Article
Adrenergic Modulation Regulates the Dendritic Excitability of Layer 5 Pyramidal Neurons In Vivo Graphical Abstract
Authors Christina Labarrera, Yair Deitcher, Amir Dudai, Benjamin Weiner, Adi Kaduri Amichai, Neta Zylbermann, Michael London
Correspondence
[email protected]
In Brief Labarrera et al. show that noradrenergic neuromodulation can be an effective way to regulate the interaction between different input streams of information processed by an individual neuron. These findings may have important implications for our understanding of how adrenergic neuromodulation affects sensory integration, attention, and working memory.
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Blocking Ih increases apical tuft excitability in L5 pyramidal neurons in vivo a2A adrenoceptor agonist reduces Ih in L5 pyramidal neurons a2A adrenoceptor agonist increases apical tuft excitability in L5 pyramidal neurons Modeling shows that adrenergic modulation of Ih effectively regulates tuft excitability
Labarrera et al., 2018, Cell Reports 23, 1034–1044 April 24, 2018 ª 2018 https://doi.org/10.1016/j.celrep.2018.03.103
Cell Reports
Article Adrenergic Modulation Regulates the Dendritic Excitability of Layer 5 Pyramidal Neurons In Vivo Christina Labarrera,1,2 Yair Deitcher,1,2 Amir Dudai,1 Benjamin Weiner,1 Adi Kaduri Amichai,1 Neta Zylbermann,1 and Michael London1,3,4,* 1Edmond and Lily Safra Center for Brain Sciences and Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel 2These authors contributed equally 3Twitter: @mikilon 4Lead Contact *Correspondence:
[email protected] https://doi.org/10.1016/j.celrep.2018.03.103
SUMMARY
The excitability of the apical tuft of layer 5 pyramidal neurons is thought to play a crucial role in behavioral performance and synaptic plasticity. We show that the excitability of the apical tuft is sensitive to adrenergic neuromodulation. Using two-photon dendritic Ca2+ imaging and in vivo whole-cell and extracellular recordings in awake mice, we show that application of the a2A-adrenoceptor agonist guanfacine increases the probability of dendritic Ca2+ events in the tuft and lowers the threshold for dendritic Ca2+ spikes. We further show that these effects are likely to be mediated by the dendritic current Ih. Modulation of Ih in a realistic compartmental model controlled both the generation and magnitude of dendritic calcium spikes in the apical tuft. These findings suggest that adrenergic neuromodulation may affect cognitive processes such as sensory integration, attention, and working memory by regulating the sensitivity of layer 5 pyramidal neurons to top-down inputs. INTRODUCTION Neuromodulation is a fundamental process by which a handful of neurotransmitters and numerous peptides can profoundly alter the behavior of individual neurons, neuronal networks, neural systems, and, consequently, the behavior of the organism. The noradrenergic system is one such important neuromodulatory system. Norepinephrine (NE) in the CNS plays key roles in many executive functions, such as attention and working memory, as well as in sensory processing and coding (Hurley et al., 2004; Arnsten and Li, 2005; Aston-Jones and Cohen, 2005; Briand et al., 2007; Sara, 2009; Sara and Bouret, 2012; Chamberlain and Robbins, 2013). Mechanistically, adrenoceptors have been identified as G protein-coupled receptors (GPCRs), and their downstream pathways are well characterized. However, our understanding of how these processes affect synaptic
integration in individual neurons and in the neuronal network is still lacking. In the cortex, the excitable processes in the dendrites of pyramidal neurons have been demonstrated to play fundamental roles in synaptic integration and plasticity and to have major effects on perception and behavior (Stuart and Sakmann, 1994; Schiller et al., 1997; Larkum et al., 1999b; 2009; Branco et al., 2010; Jia et al., 2010; Xu et al., 2012; Smith et al., 2013; Cichon and Gan, 2015; Palmer et al., 2016; Takahashi et al., 2016; Li et al., 2017). A salient example for such excitable dendritic mechanisms is Ca2+-dependent active currents in the apical tuft of layer 5 pyramidal cells (L5PCs), which are the main carriers of the dendritic Ca2+ spike. In vitro