Distal spike initiation zone location estimation by morphological simulation of ionic current filtering demonstrated in a novel model of an identified Drosophila motoneuron.

Studying ion channel currents generated distally from the recording site is difficult because of artifacts caused by poor space clamp and membrane filtering. A computational model can quantify artifact parameters for correction by simulating the currents only if their exact anatomical location is known. We propose that the same artifacts that confound current recordings can help pinpoint the source of those currents by providing a signature of the neuron's morphology. This method can improve the recording quality of currents initiated at the spike initiation zone (SIZ) that are often distal to the soma in invertebrate neurons. Drosophila being a valuable tool for characterizing ion currents, we estimated the SIZ location and quantified artifacts in an identified motoneuron, aCC/MN1-Ib, by constructing a novel multicompartmental model. Initial simulation of the measured biophysical channel properties in an isopotential Hodgkin-Huxley type neuron model partially replicated firing characteristics. Adding a second distal compartment, which contained spike-generating Na+ and K+ currents, was sufficient to simulate aCC's in vivo activity signature. Matching this signature using a reconstructed morphology predicted that the SIZ is on aCC's primary axon, 70 μm after the most distal dendritic branching point. From SIZ to soma, we observed and quantified selective morphological filtering of fast activating currents. Non-inactivating K+ currents are filtered ∼3 times less and despite their large magnitude at the soma they could be as distal as Na+ currents. The peak of transient component (NaT) of the voltage-activated Na+ current is also filtered more than the magnitude of slower persistent component (NaP), which can contribute to seizures. The corrected NaP/NaT ratio explains the previously observed discrepancy when the same channel is expressed in different cells. In summary, we used an in vivo signature to estimate ion channel location and recording artifacts, which can be applied to other neurons

Morphology passive parameters fitted to 3rd instar aCC responses to passive voltage clamp protocol recordings (<i>n</i> = 5).

<p>Morphology passive parameters fitted to 3rd instar aCC responses to passive voltage clamp protocol recordings (<i>n</i> = 5).</p

Symmetry of voltage attenuation in the morphology.

<p>Each row indicates a morphological location for which we simulated the ratio of voltage amplitude at measurement site over voltage at current injection site for two conditions: injecting distally and measuring at somatic electrode (elec), and injecting at the electrode and measuring distally (distal). Experiments were repeated for direct current (DC, 0 Hz) and high frequency (sinusoidal, 100 Hz) stimuli. In addition to the two dendritic locations (dend1 & 2) in the ipsilateral arbor shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004189#pcbi.1004189.g004" target="_blank">Fig 4A</a>, we also tested the distal-most tip of the contralateral dendritic arbor (contra tip) and the spike initiation zone (SIZ—see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004189#pcbi.1004189.g006" target="_blank">Fig 6D</a>).</p><p>Symmetry of voltage attenuation in the morphology.</p

Isopotential and two-compartment models compared to recordings.

<p>(<b>A</b>) Third instar aCC motoneuron recording (i.) in presence of 0.2 mM Cd⁺² to block Ca⁺² channels missing from models. Voltage response to three current injection levels. Isopotential (ii.) and two-compartment (iii.) models’ voltage responses to same current injection. (<b>B</b>) Recorded and simulated firing responses to current injection were similar. Firing rate was calculated as the inverse of mean interspike interval (ISI). iso-model: isopotential model; 2c-model: two-compartment model. (<b>C</b>) Models also approximated aCC motoneuron delay to first spike in response to current injection. (<b>D</b>) The firing rate-membrane voltage (f-V) response of only the two-compartment model qualitatively mimicked the inter-spike voltage depolarizations that appeared for high firing rates.</p

Simulations of the passive morphology showed its compactness and filtering properties.

<p>(<b>A</b>) Simulation setup for the ipsilateral dendritic field of the reconstructed morphology (thick line is the primary neurite), where a 50 pA current clamp (CC) stimulus was applied at the model electrode compartment. (<b>B</b>) Voltage across model morphology locations marked in panel <b>A</b> in response to the current stimulus. (<b>C</b>) Electrotonic structure of the morphology compared at different stimulus frequencies. Lambda scale bar shows one length constant, at which voltage changes to about 37% of its initial value. For a step input (0 Hz), the length constant was several times larger than the arbor size, meaning voltages will travel across the arbor unimpeded. At a higher input frequency of 100 Hz, the span across the morphology was larger than one electrotonic length. (<b>D</b>) Ionic-like currents were simulated on the passive morphology by varying current injection location and responses were recorded holding the soma in voltage clamp at -60 mV. (<b>E</b>) Voltage clamp current evoked in response to exponentially decaying stimulus of 2 nA magnitude and 2 ms time constant injected at six locations in panel <b>A</b> starting at the electrode, and numbered in aquamarine as they went distally. (<b>F</b>) When same stimulus is injected at middle of compartment axon (prox), compartments far from the electrode escaped from the voltage clamp to various degrees. (<b>G</b>) By varying the time-constant of the same exponentially decaying stimulus applied to a fixed location axon (prox) in simulated morphology, we showed that the membrane response is filtered dependent on frequency. Dashed gray line shows the fastest (<i>τ</i> = 0.3 ms) current stimulus applied distally.</p

Morphological reconstruction of the 3rd instar aCC motoneuron.

<p>(<b>A</b>) Schematic muscle projections of aCC motoneurons based on their location in the nerve cord segment. (<b>B</b>) Stack of microscope images was reconstructed using Amira software (Visage Imaging GmbH, Berlin, Germany) and then imported into the Neuron simulator [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004189#pcbi.1004189.ref072" target="_blank">72</a>]. The rightmost schema indicates the major morphological components in an idealized depiction. “ext. axon” indicates the missing extended axon from the reconstruction (not drawn to scale). (<b>C</b>) Equivalent circuit of the measured passive properties including an electrode model for voltage clamp. (<b>D</b>) Passive response to voltage-clamp step to −90 mV from a holding potential of −60 mV is simulated in Neuron with the fitted parameters (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004189#pcbi.1004189.t002" target="_blank">Table 2</a>).</p