Tunable Oscillations in the Purkinje Neuron

In this paper, we study the dynamics of slow oscillations in Purkinje neurons in vitro, and derive a strong association with a forced parametric oscillator model. We demonstrate the precise rhythmicity of the oscillations in Purkinje neurons, as well as a dynamic tunability of this oscillation using a photo-switchable compound. We show that this slow oscillation can be induced in every Purkinje neuron, having periods ranging between 10-25 seconds. Starting from a Hodgkin-Huxley model, we also demonstrate that this oscillation can be externally modulated, and that the neurons will return to their intrinsic firing frequency after the forced oscillation is concluded. These results signify an additional functional role of tunable oscillations within the cerebellum, as well as a dynamic control of a time scale in the brain in the range of seconds.Comment: 12 pages, 5 figure

A Signal Processing Analysis of Purkinje Cells in vitro

Cerebellar Purkinje cells in vitro fire recurrent sequences of Sodium and Calcium spikes. Here, we analyze the Purkinje cell using harmonic analysis, and our experiments reveal that its output signal is comprised of three distinct frequency bands, which are combined using Amplitude and Frequency Modulation (AM/FM). We find that the three characteristic frequencies – Sodium, Calcium and Switching – occur in various combinations in all waveforms observed using whole-cell current clamp recordings. We found that the Calcium frequency can display a frequency doubling of its frequency mode, and the Switching frequency can act as a possible generator of pauses that are typically seen in Purkinje output recordings. Using a reversibly photo-switchable kainate receptor agonist, we demonstrate the external modulation of the Calcium and Switching frequencies. These experiments and Fourier analysis suggest that the Purkinje cell can be understood as a harmonic signal oscillator, enabling a higher level of interpretation of Purkinje signaling based on modern signal processing techniques

Gradient lithography of engineered proteins to fabricate 2D and 3D cell culture microenvironments

Spatial patterning of proteins is a valuable technique for many biological applications and is the prevailing tool for defining microenvironments for cells in culture, a required procedure in developmental biology and tissue engineering research. However, it is still challenging to achieve protein patterns that closely mimic native microenvironments, such as gradient protein distributions with desirable mechanical properties. By combining projection dynamic mask lithography and protein engineering with non-canonical photosensitive amino acids, we demonstrate a simple, scalable strategy to fabricate any user-defined 2D or 3D stable gradient pattern with complex geometries from an artificial extracellular matrix (aECM) protein. We show that the elastic modulus and chemical nature of the gradient profile are biocompatible and allow useful applications in cell biological research

Local plasticity of dendritic excitability can be autonomous of synaptic plasticity and regulated by activity-based phosphorylation of Kv4.2.

While plasticity is typically associated with persistent modifications of synaptic strengths, recent studies indicated that modulations of dendritic excitability may form the other part of the engram and dynamically affect computational processing and output of neuronal circuits. However it remains unknown whether modulation of dendritic excitability is controlled by synaptic changes or whether it can be distinct from them. Here we report the first observation of the induction of a persistent plastic decrease in dendritic excitability decoupled from synaptic stimulation, which is localized and purely activity-based. In rats this local plasticity decrease is conferred by CamKII mediated phosphorylation of A-type potassium channels upon interaction of a back propagating action potential (bAP) with dendritic depolarization

Kv4.2 channels are responsible for DED and the process requires Ca2+ and MEK.

