1. A Novel Form of Local Plasticity in Tuft Dendrites of Neocortical Somatosensory Layer 5 Pyramidal Neurons 
Maya Sandler, Yoav Shulman, Jackie Schiller 
Neuron 
Volume 90, Issue 5, Pages (June 2016)
DOI: /j.neuron
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Figure 1
Synaptic Spasticity Evoked by Low-Frequency Unpaired EPSP Stimulation in Tuft Dendrites
(A) Fluorescence image of a layer 5 pyramidal neuron loaded with CF-633 (200 μM) showing the experimental setup. A recording electrode located distal to the first bifurcation (red; 525 μm from soma) and a stimulating electrode located in distal tuft dendrite (blue; 910 μm from soma) are shown.
(B) Amplitude of single EPSPs is represented over time during 0.1 Hz stimulation frequency showing fast potentiation. Example EPSPs from two different time points (average of five repetitions; blue and cyan) during the stimulation are presented below.
(C) Fluorescent image of a layer 5 pyramidal neuron showing a recording electrode located at the first bifurcation (red; 800 μm from soma) and a stimulating electrode located in distal tuft dendrite (blue; 1,040 μm from soma).
(D) Amplitude of single EPSPs is represented over time during 0.03 Hz stimulation frequency followed by 0.1 Hz stimulation frequency. Example EPSPs from two different time points (average of five repetitions; blue and cyan) during the stimulation are presented below.
(E) Reconstruction of biocytin-filled layer 5 pyramidal neuron showing the sites of stimulation (blue electrode; 1,000 μm from the soma) and recording (red electrode; 800 μm from the soma) electrodes.
(F) Amplitude of single EPSPs is represented over time during 0.03 Hz stimulation frequency, followed by 0.1 Hz stimulation frequency and later 0.005 Hz testing stimulation frequency. EPSP amplification lasted 78 min in this example.
(G) Example of EPSPs (mean EPSPs of 15 traces) during the 0.03-Hz stimulation frequency (black trace) and post-potentiation during the 0.1-Hz stimulation frequency (gray trace).
(H and I) A summary plot of the percent change during 0.01 Hz stimulation of the EPSP amplitude (left) and rise time (right). Averages ± SEM; n = 57 dendritic segments in 53 cells. A significant potentiation was observed in mean amplitude change (h; p = e−10) as well as in rise slope change (f; p = e−05).
(J) A summary plot of the percent change during 0.01 Hz stimulation of the EPSP amplitude as recorded at the soma (n = 16 dendrites; p = 4.8e−5).
(K) Summary plot of the duration (in minutes) of the potentiation from six stimulated dendrites (black) and the mean duration (red).
See also Figures S1 and S2.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 2
Low-Frequency 0.1 Stimulation at Basal Dendrites of Layer 5 Pyramidal Neurons Did Not Induce Potentiation
(A) Reconstruction of biocytin-filled layer 5 pyramidal neuron showing the somatic recording electrode (red) and two stimulating electrodes in proximal (blue) and distal (green) basal dendritic locations.
(B) Amplitude of single EPSPs of proximal (blue; 80 μm from the soma) and distal (green; 220 μm from the soma) stimulations is represented over time during 0.03 Hz followed by 0.1 Hz stimulation frequencies, showing no significant increase in EPSP amplitude.
(C) Example EPSPs recorded at proximal and distal dendrites at 0.03 Hz (blue and green) versus 0.1 Hz (light blue and light green).
(D and E) Summary plot of the mean (±SEM) change in EPSP amplitude at proximal (D) and distal (E) locations at 0.03 Hz and 0.1 Hz stimulation frequencies (n = 12 dendrites).
See also Figure S3.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

