A Novel Form of Local Plasticity in Tuft Dendrites of Neocortical Somatosensory Layer 5 Pyramidal Neurons

Article

A Novel Form of Local Plasticity in Tuft Dendrites of Neocortical Somatosensory Layer 5 Pyramidal Neurons Highlights

Authors

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Unpaired low-frequency stimulation results in potentiation of tuft dendritic EPSPs

Maya Sandler, Yoav Shulman, Jackie Schiller

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Plasticity was accompanied by enhanced excitability in the activated tuft branches

Correspondence

Kv4.2 channels, NMDAR, membrane internalization, and AMPAR insertion are required

In Brief

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d

This plasticity was unique to tuft dendrites and did not occur in basal dendrites

Sandler et al., 2016, Neuron 90, 1–15 June 1, 2016 ª 2016 Elsevier Inc. http://dx.doi.org/10.1016/j.neuron.2016.04.032

[email protected]

Sandler et al. describe a novel form of synaptic and dendritic plasticity unique to tuft dendrites, requiring Kv4.2, NMDAR, membrane internalization, and AMPAR insertion. This modulates coupling efficiency of forward and backward signals of activated tuft branches with other compartments.

Please cite this article in press as: Sandler et al., A Novel Form of Local Plasticity in Tuft Dendrites of Neocortical Somatosensory Layer 5 Pyramidal Neurons, Neuron (2016), http://dx.doi.org/10.1016/j.neuron.2016.04.032

Neuron

Article A Novel Form of Local Plasticity in Tuft Dendrites of Neocortical Somatosensory Layer 5 Pyramidal Neurons Maya Sandler,1 Yoav Shulman,1 and Jackie Schiller1,* 1Department of Physiology, The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa 35254, Israel *Correspondence: [email protected] http://dx.doi.org/10.1016/j.neuron.2016.04.032

SUMMARY

Tuft dendrites of layer 5 pyramidal neurons form a separate biophysical and processing compartment. Presently, little is known about plasticity mechanisms in this isolated compartment. Here, we describe a novel form of plasticity in which unpaired low-frequency (0.1 Hz) stimulation of tuft inputs resulted in prolonged transient (86.3 ± 7.3 min) potentiation of EPSPs (286.1% ± 30.5%) and enhanced local excitability that enabled more-efficient back-propagation of axo-somatic action potentials and dendritic calcium spikes selectively into the activated dendritic segments. This plasticity was exclusive to tuft dendrites and did not occur in basal dendrites. Induction of this plasticity depended on activation of Kv4.2 potassium and NMDAR channels, internalization of membrane proteins, and insertion of AMPAR. This unique form of tuft plasticity increases proximaldistal electrical coupling of activated tuft dendrites and opens a prolonged time window for binding and storing feedforward and feedback information in a branch-specific manner.

INTRODUCTION Neocortical pyramidal neurons, which are the main processing units of the cortex, possess three separate dendritic arborizations: the basal tree that branches directly from the soma; oblique dendrites branching from the proximal apical dendrite; and the tuft tree that branches from the distal apical trunk in layers 1 and 2. These dendrites contain active dendritic conductances including voltage-gated sodium, calcium, N-methyl-D-aspartate receptor (NMDAR), and potassium channels, which participate in shaping the local voltage and provide the neuron with rich nonlinear capabilities to process incoming inputs (London and Ha¨usser, 2005; Major et al., 2013). Tuft dendrites of layer 5 pyramidal neurons are weakly excitable dendrites forming a special biophysical and integration compartment (Larkum et al., 2009; Harnett et al., 2013). They were shown to contain unique dendritic conductances such as Ih channels and Kv4.2 potassium

channels (Berger et al., 2001; Harnett et al., 2013) and are selectively innervated by long-range cortico-cortical feedback inputs and neuromodulatory fibers (Rockland and Pandya, 1979; Lysakowski et al., 1986; Cauller and Connors, 1994; Cauller et al., 1998; Petreanu et al., 2009; Larkum, 2013; Major et al., 2013). A prominent feature of tuft dendrites is their relative isolation from the rest of the dendritic tree and soma, with respect to both forward and backward propagation of information. Axo-somatic back-propagating action potentials (BAPs) and apical calcium spikes severely attenuate as they spread to tuft dendritic compartment. Furthermore, tuft excitatory postsynaptic potentials (EPSPs) contribute very little to the apical calcium initiation zone and the soma (Larkum et al., 2001, 2009; Harnett et al., 2013). Interestingly, previous findings reported a high degree of local intra-branch interaction but little inter-branch interactions, indicating a high degree of compartmentalization within the tuft tree (Larkum et al., 2009; Harnett et al., 2013). Synaptic plasticity rules are important in determining how cortical networks organize, acquire, and store information. It has been previously shown that plasticity rules differ along different locations of the dendritic tree of pyramidal neurons (Froemke et al., 2005; Gordon et al., 2006; Letzkus et al., 2006; Sjo¨stro¨m and Ha¨usser, 2006). For example, whereas synapses innervating proximal basal dendrite undergo spike-timingdependent plasticity (STDP) when paired with BAPs, synapses innervating distal basal dendrites undergo plasticity when paired with local NMDA spikes and in the presence of brain-derived neurotrophic factor (BDNF) (Gordon et al., 2006). In addition to long-term changes in synaptic strength, recent studies indicated that intrinsic dendritic excitability may also undergo plasticity changes that can serve as a powerful mechanism for modulating integration of synaptic inputs and ultimately dynamically change the neuronal output (Frick et al., 2004; Remy et al., 2010; Kastellakis et al., 2015). Presently, very little is known about the plasticity mechanisms of synapses located in tuft dendrites of layer 5 pyramidal neurons. Yet, from recent in vivo studies, it is clear that tuft dendrites undergo structural and functional plasticity changes that are important for sensory motor learning of novel sensory motor skills (Fu et al., 2012; Cichon and Gan, 2015; Hayashi-Takagi et al., 2015). For example, NMDA-dependent branch-specific plasticity mechanisms in the tuft were shown to occur during novel learning tasks (Cichon and Gan, 2015). Moreover, during Neuron 90, 1–15, June 1, 2016 ª 2016 Elsevier Inc. 1