studies have shown a strong effect of a2-adrenoceptor family agonists (mainly clonidine and guanfacine) on the integration properties of pyramidal neurons (Barth et al., 2008; Sheets et al., 2011), and in vivo guanfacine (an a2A-adrenoceptor agonist) application has been shown to enhance persistent activity in the prefrontal cortex (PFC) and improve working memory (Wang et al., 2007). With the importance of active dendritic integration in mind, and encouraged by these findings, we set out to test whether these effects could stem from NE action on dendritic active mechanisms. Because in in vitro studies the ambient level of the neuromodulator is unknown, and in in vivo studies dendritic excitability has not been investigated, we explored the baseline tuft Ca2+ activity and excitability in vivo in the awake state. We further tested whether dendritic Ca2+ events in the tuft are affected by an increase in the concentration of the adrenergic modulator and investigated the underlying mechanism. To this end, we performed two-photon imaging from dendrites and intracellular recordings from L5PCs in the somatosensory cortex of awake mice. RESULTS Increased Ca2+ Activity in the Apical Tuft of L5PCs following Application of Guanfacine To test the effect of adrenergic modulation on dendritic activity, we have expressed the genetically encoded Ca2+ indicator GCaMP6s (Xu et al., 2012; Chen et al., 2013) using adeno-associated virus (AAV) vectors in a sparse subpopulation of L5PCs in
1034 Cell Reports 23, 1034–1044, April 24, 2018 ª 2018 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Figure 1. a2A-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 mm). (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 mm). (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 a2A-adrenoceptor agonist). (H) Histograms of all DF/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 DF/F values larger than 25%. (I) The area under the tail in the histograms of all ROIs is significantly different under both conditions.
the somatosensory cortex (Figure 1A; Experimental Procedures). The sparse expression enabled identification of individual L5PC dendrites by tracing them with two-photon imaging from layer 5 up to layer 1 (Figure 1B). To estimate the baseline Ca2+ activity in the tuft, we measured dendritic Ca2+ transients in an awake head-restrained mouse from a plane of focus above the region of the main bifurcation point of L5PCs under baseline conditions (Figures 1B and 1C) (depth from the pia between 200 and 350 mm, n = 41 imaging regions of interest [ROIs] from 3 mice). The recordings showed Ca2+ transients in some dendrites, as can be seen in Figures 1D and 1E. Ten minutes after obtaining baseline activity, we replaced the solution over the craniotomy with an artificial cerebrospinal fluid (ACSF) solution containing 5 mM guanfacine (an a2A-adrenoceptor agonist) (Wang et al.,
2007; Sheets et al., 2011). We then continued to measure Ca2+ activity in the dendrites. Figures 1F and 1G depict examples of DF/F traces, recorded 10 min after the application of guanfacine from multiple ROIs that cover dendritic branches of L5PCs. These data indicate an increase in the frequency and amplitude of Ca2+ transients following the application of guanfacine. To quantify the effect of guanfacine on Ca2+ activity in the dendrites, we computed the histograms of DF/F for 100-s segments of the experiment before and after the application of guanfacine (Figure 1H). A rightward shift in the amplitude histogram is observed following the application of guanfacine, indicating an increase in the number of high-amplitude events. We measured the area under the histogram at DF/F values larger than 25% (Figure 1H, gray rectangle) and compared this area for the two experimental conditions. Figure 1I shows the comparison for the population (n = 41 ROIs; before application, 0.05% ± 0.02%; after application, 0.19% ± 0.03%; p < 0.0005, paired t test), indicating a significant change in Ca2+ activity before and after the application of guanfacine. The results show that activation of a2A-adrenoceptors increases the Ca2+ activity in the apical dendrites of L5PCs. To control for the possibility that the changes in dendritic