1. Volume 23, Issue 4, Pages 1034-1044 (April 2018)
Adrenergic Modulation Regulates the Dendritic Excitability of Layer 5 Pyramidal Neurons In Vivo 
Christina Labarrera, Yair Deitcher, Amir Dudai, Benjamin Weiner, Adi Kaduri Amichai, Neta Zylbermann, Michael London 
Cell Reports 
Volume 23, Issue 4, Pages (April 2018)
DOI: /j.celrep
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2. Cell Reports 2018 23, 1034-1044DOI: (10.1016/j.celrep.2018.03.103)
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3. Figure 1
α2A-Adrenoceptor Activation Increases Ca2+ Activity in L5PC Apical Tuft Dendrites
(A) L5PCs expressing GCaMP6s, a genetically encoded Ca2+ indicator in the somatosensory cortex (scale bar, 100 μm).
(B) Schematic illustration of the two-photon Ca2+ imaging configuration from dendrites in a head-restrained mouse walking on a wheel. Inset: 3D projection of an image stack, showing sparse labeling of L5PCs dendrites (scale bar, 100 μm).
(C) Example of the horizontal imaging plane showing three ROIs recorded from the same experiment.
(D) Ca2+ fluorescence traces of the three ROIs indicated in (C) under baseline conditions over 100 s.
(E) Color raster plot of Ca2+ signals from all ROIs from the same experiment under control conditions (n = 27 ROIs). Each row represents one ROI.
(F and G) Same as in (D) and (E), respectively, but after topical application of 5 mM guanfacine (an α2A-adrenoceptor agonist).
(H) Histograms of all ΔF/F traces of the same experiment under control conditions (black) and after application of guanfacine (purple). The grey rectangle and dashed line depict the area under the histogram at ΔF/F values larger than 25%.
(I) The area under the tail in the histograms of all ROIs is significantly different under both conditions.
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4. Figure 2
Targeted α2A-Adrenoceptor Activation Increases Ca2+ Activity in L5PC Apical Tuft Dendrites
(A) Left: schematic illustration of the two-photon Ca2+ imaging configuration from lightly anaesthetized mice. Inset: a glass pipette loaded with red fluorescent dye (Alexa 594) and 10 mM guanfacine was placed near the main apical bifurcation of an L5PC.
(B) Ca2+ imaging of apical dendrites at three different time points (gray rectangles in C). The three frames represent an average of 3 s at different time points along the experiment: before (B1), during (B2), and after (B3) local application of Alexa and guanfacine (scale bar, 10 μm). One ROI from the red channel (dashed red circles) and additional three ROIs from the green channel (solid colored circles) are shown.
(C) Top: ΔF/F traces of the red channel close to the pipette tip (dashed red circles in B), indicating the flow of Alexa and guanfacine out of the pipette. Bottom: ΔF/F traces of the green channel in three ROIs (solid colored circles in B).
(D) Left: histograms of all ΔF/F traces under baseline conditions (“pre,” black) and after application of guanfacine (purple). Right: the area under the tail in the histograms of all ROIs is significantly different under both conditions.
(E) Same as (D) but for application of ACSF (gray).
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5. Figure 3 α2A-Adrenoceptor Activation Increases Dendritic Excitability
(A) Schematic illustration of the recording configuration; whole-cell recordings from head-restrained mice walking on a wheel.
(B) Neurobiotin 488-filled and reconstructed neuron (scale bar, 100 μm).
(C and D) Example of membrane potential responses to current injections (C) and frequency-current (F-I) curve (D).
(E and F) Schematic illustration of the critical frequency protocol. Trains of 4 current injections of frequencies between 50 and 260 Hz were injected into neurons to evoke back-propagating action potentials and dendritic calcium spikes. Four current injections at 50 Hz (E) and 200 Hz (F) are shown.
(G) Overlay of traces from the lower and higher 3 frequencies, illustrating two populations of responses (traces from all frequencies are presented in Figure S3). The area between the dashed lines was used for integration of voltage to calculate (H). Low frequencies are illustrated in light blue and high frequencies in red.
(H) Example of a critical frequency plot; the critical frequency is illustrated by a dashed lined at 93 Hz.
(I) Distribution of critical frequency values for a population of recorded neurons (CF = 101 ± 4 Hz, n = 36).
(J) Critical frequency was measured in the same neurons before and after topical application of 5 mM guanfacine, and a significant drop in CF was measured.
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6. Figure 4
Ih Blockage Results in a Similar Effect as that of α2A-Adrenoceptor Activation
(A) Membrane potential response to a hyperpolarization current injection (25 repetitions and average, black) shows a clear sag response under control conditions. Dashed lines mark the resting membrane potential, peak amplitude of the sag, and the steady-state membrane potential used to measure the sag ratio (Experimental Procedures).