4. Figure 3
Low-Frequency Potentiation in Tuft Dendrites Is Dependent on NMDAR, Kv4.2, Insertion of AMPAR Channels, and Insertion of GluR1 Subunits of the AMPAR to the Postsynaptic Membrane
(A) NMDAR channel activation is necessary for low-frequency induction of potentiation in tuft dendrites. An example experiment shows the amplitude of single EPSPs represented over time during 0.03 Hz followed by 0.1 Hz stimulation in the presence of the NMDAR blocker APV (100 μM). Examples of EPSPs (mean EPSPs of 15 traces) during the 0.03 Hz (cyan trace) and 0.1 Hz (blue) stimulation are presented below the graph.
(B) Kv4.2 channels are essential for low-frequency induction of potentiation in tuft dendrites. An example experiment shows the amplitude of single EPSPs represented over time during 0.03 Hz followed by 0.1 Hz stimulation frequencies in the presence of Kv4.2 blocker heteropodatoxin-2 (0.5 μM). Examples of EPSPs (mean EPSPs of 15 traces) during the 0.03 Hz (cyan trace) and 0.1 Hz (blue) stimulation are presented below the graph.
(C) Experimental setup. A fluorescence image of a layer 5 pyramidal neuron shows the dendritic recording electrode at first bifurcation containing intracellular blocker DYN, which spreads through the cell (teal; 630 μm from soma) and focal stimulating electrode (blue; 860 μm from soma) nearby a tuft dendrite.
(D) Internalization of membrane proteins is required for low-frequency induction of potentiation in tuft dendrites. An example experiment shows the amplitude of single EPSPs represented over time during 0.03 Hz followed by 0.1 Hz stimulation frequencies in the presence of intracellular DYN (100 μg/ml). Examples of EPSPs (mean EPSPs of 15 traces) during the 0.03 Hz (cyan trace) and 0.1 Hz (blue) stimulation are presented below the graph.
(E) Insertion of AMPAR is required for the low-frequency potentiation. An example experiment shows the amplitude of single EPSPs recorded in the presence of PEP-1 (100 μM) in the patch electrode and represented over time during 0.03 Hz followed by 0.1 Hz stimulation frequencies. Examples of EPSPs (mean EPSPs of 15 traces) during the 0.03 Hz (cyan trace) and 0.1 Hz (blue) stimulation are presented below the graph.
(F) A summary plot of the EPSP amplitude change (%) following 0.1 Hz stimulation in control conditions (n = 57 dendrites) compared with APV (blue; n = 9 dendrites), Kv4.2 blocker heteropodatoxin-2 (green; n = 19 dendrites), DYN (teal; n = 13 dendrites), and PEP-1 (purple; n = 17 dendrites). Statistical significance was calculated from two-tails, unpaired t test. Note the complete blockade in the presence of the blockers.
See also Figure S4.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

5. Figure 4
Number and Distribution of Spines Activated during the Low-Frequency Stimulation Protocol
(A) A fluorescence image of a tuft dendrite of layer 5 pyramidal neuron loaded with CF-633 (200 μM) and OGB1 (100 μM). Inset shows the stimulated segment (blue electrode 950 μM from soma) in high resolution.
(B) Line scan (2 ms per line; red line in A) through spine and neighboring dendritic shaft showing calcium transient evoked by dendritic EPSPs (upper right panel; recording electrode 600 μM from soma) is presented. Calcium transients shown in the line scan are presented as ΔF/F for spine and corresponding shaft (lower right panel).
(C) A dendritic tuft segment showing activated spines (red circles; stimulating electrode in blue).
(D) Summary plot showing the average total number of spines activated per dendritic segment. The average number of spines activated within ±5 μm, ±10 μm, ±20 μm, and larger than ±30 μm of the center of stimulus site and the average number of spines activated per dendritic segment as calculated by our neuron simulations are shown.
(E) Example of calcium transients in spine (left) and corresponding shaft (right) presented as ΔF/F in control and following potentiation with 0.1 Hz stimulation.
(F) Summary plot of ΔF/F change (%) in amplitude (±SEM) in spine and shaft following the potentiation with 0.1 Hz stimulation.
(G) Distribution of the % potentiation with 0.1 Hz stimulation as a function of the initial EPSP amplitude.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

6. Figure 5
Increased Propagation of Axo-somatic BAPs following Low-Frequency Plasticity in Tuft Dendrites
(A) Fluorescence reconstruction of layer 5 pyramidal neuron showing the site of stimulation (blue electrode; 850 μm from the soma), dendritic recording (red electrode; 770 μm from the soma), and somatic recording (gray electrode).
(B) Axo-somatic APs were evoked by current injection (50 ms; 0.5 nA at the somatic electrode) and recorded simultaneously in dendritic and somatic electrodes in control (black) and post-0.1 Hz frequency stimulation (red).
(C) Summary plot of mean (±SEM) BAPs evoked calcium transients (ΔF/F %; n = 35 cells) presented as a function of distance from the soma (black). Each value is an average of 100 μm dendritic segments. The first 10–50 μm are referred to as “somatic” region. Gray bars show mean ΔF/F of noise values (±SEM).
(D) Calcium transients (ΔF/F %) evoked by BAPs recorded in the stimulated dendrite (same neuron as in A; blue dot illustrates the location of stimulated site). Calcium transients are shown for control (black traces) and post-low-frequency stimulation protocol (red traces). Note a significant increase in calcium transient maximal at the activated site (820–995 μm from soma).
(E) Average calcium transients (ΔF/F) evoked by BAPs presented along the apical dendrite in control (black bars) and post-0.1 Hz stimulation (red bars). Blue dot represents the stimulus site. Same dendrite as in (D) is shown.
(F) Summary plot of the mean change in BAPs-evoked calcium transients (±SEM) presented as a function the distance from the stimulus site (0) in 100 μm segments, from proximal (to stimulated segment; negative values) to distal (from stimulated segment; positive values) locations (n = 12 cells).
(G) Mean change in BAPs-evoked calcium transients (±SEM) for stimulated (0.1 Hz; black; n = 10) and unstimulated (blue; n = 7) tuft dendrites. p = between stimulated and unstimulated tuft dendrites.
(H) Mean BAP amplitude as recorded at the dendritic electrode (±SEM) for control (black) and post-0.1 Hz stimulation (red).
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

