisms: 1) a frequency independent reduction in the conductance-to-irradiance yield due to the ChR2 light adaptation process and 2) a frequency dependent reduction in the conductance-to-current yield due to a decrease in membrane re-polarization level between spikes that weakens the ionic driving force. The effect of the first mechanism can be minimized by using ChR2 mutants with lower irradiance threshold. In contrast the effect of the second mechanism is fundamentally limited by the rate the native ion channels re-polarize the membrane potential.

Synaptic plasticity: rush hour traffic in the AMPA lanes.

Recent experiments indicate that modification of synaptic strength may involve rapid regulation of vesicular traffic on the postsynaptic side of the synapse. The specific vesicular trafficking route taken by postsynaptic receptors appears to depend on the stimulus.

Synaptic depression and the kinetics of exocytosis in retinal bipolar cells.

The capacitance technique was used to investigate exocytosis at the ribbon synapse of depolarizing bipolar cells from the goldfish retina. When the Ca(2+) current was activated strongly, the rapidly releasable pool of vesicles (RRP) was released with a single rate-constant of approximately 300-500 sec(-1). However, when the Ca(2+) current was activated weakly by depolarization in the physiological range (-45 to -25 mV), exocytosis from the RRP occurred in two phases. After the release of 20% or more of the RRP, the rate-constant of exocytosis fell by a factor of 4-10. Thus, synaptic depression was caused by a reduced sensitivity to Ca(2+) influx, as well as simple depletion of the RRP. In the resting state, the rate of exocytosis varied with the amplitude of the Ca(2+) current raised to the power of 2. In the depressed state, the sensitivity to Ca(2+) influx was reduced approximately fourfold. The initial phase of exocytosis accelerated e-fold for every 2.1 mV depolarization over the physiological range and averaged 120 sec(-1) at -25 mV. The synapse of depolarizing bipolar cells therefore responds to a step depolarization in a manner similar to a high-pass filter. This transformation appears to be determined by the presence of rapidly releasable vesicles with differing sensitivities to Ca(2+) influx. This might occur if vesicles were docked to the plasma membrane at different distances from Ca(2+) channels. These results suggest that the ribbon synapse of depolarizing bipolar cells may be a site of adaptation in the retina.

Two actions of calcium regulate the supply of releasable vesicles at the ribbon synapse of retinal bipolar cells.

Ribbon synapses of sensory neurons are able to sustain high rates of exocytosis in response to maintained depolarization, but it is not known how this is achieved. Using the capacitance technique, we have found that Ca(2+) regulates the supply of releasable vesicles at the ribbon synapse of depolarizing bipolar cells from the retina of goldfish. Ca(2+) had two actions that could be differentiated by introduction of the Ca(2+) chelator EGTA; one action stimulated refilling of the rapidly releasable pool of vesicles from a reserve pool, and a second action stimulated recruitment of vesicles to the reserve pool. The capacity of the reserve pool was approximately 3500 vesicles, which is similar to the number that can attach to the ribbons. These results suggest that continuous exocytosis at ribbon synapses is maintained by the Ca(2+)-dependent translocation of vesicles from the cytoplasm, through the ribbon, to release sites on the plasma membrane.

Electrical resonance and Ca2+ influx in the synaptic terminal of depolarizing bipolar cells from the goldfish retina.

1. Whole-cell recordings and fura-2 measurements of cytoplasmic [Ca2+] were made in depolarizing bipolar cells isolated from the retinae of goldfish. The aim was to study the voltage signal that regulates Ca2+ influx in the synaptic terminal. 2. The current-voltage relation was linear up to about -44 mV. At this threshold, the injection of 1 pA of current triggered a maintained 'all-or-none' depolarization to a plateau of -34 mV, associated with a decrease in input resistance and a damped voltage oscillation with a frequency of 50-70 Hz and initial amplitude of 4-10 mV. A second frequency component of 5-10 Hz was often observed. In a minority of cells the response to current injection was transient, recovering with an undershoot. 3. Unstimulated bipolar cells generated similar voltage signals, driven by current entering the cell through a non-specific cation conductance that continuously varied in amplitude. 4. The threshold for activation of the Ca2+ current was -43 mV and free [Ca2+]i in the synaptic terminal rose during a depolarizing response. Simultaneous measurements of the fluorescence associated with the membrane marker FM1-43 demonstrated that these Ca2+ signals stimulated exocytosis. Regenerative depolarizations and associated rises in [Ca2+]i were blocked by inhibiting L-type Ca2+ channels with 30 microM nifedipine. 5. Depolarization beyond -40 mV also elicited an outwardly rectifying K+ current. Blocking this current by replacing external Ca2+ with Ba2+ caused the voltage reached during a depolarizing response to increase to +10 mV. 6. The majority of the K+ current was blocked by 100 nM charybdotoxin, indicating that it was carried by large-conductance Ca2+ activated K+ channels. A transient voltage-gated K+ current remained, which began to activate at -40 mV. High-frequency voltage oscillations were blocked by 100 nM charybdotoxin, but low-frequency oscillations remained. 7. These results indicate that the voltage response of depolarizing bipolar cells is shaped by L-type Ca2+ channels, Ca(2+)-activated K+ channels and voltage-dependent K+ channels. This combination of conductances regulates Ca2+ influx into the synaptic terminal and confers an electrical resonance on the bipolar cell.