Please cite this article in press as: Sandler et al., A Novel Form of Local Plasticity in Tuft Dendrites of Neocortical Somatosensory Layer 5 Pyramidal Neurons, Neuron (2016), http://dx.doi.org/10.1016/j.neuron.2016.04.032

motor learning, new spines are formed in short stretches of tuft dendrites, creating anatomical clusters (Fu et al., 2012). It is possible that tuft dendrites of neocortical pyramidal neurons form a special plasticity compartment especially in face of their relative isolation from axo-somatic BAPs and apical calcium spikes (Larkum et al., 2009; Harnett et al., 2013) and their special role in integrating feedback information (Larkum, 2013). An intriguing possibility for better coupling of the activity in tuft dendrites with the activity in the other dendritic locations and soma is via plasticity of dendritic excitability in tuft branches. One important candidate contributing to induction of localized plasticity in intrinsic dendritic excitability is the voltage-gated potassium channel Kv4.2, which is the dominant fast inactivating (Ia) potassium channel subtype in pyramidal neurons (Magee and Johnston, 2005; Kim et al., 2007; Kim and Hoffman, 2008). Previous studies have shown that Ia channels in general and Kv4.2 channels in particular play an important role in local dendritic plasticity and as such critically participate in regulating intrinsic excitability of dendrites in CA1 pyramidal neurons (Cai et al., 2004; Kim et al., 2007; Losonczy et al., 2008). In contrast to CA1 neurons, little is known about the density and possible role in plasticity of Kv4.2 channels in layer 5 pyramidal neurons. Current electrophysiological data point to a steep decrease in the density of Ia currents along the proximal apical trunk of layer 5 pyramidal neurons, which stabilizes to a constant low level in distal dendrites (Korngreen and Sakmann, 2000; Almog and Korngreen, 2014). However, despite the steep decrease, a recent study showed the existence of Ia currents throughout the tuft dendrites and their contribution in controlling nonlinear tuft excitability (Harnett et al., 2013). Here, we set out to investigate plasticity mechanisms in tuft dendrites of layer 5 pyramidal neurons. Using direct dendritic recordings from tuft dendrites combined with dendritic calcium imaging, we find a novel form of plasticity unique to tuft dendrites, in which low-frequency single unpaired EPSPs resulted in NMDAR and Kv4.2-dependent potentiation of the synaptic responses as well as increased dendritic excitability, allowing for more-efficient axo-somatic BAPs and dendritic calcium spikes into the activated distal tuft branches. RESULTS Low-Frequency 0.1-Hz Activation of Single Unpaired EPSPs Induces Long-Term Potentiation To investigate the local plasticity mechanisms in tuft dendrites, we typically performed whole-cell patch-clamp voltage recordings from primary to tertiary dendritic tuft branches and focally stimulated synaptic inputs in proximity to a single tuft branch distal to recording site typically in layers 1 and 2 (Figure 1A). Repeated activation of single unpaired EPSPs at a low frequency of 0.1 Hz, which is normally used as control stimulation in various long-term potentiation (LTP) protocols (Magee and Johnston, 1997; Markram et al., 1997; Sjo¨stro¨m et al., 2001; Froemke and Dan, 2002; Gordon et al., 2006), resulted in a surprising large and robust potentiation in the amplitude of EPSPs (Figure 1B). To establish a stable control stimulation frequency in which the single unpaired EPSP amplitude remained stable over time, we further decreased the stimulation frequency. Decreasing the fre2 Neuron 90, 1–15, June 1, 2016

quency of stimulation to 0.033 Hz resulted in stable EPSP amplitude. However, upon increasing the stimulation frequency to 0.1 Hz, a rapid increase in EPSP amplitude was observed in 73.1% of stimulated dendrites (within 5.4 ± 0.51 min in 57 out of 78 stimulated dendrites in 68 recorded neurons; Figures 1C and 1D). In these neurons, the EPSP amplitude and rise slope recorded at the dendrite increased by an average of 286.1% ± 30.5% and 341.4% ± 59.5% of the control EPSP amplitude, respectively (Figures 1H and 1I; n = 57 dendrites). The EPSP amplitude as recorded at the soma increased by an average of 201.24% ± 21.5% of control EPSP (Figure 1J; n = 16). Next, we determined the duration this synaptic plasticity phenomenon lasted. After establishing the potentiation of tuft EPSPs at 0.1-Hz stimulation, we reduced the stimulation rate to 0.0083– 0.0017 Hz (Figures 1E–1G and S1). We found that synaptic potentiation induced by the 0.1-Hz unpaired stimulation was reversible after an average period of 86.3 ± 7.3 min (n = 6 cells; Figure 1K). Thus, our experiments showed that low-frequency activation of unpaired EPSPs in distal tuft dendrites resulted in a prolonged yet transient synaptic potentiation. The de-potentiation process occurred within a single stimulus in half of the cases (within 180–1,200 s, depending on our stimulation frequency) and in the other half in a more-gradual manner over several stimuli. To rule out the possibility that the abrupt depotentiation is due to a technical problem such as instability of the stimulation, we attempted to re-potentiate after the depotentiation period. As can be shown in Figure S1, we could easily re-potentiate by stimulating again with 0.1 Hz without changing the stimulus intensity or any other parameter beside the frequency of activation (n = 7). This indicates that the reduction in amplitude represents a de-potentiation process and not a technical problem in our stimulation. The plasticity phenomenon we describe here has not been reported previously in other neurons or other dendritic regions of pyramidal neurons. Many groups including our own used 0.1-Hz stimulation to establish the baseline control stimulation in LTP experiments (Magee and Johnston, 1997; Markram et al., 1997; Sjo¨stro¨m et al., 2001; Froemke and Dan, 2002; Gordon et al., 2006). To further establish the uniqueness of the phenomenon, we performed two additional sets of experiments: first, we tested whether unpaired EPSPs delivered at ‘‘traditional’’ LTP frequencies (Markram et al., 1997; Letzkus et al., 2006; Sjo¨stro¨m and Ha¨usser, 2006) at low repetition rates of 0.03 Hz can replace the 0.1-Hz single-EPSP stimulation protocol in tuft dendrites. Our protocol consisted of unpaired high-frequency train (five pulses at 50 Hz) delivered every 0.03 Hz repeated 25–50 times. With this protocol, we took care that the high-frequency train will not evoke local dendritic NMDA or calcium spikes. Under this stimulation protocol, we could not observe a significant potentiation in tuft EPSPs (Figure S2; the amplitude after the high-frequency stimulation was 97.7 ± 2.95 of the control EPSP; n = 8). Interestingly, in the same dendrites after switching to 0.1 Hz single-EPSP stimulation protocol, we could observe a robust and significant potentiation (199.2 ± 0.76; Figure S2). This result ruled out the possibility that the high-frequency stimulation damaged the activated dendrite or that these specific dendrites could not undergo plasticity changes. Taken together, increasing the number of EPSPs by

Please cite this article in press as: Sandler et al., A Novel Form of Local Plasticity in Tuft Dendrites of Neocortical Somatosensory Layer 5 Pyramidal Neurons, Neuron (2016), http://dx.doi.org/10.1016/j.neuron.2016.04.032

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 mM) showing the experimental setup. A recording electrode located distal to the first bifurcation (red; 525 mm from soma) and a stimulating electrode located in distal tuft dendrite (blue; 910 mm 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 mm from soma) and a stimulating electrode located in distal tuft dendrite (blue; 1,040 mm 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 mm from the soma) and recording (red electrode; 800 mm 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 = 6.55197e 10) as well as in rise slope change (f; p = 1.24779e 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 90, 1–15, June 1, 2016 3

Please cite this article in press as: Sandler et al., A Novel Form of Local Plasticity in Tuft Dendrites of Neocortical Somatosensory Layer 5 Pyramidal Neurons, Neuron (2016), http://dx.doi.org/10.1016/j.neuron.2016.04.032

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 mm from the soma) and distal (green; 220 mm 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.