7. Figure 6
Differential Effect of Kv4.2 Blocker on Basal Compared to Tuft Dendrites
(A) An example of BAPs-evoked calcium transient along a basal dendrite of post-0.03 Hz stimulation frequency (black) compared with post-0.1 Hz stimulation frequency (red). Stimuli were located 80 μm and 220 μm from the soma. Pair of BAPs was evoked by somatic current injection (bottom trace).
(B) Mean change in BAP -evoked calcium transients (±SEM) in basal dendrites after 0.1 Hz frequency stimulation plotted as a function of the distance from the soma in 20-μm segments (black; n = 11 cells).
(C) Summary plot of percent change (control relative to heteropodatoxin-2) of mean (±SEM) BAP-evoked calcium transients in basal dendrites (n = 9) and BCaS-evoked calcium transients in tuft dendrites (n = 12). p = e−09 between basal and tuft dendrites.
(D and E) Mean EPSP amplitude and half-width change (control relative to heteropodatoxin-2; 0.5 μM) in tuft (n = 16) and basal (n = 13) dendrites in control stimulation frequency (0.03 Hz).
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

8. Figure 7
BPCaS-Evoked Calcium Profile at Apical and Tuft Dendrites of Layer 5 Pyramidal Neurons
(A) Double patch-clamp somatic (blue) and dendritic recording (red) were performed from a layer 5 pyramidal neuron loaded with OGB1 (100 μM).
(B and D) Calcium spikes were evoked by current injections via the dendritic recording electrode, and calcium transients (ΔF/F %) were recorded from two apical branches left (yellow; B) and right (orange; D).
(C and E) Calcium transients (ΔF/F %) are presented as a function of the distance from the soma for left (C) and right (E) dendritic branches. In caption, calcium spike as recorded at the dendritic electrode (515 μm from soma) is shown.
(F) Examples of BPCaS-evoked calcium profiles from five neurons presented as ΔF/F values (%) versus the distance from the soma. Cross indicates the location of primary bifurcation of each neuron.
(G) Summary plot of mean (±SEM) BPCaS-evoked calcium profiles (ΔF/F %; 45 dendrites; 20 of which with local TTX at the soma) presented as a function of distance from the soma. Each bar is an average of 100-μm segment. Gray bars show mean ΔF/F of noise values (±SEM).
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

9. Figure 8
Branch-Specific Increased Propagation of BCaS following Low-Frequency Plasticity in Tuft Dendrites
(A) Fluorescence reconstruction of a layer 5 pyramidal neuron showing the site of stimulation (blue electrode; 1,070 μm from the soma) and dendritic recording electrode (red electrode; 860 μm from the soma). Numbers 1–9 represent selected regions of interest (ROIs) along the dendrite.
(B) Calcium transients evoked by BCaS (dendritic current injection 0.8 nA square current; 100 ms; bottom) recorded in the stimulated dendritic branch (blue dot illustrates the stimulated segment), sister branch, and mother branch. Calcium transients are shown for control (black traces) and poststimulation induction protocol (red traces). Numbers 1–9 correspond with numbers in (A). Note a significant increase in BCaS-evoked calcium transient specific to the activated site.
(C) EPSP amplitude represented over time during 0.03 Hz followed by 0.1 Hz stimulation, producing a significant increase in EPSP amplitude (same neuron as in A and B).
(D) Average calcium transients (ΔF/F %) evoked by BCaS presented for mother stimulated and sister unstimulated branches in control (black bars) and post-0.1 Hz stimulation (red bars). Lines represent the noise value (black for control; red for post-potentiation). Blue dot represents the stimulated dendritic segment. Same neuron as in (A) and (B) is shown.
(E) Summary plot of the mean change in calcium transients (ΔF/F % ± SEM) evoked by BCaS plotted as a function of the distance from the center of stimulated dendritic segment (0) in 20-μm segments, from proximal (to soma; negative values) to distal (from soma; positive values) locations.
(F) Summary plot of % change (±SEM) in BPCaS-evoked calcium transients at stimulated dendritic locations (n = 10 cells) and at sister unstimulated dendrites (all ROIs along sister dendrites; n = 7 cells; p = between stimulated and unstimulated dendrites).
(G) Summary plot of mean BPCaS-evoked calcium transients (ΔF/F % ± SEM) in the presence of the Kv4.2 blocker, heteropodatoxin-2 (0.5 μM), during 0.03 Hz and 0.1 Hz stimulation.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions