Amanda J. Foust

Action Potentials Initiate in the Axon Initial Segment and Propagate through Axon Collaterals Reliably in Cerebellar Purkinje Neurons

Purkinje neurons are the output cells of the cerebellar cortex and generate spikes in two distinct modes, known as simple and complex spikes. Revealing the point of origin of these action potentials, and how they conduct into local axon collaterals, is important for understanding local and distal neuronal processing and communication. By using a recent improvement in voltage-sensitive dye imaging technique that provided exceptional spatial and temporal resolution, we were able to resolve the region of spike initiation as well as follow spike propagation into axon collaterals for each action potential initiated on single trials. All fast action potentials, for both simple and complex spikes, whether occurring spontaneously or in response to a somatic current pulse or synaptic input, initiated in the axon initial segment. At discharge frequencies of less than ∼250 Hz, spikes propagated faithfully through the axon and axon collaterals, in a saltatory manner. Propagation failures were only observed for very high frequencies or for the spikelets associated with complex spikes. These results demonstrate that the axon initial segment is a critical decision point in Purkinje cell processing and that the properties of axon branch points are adjusted to maintain faithful transmission.

The spatio‐temporal characteristics of action potential initiation in layer 5 pyramidal neurons: a voltage imaging study

Non‐technical summary  Axons are long process of nerve cells that mediate communication and control within the organism. Mammalian neurons have developed a complex ion channel clustering mechanism in axons to optimize rapid signalling. It has been discovered recently that intricate details of the spatial pattern of channel clustering play a critical role in signal processing in the axon. The electrical properties of axons, however, have been difficult to study using electrodes because axons are very small in diameter. We took advantage of a critical methodological improvement in the high sensitivity membrane potential imaging (Vm imaging) technique to study electrical correlates of channel clustering in the axon of cortical neurons. Voltage imaging revealed the location and length of the axonal site for nerve impulse initiation as well as the pattern of saltatory conduction in myelinated axons in the form of dynamic spatial maps of transmembrane potential.

Optically teasing apart neural swelling and depolarization

We measured birefringence, 90 degree scattered light, and voltage sensitive dye changes from lobster walking leg nerves. Systematic application of key chemical agents revealed separate cellular mechanisms underlying fast optical signals. Each agent exhibited mixed effects, some having a greater effect on cellular swelling and refractive index, and some altering membrane potential. Birefringence changes were tightly correlated with voltage sensitive dye signals and were perturbed by those agents that altered membrane potential. Signals from light scattered at 90 degrees corroborated the hypothesis that large angle scattering signals arise from changes in the interstitial spaces and were perturbed by those agents that altered cellular swelling and refractive index. We conclude that multiple cellular mechanisms can be exploited to measure rapid optical signals. Since birefringence produces much larger changes than scattering, the use of polarized light might lead to improvements in imaging neural activity with high temporal resolution, especially since birefringence changes corresponded closely to membrane potential.

Somatic Membrane Potential and Kv1 Channels Control Spike Repolarization in Cortical Axon Collaterals and Presynaptic Boutons

The shape of action potentials invading presynaptic terminals, which can vary significantly from spike waveforms recorded at the soma, may critically influence the probability of synaptic neurotransmitter release. Revealing the conductances that determine spike shape in presynaptic boutons is important for understanding how changes in the electrochemical context in which a spike is generated, such as subthreshold depolarization spreading from the soma, can modulate synaptic strength. Utilizing recent improvements in the signal-to-noise ratio of voltage-sensitive dye imaging in mouse brain slices, we demonstrate that intracortical axon collaterals and en passant presynaptic terminals of layer 5 pyramidal cells exhibit a high density of Kv1 subunit-containing ion channels, which generate a slowly inactivating K+ current critically important for spike repolarization in these compartments. Blockade of the current by low doses of 4-aminopyridine or α-dendrotoxin dramatically slows the falling phase of action potentials in axon collaterals and presynaptic boutons. Furthermore, subthreshold depolarization of the soma broadened action potentials in collaterals bearing presynaptic boutons, an effect abolished by blocking Kv1 channels with α-dendrotoxin. These results indicate that action potential-induced synaptic transmission may operate through a mix of analog–digital transmission owing to the properties of Kv1 channels in axon collaterals and presynaptic boutons.

Action potential propagation imaged with high temporal resolution near-infrared video microscopy and polarized light

To identify the neural constituents responsible for generating polarized light changes, we created spatially resolved movies of propagating action potentials from stimulated lobster leg nerves using both reflection and transmission imaging modalities. Changes in light polarization are associated with membrane depolarization and provide sub-millisecond temporal resolution. Typically, signals are detected using light transmitted through tissue; however, because we eventually would like to apply polarization techniques in-vivo, reflected light is required. In transmission mode, the optical signal was largest throughout the center of the nerve, suggesting that most of the optical signal arose from the inner nerve bundle. In reflection mode, polarization changes were largest near the edges, suggesting that most of the optical signal arose from the outer sheath. In support of these observations, an optical model of the tissue showed that the outer sheath is more reflective while the inner nerve bundle is more transmissive. In order to apply these techniques in-vivo, we must consider that brain tissue does not have a regular orientation of processes as in the lobster nerve. We tested the effect of randomizing cell orientation by tying the nerve in an overhand knot prior to imaging, producing polarization changes that can be imaged even without regular cell orientations.