applying trains of EPSPs at 0.03 Hz does not evoke the low-frequency tuft potentiation. Second, we tested the uniqueness of the phenomenon to tuft dendrites over basal dendrites. We repeated the low-frequency stimulation protocol we used for tuft dendrites while recording from the soma of layer 5 pyramidal neurons and stimulating at two dendritic locations along basal dendrites: proximal (70 ± 2.86 mm from soma) and distal (193 ± 13.75 mm from soma; Figure 2A). Contrary to tuft dendrites and in agreement with previous published data (Gordon et al., 2006), we did not observe a significant change in EPSP amplitudes both for proximal and distal dendritic stimulation locations (Figures 2B–2E). The average change in EPSP amplitude before and after the 0.1-Hz stimulation was 102.9% ± 1.4% and 100.1% ± 0.6% for proximal (p = 0.145) and distal (p = 0.717) dendritic locations, respectively (n = 12 dendrites; Figures 2D and 2E). To address the question whether the differences we observed between basal and tuft dendrites reflect higher plasticity threshold for basal dendrites in the frequency domain, we performed additional experiments in both proximal and distal basal dendrites using a series of increasing frequency stimulations. We stimulated focally inputs to basal dendrites starting with control frequency of 0.03 Hz and increased the frequency gradually to 0.1, 0.2, 0.5, 1, and 2 Hz unpaired single-EPSP stimulation trains. At all frequencies tested, we did not observe a significant potentiation of the EPSP (Figure S3). On the contrary, we observed a significant depression of EPSP amplitude for all frequencies tested in distal dendrite inputs and for 1 and 2 Hz stimulation in proximal basal dendrite inputs (Figure S3E). However, when at the end of the stimulation series we paired the single EPSP with a short train of BAPs, we observed a significant LTP at the proximal dendritic location (293% ± 69.9% of control for proximal EPSPs and 97.76% ± 23.9% for distal EPSPs). These results are in line with previous published data (Gordon et al., 2006) and indicate that the proximal dendrites possess the capa4 Neuron 90, 1–15, June 1, 2016

bility to undergo LTP given the appropriate stimulation paradigm. In conclusion, our results support the notion that tuft dendrites are fundamentally different from basal dendrites with respect to their capability to undergo low-frequency potentiation with unpaired EPSPs. Mechanisms Responsible for the Induction of the LowFrequency Unpaired Potentiation in Tuft Dendrites To get insight about the mechanisms underlying this special form of potentiation in tuft dendrites, we used specific blockers. We find that NMDAR activation is essential for this type of plasticity changes. In the presence of the NMDAR blocker, APV (100 mM in the bath solution), we could not induce potentiation of EPSPs in tuft dendrites using 0.1-Hz frequency stimulation (Figures 3A and 3F). On average, EPSP amplitude after 0.1-Hz stimulation in the presence of APV was 87.2% ± 7.6% of control EPSPs (n = 9 dendrites; p = 3.86091e 08 compared to control bath solution). We further tested whether mGluR5, which was shown to potentiate NMDAR (Rook et al., 2015) and participate in various forms of long-term plasticity process (Anwyl, 2009; Niswender and Conn, 2010), also participates in the low-frequency potentiation we describe here. Addition of the mGluR5 blocker 2-methyl6-(phenylethynyl) pyridine (MPEP-hydrochloride; 10 mM) did not prevent the potentiation in tuft dendrites using the 0.1-Hz-frequency stimulation (Figure S4). On average, we observed EPSP potentiation of 170.5% ± 33.25% in the presence of MPEP (n = 4). These results indicate that mGluR5 does not play a major role in the low-frequency potentiation we describe here. Based on previous studies performed in CA1 pyramidal neurons, a possible candidate for participating in NMDAR-dependent local dendritic plasticity changes are the Kv4.2 potassium channels (Magee and Johnston, 2005; Kim and Hoffman, 2008). To test this hypothesis, we repeated the low-frequency unpaired plasticity protocol in the presence of the specific Kv4.2 channel blocker heteropodatoxin-2 (0.5 mM). We found

Please cite this article in press as: Sandler et al., A Novel Form of Local Plasticity in Tuft Dendrites of Neocortical Somatosensory Layer 5 Pyramidal Neurons, Neuron (2016), http://dx.doi.org/10.1016/j.neuron.2016.04.032

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 mM). 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 mM). 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 mm from soma) and focal stimulating electrode (blue; 860 mm 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 mg/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 mM) 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.

that, in the presence of the specific Kv4.2 blocker, we were unable to induce EPSP potentiation in any of the cases tested (Figure 3B; n = 19 dendrites). In these experiments, following unpaired low-frequency stimulation, the average EPSP amplitude reached an average of 93.6% ± 3.9% of the control value (Figures 3B and 3F; p = 6.42044e 08 compared to control bath solution). Thus, we concluded that tuft potentiation induced by low-frequency unpaired stimulation was dependent on activation of both NMDAR and Kv4.2 channels.

To investigate whether the novel potentiation we described in distal tuft dendrites is dependent on internalization mechanisms similar to CA1 neurons, we introduced the synthetic dynaminderived peptide to the patch electrode to inhibit the clathrinmediated endocytosis (Kim et al., 2007) and examined its effect on potentiation of tuft EPSPs. Intracellular addition of the synthetic dynamin-derived peptide to the patch electrode (membrane impermeable DYN; 100 mg/ml; Figures 3C and 3D) blocked altogether the potentiation of the tuft EPSPs (Figures 3D and 3F). Neuron 90, 1–15, June 1, 2016 5

Please cite this article in press as: Sandler et al., A Novel Form of Local Plasticity in Tuft Dendrites of Neocortical Somatosensory Layer 5 Pyramidal Neurons, Neuron (2016), http://dx.doi.org/10.1016/j.neuron.2016.04.032