<p>(A–D) Example current measurements before (black) and after stimulation (grey) showing that DED is blocked by HpTx (A) and TTX (B). Moreover DED does not happen in the absence of Ca2+ (C) and is blocked by MEK inhibitor U0126 (D). (E) Bar graph summarizing effect of different drugs. When no drugs are applied 13%±1.7% DED is observed. Application of TTX (1 uM), TEA (20 mM) and Stromatotoxin-II (100 nM) reduced the amount of DED to ∼3% while application of Hptx (100 mM) or U0126 (10 uM) or removal of calcium from external solution completely abolishes DED. The dendritic excitability was measured using 2 s pulses and the DED was quantified as a percentage change between the averaged peak current before and after stimulation. For each treatment significance of differences between the excitability before and after the stimulation was tested with two tailed heteroscedastic Student's t-test. Comparison between conditions was conducted by one-way ANOVA with Tukey post hoc test and showed no significant difference between control and APV (p = 0.08) and a statistically significant difference between control and all other conditions with p<0.001.</p

Persistent decrease in dendritic excitability following paired stimulation.

<p>(<b>A</b>)EYFP-ChR2 expressing hippocampal neuron is stimulated with ∼28 um light spot (blue circle) applied on the proximal dendrites coupled with somatically induced spike. The insert shows the detection protocol (top) where 2 s photostimulation was used to determine dendritic excitability and stimulation protocol (bottom) where the same 2 s photostimulation of proximal dendrite is coupled with a spike (<b>B</b>) Schematics of the optical setup. Patterned ChR2 stimulation is achieved by a 470 nm LED is collimated by a lens (CL) and directed using a mirror (M) to Digital Micromirror Device (DMD). The light reflected from DMD is collimated and scaled using a lens system (L) and coupled into the microscope via a beam splitter (BS). (<b>C</b>) An example trace for 2 s report photo depolarization before (black trace) and after (blue trace) the treatment. When no stimulation was applied (top) no change in dendritic excitability is observed, however after paired stimulation (bottom) excitability decreased (<b>D</b>) Dendritic excitability prior to paired stimulation, shows a steady basal level of dendritic excitability as assessed by measuring the peak magnitude of the ChR2-induced photo current. After paired stimulation the current decreases by 13% for 2 s report as compared to 0.74% for no stimulation. (<b>E</b>) Only stimulation by paired APs and dendritic depolarization causes DED = 12.6% (p<0.05)). Controls, where no stimulation takes place or either APs or light alone are delivered show no significant DED (p>0.05 in all cases). (<b>F</b>) There is no change in spike-current relationship between stimulated (□) and unstimulated neurons (o) or neurons stimulated only with APs (▴) indicating no change in intrinsic excitability. The excitability was measured by applying depolarizing current pulses (2 s in duration) in 0.1-nA increments. For each depolarizing step, the number of evoked action potentials was counted and plotted against injected current amplitude. Inset shows a representative current clamp trace.</p

DED is spatially localized.

<p>(A, B, C and D) Top panels display the dendritic location used for dendritic stimulation (blue circle) and the range of possible report locations (white dotted lines). Bottom panels show example current measurements before (black) and after stimulation (grey or blue) as well normalized current before and after stimulation. Following proximal dendritic stimulation, (A) proximal dendritic current decreases (DED = 2.94%±2.19%, p<001, n = 11/12) (B) somatic current shows increase in excitability by 4.89%±1.89 (p<0.001 n = 10/10) (C) Un-stimulated dendrites don't show DED (%DED = 0.05%±2.93%, p = 0.22, n = 10/10) and (D) Current resulting from whole cell photo-stimulation does not change significantly (DED = −0.4%±1.9, n = 9/9, p = 0.10).</p

Kv4.2 phosphorylation is enhanced along the stimulated dendrite.

<p>(A) Immunostaining against Kv4.2 phosphoylated at Ser 438 shows enhanced phosphorylation at the stimulated dendrite as compared with the un-stimulated dendrite of the same cell. The dendrites have comparable amounts of ChR-YFP. Dendrites from two representative cells are shown (top and bottom). The white circles indicate the location of photostimulation. (B) Mean immunofluorescence of pKv4.2 along the stimulated dendrites is much higher (mean fluorescence  = 36.5±2.84) than along the non-stimulated dendrites (mean fluorescence  = 19.8±3.17), p = 0.01, n = 5/5.</p