(B) Sag amplitude distribution (n = 49 cells).
(C) Sag ratio distribution (n = 49 cells).
(D) Membrane potential response to the same hyperpolarization current injections from the same neuron as in (A) following topical application of ZD7288 (20 μL of 0.5 mM the scale is the same as in A). Also depicted is the average response in control (solid black) as well as the normalized and shifted average in ZD to compare the sag ratio (dashed green).
(E and F) Increase in input resistance (E), decrease in resting membrane potential (F), and reduction in sag ratio are significant in the population.
(G) The critical frequency is significantly reduced following application of ZD7288.
(H) Two-photon imaging of Ca2+ traces from L5PC dendrites shows an increase in Ca2+ activity. Shown are example traces from two ROIs before (top) and after application of ZD7288 (bottom).
(I) Color raster plot of Ca2+ activity from 17 ROIs from the same experiment.
(J) The distribution of ΔF/F from all ROIs shows a decrease in peak amplitude and a rightward tail, indicating stronger and more frequent Ca2+ events.
(K) The area under the tail in the histogram in (J) is significantly different under the two conditions.
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7. Figure 5
α2A-Adrenoceptor Activation Modulates Ih in Layer 5 Pyramidal Neurons
(A) Example of the membrane potential response (50 repetitions average) to hyper-polarization current steps (−300, −400, and −500 pA) before and after application of guanfacine (black, control; purple, guanfacine; throughout, spikelets are action potentials reduced because of averaging).
(B) Population data show a significant decrease in sag ratio for current- injected steps of −500 pA.
(C) Sag after guanfacine application tends to slow down; rise and decay time constants significantly increase.
(D) Example Zap current chirp (0–20 Hz over 24 s, logarithmic increase of frequency with time) and voltage response (average of 12 and 17 repetitions in the control and after application of guanfacine, respectively).
(E) Example of an impedance curve derived from the recordings depicted in (D). Top: the impedance in the control shows a resonance peak, whereas the resonance is gone after application of guanfacine. Bottom: average impedance curve for the population under both control and guanfacine conditions (normalized to the impedance at 1 Hz). The curves show that, in the control, there is a resonance frequency, and the degree of resonance is greatly reduced by application of guanfacine.
(F) Population data for the difference between the impedance at peak frequency and impedance at 1 Hz normalized by the latter.
(G–J) Modeling Zap responses.
(G) Activation curve of the Ih model used in the simulation. The black line is the original model and the purple after 20 mV shift of the activation curve to the left (a leftward shift in the activation curve translates into a drop of the percentage of open conductance, which results in a change in impedance as well as the time constant of the cell). Also shown is the voltage dependence for the Ih activation time constant (a leftward shift leads to slowdown of Ih) See also (C).
(H) Example voltage responses of the isopotential model to the current used in (D) before and after 15 mV leftward shift of the activation curve.
(I) The impedance curves for the model under the two conditions (dashed lines) as well as the impedance curves from the data presented in (E) (solid lines).
(J) The results of a simulation of a full compartmental model, including an exponential increase in the density of Ih with distance toward the apical dendrites, shows a better match to the experimentally measured impedance curves.
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8. Figure 6 Modulation of Ih on the Excitability of the Apical Tuft
(A) A schematic illustration of a full compartmental model of L5PC.
(B) Voltage responses in the nexus in response to a local current pulse. Three conditions are shown: control (black), V50 of the activation curve of Ih was negatively shifted by 15 mV (purple), and Ih was removed from the model (green). In all cases, the membrane potential response was regenerative but to various degrees.
(C) The peak of the voltage response in the nexus is plotted against the degree of shift of the V50 of Ih activation curve. Also shown are the boundary conditions of no shift (black) and complete removal of Ih (green).
(D–F) Schematic illustration of the functional consequences of NE modulation on dendritic excitability.
(D) When NE levels are low, dendritic HCN channels are open, which increases the electrotonic length of the apical dendrites, reduces temporal summation of back-propagating action potentials, and increases the threshold for dendritic Ca2+ spikes. Functionally, this reduces the interaction between feedforward inputs arriving at the perisomatic region and feedback inputs arriving at the apical tuft.
(E) Increased levels of NE modulate Ih (shift the activation curve of Ih toward a more hyperpolarized potential) and reduce its effect on excitability of the tuft, enabling dendritic spikes and facilitating interaction between somatic and dendritic compartments.
(F) NE enables gradual changes in the excitability of the tuft. This translates into different “states” corresponding to different levels of NE. With a high level of NE, the population is highly responding to feedback inputs, whereas with low levels, they are essentially muted.
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