In the presence of intracellular DYN after the 0.1-Hz unpaired induction protocol, the average EPSP was 95.9% ± 1.98% of the control EPSP amplitude acquired at 0.033 Hz (n = 13 dendrites; p = 7.75094e 08 compared to control intracellular solution). Thus, we concluded that clathrin-mediated endocytosis possibly of Kv4.2 channels (Kim et al., 2007) plays an important role in the observed potentiation of distal tuft inputs. It is important to stress that the increase in the tuft EPSP amplitude following the unpaired low-frequency stimulation could not be fully accounted for by a direct effect of Kv4.2 channels on the EPSP amplitude, as application of the specific Kv4.2 blocker heteropodatoxin-2 (0.5 mM) under control stimulation conditions (0.033 Hz) caused only a small but significant increase in the EPSP amplitude and half-width (109.4% ± 5%; p = 0.041 and 109.77% ± 2.13%; p = 0.00124, respectively; n = 16 dendrites; see also Figures 6D and 6E). However, in line with our conclusion, addition of the Kv4.2 blocker to the already potentiated EPSPs did not cause a significant change in the amplitude of the EPSPs (102.5% ± 4.5%). Similarly, dendrites that did not undergo potentiation with 0.1 Hz also showed no significant change in the EPSP amplitude after addition of the Kv4.2 blocker (103.2% ± 5.2%). As blockade of Kv4.2 could not account for the large potentiation of EPSPs we observed, we next examined whether the large potentiation in distal tuft induced by 0.1-Hz unpaired induction protocol is related to insertion of a-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid receptor (AMPAR) channels to the postsynaptic membrane (Malinow and Malenka, 2002; Kim et al., 2007). To investigate this question, we introduced Pep1-TGL (100 mM) to the patch recording electrode, which was shown to disrupt the trafficking and insertion of GluR1 subunits of the AMPAR to the postsynaptic membrane (Hayashi et al., 2000; Edelmann et al., 2015). In the presence of intracellular Pep-1-TGL, the potentiation was blocked altogether (Figures 3E and 3F). The average change in EPSPs amplitude following 0.1-Hz unpaired stimulation in the presence of Pep1TGL was 93.1% ± 2.97% (n = 17 dendrites; p = 5.76936e 08 compared to control intracellular solution). Taken together, our findings indicated that low-frequency (0.1-Hz) unpaired activation of distal tuft inputs resulted in a large potentiation of local EPSPs that was dependent on both NMDAR and Kv4.2, required internalization of membrane proteins in the postsynaptic neuron, probably Kv4.2 channels, and resulted in insertion of AMPAR to the postsynaptic membrane-activated dendrites. Spine Imaging during the Induction of the Low-Frequency Unpaired Potentiation in Tuft Dendrites To further study the low-frequency unpaired potentiation in tuft dendrites, we performed calcium imaging of the spines in the activated dendritic segment both during the control (0.03-Hz) and the 0.1-Hz synaptic stimulation. We first counted the number of spines that were activated by our focal synaptic stimulation. Active spines were defined as spines in which the calcium transients had a higher amplitude, faster rise time, and earlier onset than the calcium transients in the adjacent shaft (see Figures 4A and 4B; Yuste and Denk, 1995; Schiller et al., 1998; Kovalchuk et al., 2000). We found that, on average, 7.75 ± 0.95 spines (Figures 4C and 4D; n = 8 neurons) were activated in our stimulation 6 Neuron 90, 1–15, June 1, 2016

protocol, yielding an average dendritic EPSP amplitude of 1.86 ± 0.32 mV (recorded at average dendritic distance of 695 ± 29.3 mm from soma). It is important to stress that, whereas our stimulation can activate this number of synapses, because of the stochastic nature of synaptic transmission, the number of active synapses during individual stimuli may be smaller. The vast majority (64.7% ± 7.7%) of the activated spines were found within ±5 mm of our stimulating electrode (Figure 4D). Within this 10-mm segment, 59.7% ± 7.2% of the total spines were activated. It is important to stress that we identified 1.04 ± 0.09 spines per 1-mm dendritic length, indicating that we were able to reliably identify majority of spines in the tested segments (Larkman, 1991). To further confirm the number of activated spines during our focal synaptic stimulation, we used computer simulations (neuron simulation platform; see Supplemental Experimental Procedures) based on our previous direct measurements of unitary events in tuft dendrites of layer 5 pyramidal neurons (Larkum et al., 2009). Our simulations showed that we activated 7.87 ± 0.74 synapses (n = 8 neurons), which is well within the range of our experimental results (Figure 4D). Interestingly, after potentiation with the 0.1-Hz protocol, the number of activated spines did not change, rather the amplitude of their calcium transients (DF/F) increased significantly (263% ± 58% compared to control conditions; Figures 4E and 4F). These findings further show that the plasticity we observe is a postsynaptic process involving potentiation within activated spines. To examine whether the degree of potentiation we observe was dependent on the number of synapses activated, we correlated between the initial amplitude of the EPSP during control stimulation and the degree of potentiation (Figure 4G). The degree of potentiation deceased when the amplitude of the initial EPSP was >1 mV, indicating that the phenomenon we describe occurs with small number of synapses and does not require large activations. The decrease in potentiation with larger-EPSP amplitudes may result from the fact that higher-amplitude EPSPs consist of more pre-potentiated synapses, and in turn, the degree of their potentiation is expected to be smaller. Low-Frequency Unpaired Stimulation Increases the Propagation of Axo-somatic BAPs into Activated Tuft Dendrites The involvement of Kv4.2 potassium channels in the low-frequency unpaired tuft plasticity raises the possibility that the EPSPs potentiation is accompanied by excitability changes locally in tuft dendrites that in turn can improve backpropagation of both axo-somatic action potentials (APs) and apical dendritic calcium spikes (Larkum et al., 2009; Harnett et al., 2013). To test this possibility, we performed simultaneous patchclamp recordings from soma and distal apical dendrite of layer 5 pyramidal neurons combined with calcium imaging while focally stimulating a tuft branch (Figure 5A). We first characterized axo-somatic BAPs-evoked calcium transients into distal tuft dendrites under control conditions. In these experiments, axo-somatic BAPs were evoked by short somatic current injections (50–80 ms), which typically evoked a pair of axo-somatic APs (Figure 5B). We observed a significant gradual reduction of BAPs-evoked calcium transients along the apical dendrites and especially in distal apical tuft branches >900 mm from the

Please cite this article in press as: Sandler et al., A Novel Form of Local Plasticity in Tuft Dendrites of Neocortical Somatosensory Layer 5 Pyramidal Neurons, Neuron (2016), http://dx.doi.org/10.1016/j.neuron.2016.04.032

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 mM) and OGB1 (100 mM). Inset shows the stimulated segment (blue electrode 950 mM 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 mM from soma) is presented. Calcium transients shown in the line scan are presented as DF/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 mm, ±10 mm, ±20 mm, and larger than ±30 mm 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 DF/F in control and following potentiation with 0.1 Hz stimulation. (F) Summary plot of DF/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.

soma (n = 35 cells). The BAPs evoked calcium transients decreased from 74.7% ± 5.9% DF/F at the highest point (301– 400 mm from the soma) to a small value of 20.2% ± 1.4% DF/F at the most-distal tuft locations measured (1,000–1,100 mm from the soma). The DF/F values we report here may be an underestimate of the calcium transients because of nonlinearities of the high-affinity dye we use (OGB1). On the other hand, due to surface-to-volume considerations, similar calcium transients are expected to evoke higher calcium transients in distal dendrites. Following the 0.1-Hz unpaired stimulation, in addition to the large potentiation of EPSPs, we observed a significant increase in the voltage of axo-somatic BAPs as recorded by the distal dendritic recording electrode (Figure 5B), as well as a significant increase in the calcium transients evoked by axo-somatic BAPs specifically to the activated tuft regions (Figures 5D–5G). On average, the amplitude of the axo-somatic BAP voltage increased by 165.3% ± 44.5% after stimulation (Figure 5H) and the average calcium transients evoked by axo-somatic BAPs increased by 242.2% ± 25.54% (n = 10 cells) at the activated

tuft dendrites (Figures 5D–5G). We observed a maximal effect over a dendritic segment of several tens of microns around the stimulation site (Figure 5F). Yet, the BAP-evoked calcium transients were increased to a lesser degree up to the most-distal dendritic segments measured. Interestingly, the increase in calcium transient amplitude lasted 97 ± 11.4 min (n = 5), slightly longer than the average time course of EPSP potentiation. At the non-activated dendrites, the average BAP-evoked calcium transient amplitudes before and after 0.1-Hz frequency stimulation were not significantly changed (Figure 5G; 99.74% ± 0.8%; n = 7 dendrites). Similar to synaptic potentials, plasticity of excitability following the 0.1-Hz unpaired stimulation was also unique to tuft dendrites. In basal dendrites, we did not observe a significant change in BAPs-evoked calcium transients following 0.1-Hz unpaired stimulation, confirming the lack of excitability changes in basal dendrites following the low-frequency activation protocol (Figures 6A and 6B). On average, following 0.1-Hz unpaired stimulation, BAP-evoked calcium transients in basal dendrites were 99.7% ± 0.6% of the prestimulus control value (n = 11 cells; p = 0.37). One possible reason for the differential effect between basal and tuft dendrites is the difference in the density of Kv4.2 Neuron 90, 1–15, June 1, 2016 7

Please cite this article in press as: Sandler et al., A Novel Form of Local Plasticity in Tuft Dendrites of Neocortical Somatosensory Layer 5 Pyramidal Neurons, Neuron (2016), http://dx.doi.org/10.1016/j.neuron.2016.04.032

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 mm from the soma), dendritic recording (red electrode; 770 mm 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 (DF/F %; n = 35 cells) presented as a function of distance from the soma (black). Each value is an average of 100 mm dendritic segments. The first 10–50 mm are referred to as ‘‘somatic’’ region. Gray bars show mean DF/F of noise values (±SEM). (D) Calcium transients (DF/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 mm from soma). (E) Average calcium transients (DF/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 mm 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 = 0.000692 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).

channels in tuft compared to basal dendrites. To investigate this possibility, we examined the effect of blocking Kv4.2 channels on EPSPs voltage and on BAP-evoked calcium transients in basal compared to tuft dendrites in control stimulation conditions. Whereas in basal dendrites, BAP-evoked calcium transients remained unchanged following blockade of Kv4.2 with heteropodatoxin-2 (0.5 mM), calcium transients in tuft dendrites were increased by blockade of Kv4.2 channels (Figure 6C; 99.5% ± 1.1%; n = 9 cells; p = 0.78 for basal dendrites and 172.844% ± 10.15%; n = 12; p = 1.58e 06 for tuft dendrites). Similar results were observed for EPSP voltage in tuft versus basal dendrites (Figures 6D and 6E). In contrast to tuft dendrites, we did not observe a significant change in EPSP amplitude and half-width in basal dendrites following application of heteropodatoxin-2 in control stimulation conditions. The average change in EPSP amplitude and half-width in basal dendrites in the presence of heteropodatoxin-2 was 97.8% ± 1.6% (n = 13 cells; p = 8 Neuron 90, 1–15, June 1, 2016

0.184) and 99.13% ± 2.0% (n = 13 cells; p = 0.478), respectively, compared to control. These results indicate a significant difference between the effect of Kv4.2 channels in tuft versus basal dendrites (p = 0.04 for EPSP amplitude and p = 0.0012 for EPSP half-width) and a minimal effect of Kv4.2 channels on calcium transients in basal compared to tuft dendrites (p = 1.43e 09), which in turn may be responsible for the differential effect of low-frequency unpaired plasticity in basal versus tuft dendrites. Low-Frequency Unpaired Stimulation Increases the Back Propagating Calcium Spike-Evoked Calcium Transients into Activated Tuft Dendrites Similar to axo-somatic BAPs, we examined the effect of low-frequency unpaired tuft plasticity protocol on the extent of back propagation of apical calcium spikes into tuft dendrites. Toward this end, we acquired back propagating calcium spike

Please cite this article in press as: Sandler et al., A Novel Form of Local Plasticity in Tuft Dendrites of Neocortical Somatosensory Layer 5 Pyramidal Neurons, Neuron (2016), http://dx.doi.org/10.1016/j.neuron.2016.04.032

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 mm and 220 mm 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-mm 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 = 1.43088e 09 between basal and tuft dendrites. (D and E) Mean EPSP amplitude and half-width change (control relative to heteropodatoxin-2; 0.5 mM) in tuft (n = 16) and basal (n = 13) dendrites in control stimulation frequency (0.03 Hz).

(BCaS)-evoked calcium profile in control conditions (Figure 7) and after the induction of potentiation (Figure 8). In these experiments, calcium spikes were evoked by current injection through the dendritic recording electrode typically positioned at the main bifurcation point, whereas in a fraction of the cases, the axo-somatic APs were eliminated by local application of TTX (2 mM) at the axo-somatic region. The BCaS-evoked calcium profile is presented for a single neuron and for all recorded neurons (n = 45 dendrites; 20 of them with local TTX application at the soma; Figure 7). Similar to axo-somatic BAPs, BCaS-evoked calcium transients also attenuated along the tuft dendrites, especially in the more-distal tuft branches (Figure 7G). The average BCaS-evoked calcium transients decreased from 133.8% ± 7% DF/F around the bifurcation point (600–700 mm from soma) to 32.93% ± 7.56% DF/F at the most-distal tuft locations measured (1,100–1,200 mm from the soma). Interestingly, the degree of attenuation of BCaS-evoked calcium transients differed between neurons and different tuft branches of the same neuron (Figure 7F). Following the induction of tuft plasticity, we observed a strong and significant increase in BCaS-evoked calcium transients in the activated tuft dendrites. The increase in calcium transients was focal and spanned several tens of microns of dendritic length around the activated dendritic site (Figures 8A–8E; n = 10 cells). Unstimulated branches, including sister branches of the same neuron, did not experience a significant increase in BCaS-evoked calcium transients (Figures 8B–8F). Following low-frequency unpaired tuft stimulation, the DF/F of BCaSevoked calcium transients in the activated tuft branches reached 205.94% ± 19.4% DF/F of the preinduction control value (n = 10 cells; p < 0.001) versus 103.5% ± 3.3% of the preinduction control value in sister unstimulated dendritic tuft branch (n = 7 cells; p = 0.69). Kv4.2 channels also played a crucial role in the increase of BCaS-evoked calcium transients we observe following the

0.1-Hz unpaired synaptic stimulation. When the Kv4.2 blocker, heteropodatoxin-2 (0.5 mM), was added to the bath perfusion, we observed a significant increase in BCaS-evoked calcium transients under control stimulation conditions (172.84% ± 10.156% compared to control conditions; n = 15; p = 1.57846e 06). However, in the presence of the blocker, the 0.1-Hz unpaired induction protocol resulted in only a small increase in the BCaS-evoked calcium transients in the activated branch, with an average of 103.5% ± 1.3% compared to the preinduction blocked conditions (Figure 8G; n = 10 cells; p = 0.06). These results indicate that, together with synaptic potentiation, the 0.1-Hz unpaired EPSPs induction protocol also caused a significant increase in dendritic excitability, which was dependent on Kv4.2 and was specific to the activated dendritic branches that in turn enabled better propagation of calcium spikes into the activated tuft branch. DISCUSSION We describe a novel form of plasticity in tuft dendrites of layer 5 pyramidal neurons. This local tuft plasticity is induced with low-frequency activation (0.1 Hz) of single unpaired tuft EPSPs and does not require pairing with somatic BAPs or back propagating apical calcium spikes or local NMDA spikes. This form of plasticity was exclusive to tuft dendrites, as basal dendrites did not undergo plasticity changes with this form of induction protocol. We find that induction of local tuft plasticity is dependent on both Kv4.2 potassium and NMDAR channels. Moreover, with this unique form of tuft plasticity, both synaptic inputs are amplified via insertion of AMPAR and excitability of the activated dendritic segment is increased possibly via internalization of Kv4.2 channels, enabling more efficient axo-somatic BAPs as well as dendritic BCaS to activated tuft branches. Neuron 90, 1–15, June 1, 2016 9