State Dependent Auditory Evoked Hemodynamic Responses Recorded Optically with Indwelling Photodiodes

Implantable optical technologies provide measurements of cerebral hemodynamic activity from freely behaving animals without movement constraint or anesthesia. In order to study state-dependent neural evoked responses and the consequential hemodynamic response, we simultaneously measured EEG and scattered light changes in chronically implanted rats. Recordings took place under freely behaving conditions, allowing us to compare the evoked responses across wake, sleep, and anesthetized states. The largest evoked electrical and optical responses occurred during quiet sleep compared to wake and REM sleep, while isoflurane anesthesia showed a large, late burst of electrical activity synchronized to the stimulus but an earlier optical response.

Sub-millisecond optogenetic control of neuronal firing with two-photon holographic photoactivation of Chronos

Optogenetic neuronal network manipulation promises to unravel a long-standing mystery in neuroscience: how does microcircuit activity relate causally to behavioral and pathological states? The challenge to evoke spikes with high spatial and temporal complexity necessitates further joint development of light-delivery approaches and custom opsins. Two-photon (2P) light-targeting strategies demonstrated in-depth generation of action potentials in photosensitive neurons both in vitro and in vivo, but thus far lack the temporal precision necessary to induce precisely timed spiking events. Here, we show that efficient current integration enabled by 2P holographic amplified laser illumination of Chronos, a highly light-sensitive and fast opsin, can evoke spikes with submillisecond precision and repeated firing up to 100 Hz in brain slices from Swiss male mice. These results pave the way for optogenetic manipulation with the spatial and temporal sophistication necessary to mimic natural microcircuit activity. SIGNIFICANCE STATEMENT To reveal causal links between neuronal activity and behavior, it is necessary to develop experimental strategies to induce spatially and temporally sophisticated perturbation of network microcircuits. Two-photon computer generated holography (2P-CGH) recently demonstrated 3D optogenetic control of selected pools of neurons with single-cell accuracy in depth in the brain. Here, we show that exciting the fast opsin Chronos with amplified laser 2P-CGH enables cellular-resolution targeting with unprecedented temporal control, driving spiking up to 100 Hz with submillisecond onset precision using low laser power densities. This system achieves a unique combination of spatial flexibility and temporal precision needed to pattern optogenetically inputs that mimic natural neuronal network activity patterns.

Optimized birefringence changes during isolated nerve activation

Single trial, birefringence signals associated with action potentials from isolated lobster nerves were optimized with high-intensity light-emitting diodes (LEDs) and glass polarizers. The narrow spectral output of the LEDs allowed us to select specific wavelengths, increasing the effectiveness of the polarizers and minimizing the stray light in the system. The LEDs produced intensity profiles equivalent to narrowband filtered 100-W halogen light, and birefringence signals were comparable or superior in size and clarity to halogen lamp recordings. The results support a direct correlation between signal size and polarizer extinction coefficient. Increasing the sensitivity of birefringence detection through the use of LED light sources could ameliorate noninvasive brain imaging techniques that employ fast optical consequences associated with action potential propagation.

In vitro and in vivo noise analysis for optical neural recording

Laser diodes (LD) are commonly used for optical neural recordings in chronically recorded animals and humans, primarily due to their brightness and small size. However, noise introduced by LDs may counteract the benefits of brightness when compared to low-noise light-emitting diodes (LEDs). To understand noise sources in optical recordings, we systematically compared instrument and physiological noise profiles in two recording paradigms. A better understanding of noise sources can help improve optical recordings and make them more practical with fewer averages. We stimulated lobster nerves and a rat cortex, then compared the root mean square (RMS) noise and signal-to-noise ratios (SNRs) of data obtained with LED, superluminescent diode (SLD), and LD illumination for different numbers of averages. The LED data exhibited significantly higher SNRs in fewer averages than LD data in all recordings. In the absence of tissue, LED noise increased linearly with intensity, while LD noise increased sharply in the transition to lasing and settled to noise levels significantly higher than the LEDs, suggesting that speckle noise contributed to the LDs higher noise and lower SNRs. Our data recommend low coherence and portable light sources for in vivo chronic neural recording applications.

Computer-generated holography enhances voltage dye fluorescence discrimination in adjacent neuronal structures

Abstract. Voltage-sensitive fluorescence indicators enable tracking neuronal electrical signals simultaneously in multiple neurons or neuronal subcompartments difficult to access with patch electrodes. However, efficient widefield epifluorescence detection of rapid voltage fluorescence transients necessitates that imaged cells and structures lie sufficiently far from other labeled structures to avoid contamination from out of focal plane and scattered light. We overcame this limitation by exciting dye fluorescence with one-photon computer-generated holography shapes contoured to axons or dendrites of interest, enabling widefield detection of voltage fluorescence with high spatial specificity. By shaping light onto neighboring axons and dendrites, we observed that dendritic back-propagating action potentials were broader and slowly rising compared with axonal action potentials, differences not measured in the same structures illuminated with a large “pseudowidefield” (pWF) spot of the same excitation density. Shaped illumination trials showed reduced baseline fluorescence, higher baseline noise, and fractional fluorescence transient amplitudes two times greater than trials acquired with pWF illumination of the same regions.