activity are due to a time-dependent effect rather than the effect of guanfacine, we repeated the experiment without adding the drug (Figures S1A and S1B) and observed no significant change in dendritic Ca2+ activity over the same duration (10-min gap). Additional control experiments in anesthetized mice have also shown a similar effect of guanfacine (Figures S1C and S1D). Furthermore, in dendrites that show elevated dendritic Ca2+ following the application of guanfacine, co-application of guanfacine with yohimbine (an a2-adrenoceptor antagonist) significantly reduced dendritic Ca2+ activity (Figures S1E and S1F). We performed another set of experiments to reduce the long delay imposed by diffusion of the drug and establish a more direct relationship between a2A-adrenoceptors agonist application and the observed Ca2+ dendritic signals. A glass pipette loaded with red fluorescent dye (Alexa 594) was visually guided and placed near the main bifurcation of L5PC apical dendrites (Figures 2A and 2B) of lightly anesthetized mice (0.75% isoflurane, n = 3 mice). In addition, the pipette solution contained either ACSF alone or ACSF with 10 mM guanfacine (solutions were blinded). Application of a 100-millibar (mbar) increase in pressure to the pipette for 2 s resulted in a spread of the red indicator (Figures 2B and 2C) and was followed by an increase in fluorescence of the Ca2+ indicator from nearby dendrites only in cases where guanfacine was included in the solution but not otherwise (Figures 2B–2E). Dendrites that were completely invisible before the application became very bright. Similar to the data in Figure 1, the area under the rightward tail of the fluorescence histogram was significantly higher for cases where guanfacine was included in the pipette (Figure 2D; n = 29 ROIs; before application, 0.15% ± 0.02%; after application, 0.54% ± 0.05%; p < 0.0001, paired t test) than for cases where it was not included (Figure 2E; n = 33 ROIs; before application, 0.25% ± 0.03%; after application, 0.2% ± 0.03%; p > 0.2, paired t test). To validate that the pressure application had not caused the drug to reach the soma in layer 5, altering somatic spiking (and, thereby, tuft dendritic spiking), we measured the fluorescence in the red channel (Alexa) as a function of distance from the pipette tip and showed that it decayed to Cell Reports 23, 1034–1044, April 24, 2018 1035
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(A) Left: schematic illustration of the two-photon Ca 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 mm). One ROI from the red channel (dashed red circles) and additional three ROIs from the green channel (solid colored circles) are shown. (C) Top: DF/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: DF/F traces of the green channel in three ROIs (solid colored circles in B). (D) Left: histograms of all DF/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).
baseline within 100 mm (Figure S2). These data further support the conclusion that the dendrites of the apical tuft of L5PCs increase Ca2+ activity upon activation of a2A-adrenoceptors. Application of Guanfacine Reduces the Threshold for Dendritic Excitability What causes the increase in Ca2+ signals in dendrites following activation of a2A-adrenoceptors? One possibility is that activation of a2A-adrenoceptors causes an increase in dendritic excitability, which would lead to an increase in Ca2+ signals. To further explore whether application of guanfacine affects dendritic excitability, we performed somatic whole-cell intracellular recordings from L5PCs in the somatosensory cortex of awake head-restrained mice (Figures 3A and 3B). To measure dendritic excitability, we used the critical frequency (CF) protocol proposed by Larkum et al. (1999a). This protocol enables probing the dendritic excitability from somatic recordings and was successfully applied in vitro (Berger et al., 2003; Boudewijns et al., 2013) and in vivo in anesthetized and awake rats (Potez and Lar1036 Cell Reports 23, 1034–1044, April 24, 2018
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Figure 3. a2A-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 mm). (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.