Please cite this article in press as: Sandler et al., A Novel Form of Local Plasticity in Tuft Dendrites of Neocortical Somatosensory Layer 5 Pyramidal Neurons, Neuron (2016), http://dx.doi.org/10.1016/j.neuron.2016.04.032

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 mM). (B and D) Calcium spikes were evoked by current injections via the dendritic recording electrode, and calcium transients (DF/F %) were recorded from two apical branches left (yellow; B) and right (orange; D). (C and E) Calcium transients (DF/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 mm from soma) is shown. (F) Examples of BPCaS-evoked calcium profiles from five neurons presented as DF/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 (DF/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-mm segment. Gray bars show mean DF/F of noise values (±SEM).

Comparison to Other Forms of Plasticity Kv4.2 is an important dendritic channel, which was shown previously to be involved with branch-specific changes in dendritic excitability (Magee and Johnston, 2005; Kim and Hoffman, 2008; Remy et al., 2010). However, there are fundamental differences between previously described branch plasticity and the present tuft plasticity we describe here. Long-term increase of excitability in a branch-specific manner within restricted dendritic regions required intense induction protocols such as the use of chemical LTP (Kim et al., 2007) or following theta pairing induction protocols or protocols that involved intense repetitive activation of local dendritic spikes paired with cholinergic agonists or during enriched environment (Kim et al., 2007; Losonczy et al., 2008; Makara et al., 2009). In these studies, either Kv4.2 activation range was shifted following induction of LTP in CA1 neurons (Frick et al., 2004) or downregulation of Kv4.2 was observed (Kim et al., 2007). In contrast, here, we report a strong potentiation in tuft EPSPs, which was accompanied by increased dendritic excitability and did not require pairing or special neuromodulatory mechanisms. Our data indicate that the plasticity we describe here is dependent on Kv4.2 channels and membrane internalization in the postsynaptic membrane. Thus, it is consistent with internalization of Kv4.2 into the postsynaptic neuron similar to previous reports (Kim et al., 2007). However, further studies are needed to unequivocally prove 10 Neuron 90, 1–15, June 1, 2016

that kV4.2 channels undergo internalization during the low-frequency stimulation protocol. Contrary to previous reports, the plasticity we report here is transient (lasting 86 min), with EPSPs returning to baseline values within a single stimulus in some cases. Although the mechanism by which the de-potentiation rapidly occurs is not clear to us, one can speculate that the removal of AMPARs from the membrane might be performed by internalization of vesicles containing sizeable amount of AMPARs. A vesicular de-potentiation is in line with the native packaging of AMPARs (Herring and Nicoll, 2016; Kneussel and Hausrat, 2016) and with leading theories explaining the mechanisms by which AMPARs are increased during conventional LTP (Herring and Nicoll, 2016). The frequency of activation that induced this form of plasticity was used as control stimulation in many LTP experiments in the past including by our group (Magee and Johnston, 1997; Markram et al., 1997; Sjo¨stro¨m et al., 2001; Froemke and Dan, 2002; Gordon et al., 2006), which raises the question why it was not observed previously. A possible explanation is the fact that we did not observe this form of plasticity in basal dendrites, which receive the vast majority of synaptic contacts in layer 5 pyramidal neurons (Larkman, 1991). Thus, due to the proportion of innervation of tuft dendrites compared to basal dendrites and due to the extreme attenuation of tuft inputs, it is conceivable that most synaptic activity recorded in most previous LTP experiments is almost not representing tuft synapses. It is still unclear whether oblique dendrites, which form an important processing compartment in layer 5 pyramidal neurons (Schaefer et al., 2003; Ferrante

Please cite this article in press as: Sandler et al., A Novel Form of Local Plasticity in Tuft Dendrites of Neocortical Somatosensory Layer 5 Pyramidal Neurons, Neuron (2016), http://dx.doi.org/10.1016/j.neuron.2016.04.032

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 mm from the soma) and dendritic recording electrode (red electrode; 860 mm 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 (DF/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 (DF/F % ± SEM) evoked by BCaS plotted as a function of the distance from the center of stimulated dendritic segment (0) in 20-mm segments, from proximal (to soma; negative values) to distal (from soma; positive values) locations. (F) Summary plot of % change (±SEM) in BPCaSevoked calcium transients at stimulated dendritic locations (n = 10 cells) and at sister unstimulated dendrites (all ROIs along sister dendrites; n = 7 cells; p = 0.00021 between stimulated and unstimulated dendrites). (G) Summary plot of mean BPCaS-evoked calcium transients (DF/F % ± SEM) in the presence of the Kv4.2 blocker, heteropodatoxin-2 (0.5 mM), during 0.03 Hz and 0.1 Hz stimulation.

et al., 2013), also undergo the low-frequency form of plasticity or whether they resemble more-basal dendrites. As little is known about the potassium channel composition in these dendrites, further studies are needed to specifically test their potassium channel composition and the possible occurrence of the low-frequency plasticity. The Tuft as a Unique Integration and Plasticity Compartment Tuft dendrites of layer 5 pyramidal neurons form a separate dendritic compartment both with regard to its intrinsic active

properties and the inputs it receives, rendering this dendritic compartment as a prime site for integrating top-down stream of information with bottom-up information (Siegel et al., 2000; Larkum, 2013). In addition, they are strategically situated to convey attention and state of alertness (Cauller and Connors, 1994; Cauller et al., 1998; Laberge and Kasevich, 2007; Kuhn et al., 2008). An important problem tuft dendrites need to solve is their poor access to other dendritic compartments and soma because they form a relatively isolated compartment with regard to propagation of both forward and backward voltage signals. BAPs and Neuron 90, 1–15, June 1, 2016 11

Please cite this article in press as: Sandler et al., A Novel Form of Local Plasticity in Tuft Dendrites of Neocortical Somatosensory Layer 5 Pyramidal Neurons, Neuron (2016), http://dx.doi.org/10.1016/j.neuron.2016.04.032

BCaS are markedly attenuated in distal tuft dendrites (Larkum et al., 2009; Harnett et al., 2013; Figures 5 and 7 of this study). Forward-propagating EPSPs also severely attenuate as they propagate toward the soma. The branch-specific plasticity of excitability we describe here provides an efficient mechanism for modulating the coupling efficiency of specific tuft branches with other dendritic compartments and soma. Activated tuft dendrites will better sense activation of inputs arriving to moreproximal dendritic compartments via BAPs and BCaS, and tuft inputs of activated branches will be better conveyed to moreproximal regions due to the potentiation of the EPSPs. Tuft dendrites are selectively enriched with both Ih (Berger et al., 2001) and Kv4.2 potassium channels (Burkhalter et al., 2006), which endow them with unique integrative and plasticity mechanisms. Further studies are needed to elucidate the interactions between Kv4.2 and H channels in general and the lowfrequency tuft potentiation in particular. Compartmentalized Plasticity Mechanisms Here, we show that plasticity of synaptic inputs and dendritic excitability is restricted to the activated dendritic tuft segments. In general, branch-specific plasticity can increase considerably the amount of information storage represented by single neurons and ultimately by the network (Poirazi et al., 2003a, 2003b; Kastellakis et al., 2015). A leading theory is that the anatomical dendritic structure combined with the nonlinear properties of thin dendrites and especially the ability to generate local NMDA spikes enables pyramidal neurons to perform local computations and storage of information in small dendritic sub-compartments (Mel, 1992; Polsky et al., 2004; Branco and Ha¨usser, 2010; Major et al., 2013). The size and location of these dendritic compartments are not fixed and can dynamically change according to the activation and input pattern (Branco et al., 2010; Behabadi et al., 2012; Jadi et al., 2012; Major et al., 2013). Recent studies support the notion of anatomical and functional clustering of synapses to dendritic compartments both during development and learning (Fu et al., 2012; Takahashi et al., 2012; DeBello et al., 2014; Druckmann et al., 2014; Cichon and Gan, 2015), in line with the possibility that dendritic branchlets may serve as key plasticity and storage elements during learning (Mel, 1992; Poirazi and Mel, 2001; Chklovskii et al., 2004). In the present study, we show a key mechanism available in tuft dendrites that can open a prolonged ‘‘plasticity time window’’ in activated dendritic segments. The spatial extent of plasticity depends on the activation pattern spanning from a small dendritic segment to a more-distributed and global change. Possible Physiological Significance An important question is whether the low activation frequencies that cause dramatic plasticity changes in tuft dendrites are within the physiological range of activation of tuft synapses in layer 5 pyramidal neurons in vivo. Layer 2 and 3 neurons from both the same barrel as well as from other cortical regions such as M1 and M2 are a major source of inputs to the apical tuft dendrites of layer 5 neurons in the barrel cortex (Murayama and Larkum, 2009; Petreanu et al., 2009; Mao et al., 2011; Manita et al., 2015). The low 0.03– 0.1 Hz activation frequencies we used in our experiments are well within the reported range of activity of layer 2 and 3 pyramidal 12 Neuron 90, 1–15, June 1, 2016

neurons in vivo, which showed sparse and low activity. Around a third of layer 2 and 3 pyramidal neurons in the principle barrel have been shown to be silent (<0.0083 Hz) in awake behaving mice (O’Connor et al., 2010; Peron et al., 2015), and the median response rate during task performance was only 0.18 Hz (O’Connor et al., 2010). Furthermore, layer 2 and 3 neurons only rarely fire in bursts of APs both under spontaneous and evoked activity (de Kock and Sakmann, 2009; O’Connor et al., 2010), thus matching our single EPSP activation protocol. Moreover, the mean amplitude of unitary EPSPs evoked in connected pairs of layer 2 and 3 and layer 5 neurons was 0.3 ± 0.1 mV as recorded at the soma (Reyes and Sakmann, 1999), which is also within the range of EPSP amplitudes used in our study (1.76 ± 0.25 mV locally at tuft dendrites and 0.47 ± 0.05 mV at the soma). We speculate that, under certain physiological conditions such as attentional or neuromodulatory-related cortical state changes or during task-specific activation, firing of layer 2 and 3 neurons may increase to the frequency range of the plasticity reported here. As a result, layer 2 and 3 inputs to the tuft may: (1) self-amplify in a branch-specific manner or in a more-distributed manner according to the spatial activation throughout the tuft tree; (2) in parallel, open a window of increased excitability in the activated dendritic locations, which can enable amplification of incoming feedforward posterior medial thalamic nucleus (POM) thalamocortical and feedback cortico-cortical such as M1 inputs (Xu et al., 2012); (3) enable binding of feedforward inputs (such as VPM thalamocortical inputs), which terminate on more-proximal dendritic locations such as in basal dendrites (Petreanu et al., 2009) with tuft feedback and intracortical inputs (such as M1 and layer 2 and 3) by enabling more-efficient backward-propagating axo-somatic APs and dendritic calcium spikes; and (4) finally, this mechanism may enable long-term storage of information in a branch-specific manner (Cichon and Gan, 2015). The plasticity in excitability and potentiation of EPSPs can be used as a mechanism for learning and storing novel features. Indeed, a branchspecific plasticity in tuft dendrites of layer 5 pyramidal neurons was shown to be crucial for motor learning or during exposure of rats to enriched environment (Makara et al., 2009; Cichon and Gan, 2015). EXPERIMENTAL PROCEDURES Experiments were conducted according to the animal ethics committee of the Technion. Electrophysiology Neocortical sagittal brain slices 300-mm-thick were prepared from 28- to 40-dayold male Wistar rats. Whole-cell patch-clamp recordings were performed from visually identified layer 5 pyramidal neurons using infrared (IR) Dodt-gradient contrast video microscopy. Dendritic recordings were performed from primary, secondary, and tertiary tuft branches (average distance 630 ± 15; 430–860 mm from the soma). The extracellular solution artificial cerebrospinal fluid (ACSF) contained (in mM) 125 NaCl, 25 NaHCO3, 25 glucose, 3 KCl, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2 (pH 7.4) at 35 C–36 C. The intracellular solution contained (in mM) 135 K+-gluconate, 4 KCl, 4 Mg-ATP, 10 Na2-phosphocreatine, 0.3 Na-GTP, 10 HEPES, 0.2 Oregon Green 488 Bapta-1 (OGB-1), 0.2 CF 633, and biocytin (0.2%; pH 7.2). Recording electrodes were made from thick-walled (0.25 mm) borosilicate glass capillaries on a Flaming/Brown micropipette puller (P-97; Sutter Instrument). Dual whole-cell voltage recordings were performed from the soma

Please cite this article in press as: Sandler et al., A Novel Form of Local Plasticity in Tuft Dendrites of Neocortical Somatosensory Layer 5 Pyramidal Neurons, Neuron (2016), http://dx.doi.org/10.1016/j.neuron.2016.04.032

(3–5 MU) and dendrites (10–20 MU). The electrophysiological recordings were performed using Multi-Clamp 700A and 3221A Digidata and acquired using Pclamp 10 (Molecular Devices). Focal Synaptic Stimulation Focal electrical synaptic stimulation at distal tuft dendrites (985 ± 14 mm from the soma) was performed via a theta-glass (borosilicate; Hilgenberg) pipette located in close proximity to the selected dendritic segment guided by the fluorescent image of the dendrite. The theta-stimulating electrodes were filled with CF 633 (Biotium; 0.1 mM). Current was delivered through the electrode at various intensities (3–7 mA) via stimulus isolator (ISO-Flex; AMPI). The efficacy of the stimulation was verified by simultaneous calcium imaging evoked by small EPSPs and their localization to a small segment of the stimulated dendrite. Drug Applications All experiments were performed in the presence of anion gamma-aminobutyric acid (GABAA) (1 mM bicuculline; Sigma) and metabotropic gamma-aminobutyric acid (GABAB) receptor blockers (10 mM CGP-55845) in the ACSF perfusion solution. In some experiments, NMDAR blocker (100 mM APV; Tocris Bioscience) and Kv4.2 subunit channel blocker (0.5 mM heteropodatoxin-2; Alomone) were added to the ACSF perfusion solution 40 min before the start of the recording session. The synthetic dynamin-derived peptide (membrane impermeable DYN 100 mg/ml and Pep1-TGL 100 mM; Tocris) were included in the patch electrode, confining the blockade to the postsynaptic neuron only. Pep1-TGL is a small-peptide-containing (11 amino acids) sequence targeted to the TGL motif found in the C terminus of the GluR1 subunit that binds to PDZ domain proteins (SAP-97), which was shown to be essential for the trafficking of GluR1-containing AMPAR to the membrane following LTP (Hayashi et al., 2000). Imaging Fluorescence confocal microscopy (Olympus FluoView FV1000) was combined with IR-scanning gradient contrast (IR-SGC) (Larkum et al., 2009) mounted on an upright BX61WI Olympus microscope equipped with a 603 (Olympus 0.9 NA) water objective. IR-SGC images were generated by spatially filtering the forward-scattered IR laser light with a Dodt-tube (Luigs&Neumann) and subsequent detection by a photomultiplier tube (R6357; Hamamatsu). Neurons were filled via a proximal apical dendrite (100–180 mm from soma) recording pipette containing the calcium-sensitive dye Oregon Green BAPTA-1 (OGB-1; 200 mM; Invitrogen) and CF 633 (200 mM; Biotium) to visualize the apical tuft dendritic tree. Dendrites were filled for at least 30 min before dendritic recordings were established. Fluorescence and IR-SGC images were acquired simultaneously and could be overlaid online, resulting in a full-frame rate of 5 Hz. Calcium transients were recorded in line-scan mode at 500 Hz. Data Analysis Electrophysiological data analysis was performed using Clampfit 10.3 (Axon Instruments), Igor Pro software (Wavemetrics), and a homemade Matlab software. Typically, the mean value of EPSP amplitudes was calculated for 15 EPSPs at the beginning of 0.03 Hz stimulation frequency (baseline values) and at 0.1 Hz after the EPSP amplitude was stabilized (postinduction values). When blockers were added, the EPSP amplitude was calculated at least 20 min after 0.1 Hz stimulation. Calcium images were analyzed using homemade software and Igor software. Calcium transients are presented as % DF/F. Statistical tests were performed using Excel software (Microsoft). Data are presented as average ± SEM. Computer Simulations Computer simulations were performed on biocytin-filled and reconstructed cells from our experimental study as previously described (Larkum et al., 2009); see Supplemental Experimental Procedures. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and four figures and can be found with this article online at http://dx.doi.org/ 10.1016/j.neuron.2016.04.032.

AUTHOR CONTRIBUTIONS J.S. designed and participated in the experiments and wrote the manuscript. M.S. conducted the experiments, analyzed the data, and participated in writing the manuscript. Y.S. contributed experiments to Figures 6 and S3. ACKNOWLEDGMENTS We thank Y. Schiller for helpful discussions. We thank Irena Reiter for excellent technical assistance and processing the biocytin-filled neurons. We thank Oded Schiff and Uri Dubin for assistance in analysis software. This study was supported by Israeli Science Foundation (to J.S.), the Rappaport Foundation (to J.S.), Prince Center, and by the Adelis Fund for Brain Research at the Technion (to J.S.). Received: October 25, 2015 Revised: March 24, 2016 Accepted: April 7, 2016 Published: May 19, 2016 REFERENCES Almog, M., and Korngreen, A. (2014). A quantitative description of dendritic conductances and its application to dendritic excitation in layer 5 pyramidal neurons. J. Neurosci. 34, 182–196. Anwyl, R. (2009). Metabotropic glutamate receptor-dependent long-term potentiation. Neuropharmacology 56, 735–740. Behabadi, B.F., Polsky, A., Jadi, M., Schiller, J., and Mel, B.W. (2012). Location-dependent excitatory synaptic interactions in pyramidal neuron dendrites. PLoS Comput. Biol. 8, e1002599. Berger, T., Larkum, M.E., and Lu¨scher, H.R. (2001). High I(h) channel density in the distal apical dendrite of layer V pyramidal cells increases bidirectional attenuation of EPSPs. J. Neurophysiol. 85, 855–868. Branco, T., and Ha¨usser, M. (2010). The single dendritic branch as a fundamental functional unit in the nervous system. Curr. Opin. Neurobiol. 20, 494–502. Branco, T., Clark, B.A., and Ha¨usser, M. (2010). Dendritic discrimination of temporal input sequences in cortical neurons. Science 329, 1671–1675. Burkhalter, A., Gonchar, Y., Mellor, R.L., and Nerbonne, J.M. (2006). Differential expression of I(A) channel subunits Kv4.2 and Kv4.3 in mouse visual cortical neurons and synapses. J. Neurosci. 26, 12274–12282. Cai, X., Liang, C.W., Muralidharan, S., Kao, J.P., Tang, C.M., and Thompson, S.M. (2004). Unique roles of SK and Kv4.2 potassium channels in dendritic integration. Neuron 44, 351–364. Cauller, L.J., and Connors, B.W. (1994). Synaptic physiology of horizontal afferents to layer I in slices of rat SI neocortex. J. Neurosci. 14, 751–762. Cauller, L.J., Clancy, B., and Connors, B.W. (1998). Backward cortical projections to primary somatosensory cortex in rats extend long horizontal axons in layer I. J. Comp. Neurol. 390, 297–310. Chklovskii, D.B., Mel, B.W., and Svoboda, K. (2004). Cortical rewiring and information storage. Nature 431, 782–788. Cichon, J., and Gan, W.B. (2015). Branch-specific dendritic Ca(2+) spikes cause persistent synaptic plasticity. Nature 520, 180–185. de Kock, C.P., and Sakmann, B. (2009). Spiking in primary somatosensory cortex during natural whisking in awake head-restrained rats is cell-type specific. Proc. Natl. Acad. Sci. USA 106, 16446–16450. DeBello, W.M., McBride, T.J., Nichols, G.S., Pannoni, K.E., Sanculi, D., and Totten, D.J. (2014). Input clustering and the microscale structure of local circuits. Front. Neural Circuits 8, 112. Druckmann, S., Feng, L., Lee, B., Yook, C., Zhao, T., Magee, J.C., and Kim, J. (2014). Structured synaptic connectivity between hippocampal regions. Neuron 81, 629–640. Edelmann, E., Cepeda-Prado, E., Franck, M., Lichtenecker, P., Brigadski, T., and Leßmann, V. (2015). Theta burst firing recruits BDNF release and signaling

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