kum, 2008; Boudewijns et al., 2013). A relatively hyperpolarized resting membrane potential was observed upon break-in ( 68.0 ± 2 mV, n = 51 cells), and the membrane potential response to depolarizing current injection steps resembled that of regular spiking neurons (Figures 3C and 3D); however, neurons often emitted bursts of APs riding on strong depolarization from spontaneous synaptic inputs. An example of the protocol is depicted in Figures 3E–3G. We injected regular trains of short current pulses (3 ms) at various frequencies into the soma in a random order. We then aligned the recorded membrane potential response with the peak of the last spike in the train and
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Figure 4. Ih Blockage Results in a Similar Effect as that of a2AAdrenoceptor 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 mL 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 DF/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.
integrated the area of the after-depolarization potential (ADP) in the time window between 5 and 10 ms following the peak of the last spike in the train (Figure 3G; Figure S3). Figure 3H demon-
strates that the resulting value shows a sigmoidal relationship with the frequency of the input current impulse train, implying a threshold for generating Ca2+ spikes in the dendrites. We denote CF as the frequency at which the sigmoid is at 50% of the maximum value. Figure 3I shows the distribution of the CF across all neurons under control conditions (CF = 101 ± 4 Hz, n = 36 cells). This distribution covers a similar range of critical frequencies reported in in vitro studies (98 ± 6 Hz) (Larkum et al., 1999b). For some of the neurons, we were successfully able to apply the CF protocol before and after application of guanfacine (Figure 3J). Application of guanfacine resulted in a significant drop in CF (CFbefore = 106 ± 7 Hz versus CFguan = 81 ± 5 Hz, n = 6, p < 0.05, paired t test). These results support the hypothesis that activation of the a2A-adrenoceptors increases the excitability of the apical tuft and also increases coupling between the soma and the apical tuft. Consequently, it is expected that application of guanfacine should increase the spontaneous rate of action potential (AP) bursts. To test this, we used a 16-site extracellular silicon probe and recorded spontaneous activity from layer 5 in awake head-restrained mice before and after topical application of 5 mM guanfacine (Supplemental Experimental Procedures). Figures S3E–S3G show a significant increase in bursting rate (0.028 ± 0.01 Hz under control conditions and 0.079 ± 0.01 Hz 10 min after guanfacine application; n = 76 units from 7 experiments, 4 mice; p = 0.0024, paired t test). Blocking Ih Using ZD7288 Results in Similar Effects as Application of Guanfacine Several lines of evidence suggest that the mechanism for NE modulation of dendritic excitability involves the hyperpolarization activated cation current Ih. Ih is a key voltage-dependent inward current through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. In L5PCs, HCN channels are concentrated in the apical tuft (Williams and Stuart, 2000; Berger et al., 2001; Lo¨rincz et al., 2002; Kole et al., 2006; Harnett et al., 2015), active at resting membrane potentials, further activated with hyperpolarization, and deactivated with depolarization. This reverse voltage dependence in HCN channels compared with most ion channels suggests a regulating role for Ih in which its main function is to oppose changes in membrane potential (Wahl-Schott and Biel, 2009). In addition to voltage dependence, HCN opening is modulated by the second messenger cyclic AMP (cAMP), which has a specific intracellular binding site on the channel (Wainger et al., 2001). The most prominent feature of Ih is a sag in the membrane potential response to hyperpolarization current injection (Figure 4A). In vitro studies have shown that blocking of Ih has a strong effect on the excitability of the apical dendrite (Berger et al., 2003; Harnett et al., 2015). In addition, other in vitro studies have shown that application of guanfacine and clonidine reduces the sag in membrane potential upon hyperpolarization (Carr et al., 2007; Barth et al., 2008; Sheets et al., 2011; He et al., 2014). We therefore tested whether blocking Ih results in similar effects on dendritic excitability as those described above, which followed application of guanfacine. We applied a whole-cell patch-clamp technique to L5PCs in awake mice similar to those presented in Figure 3. Repeated trials of hyperpolarization long-pulse current injection produced the characteristic sag (Figure 4A), indicating the presence of Ih. Cell Reports 23, 1034–1044, April 24, 2018 1037
At least 50 repetitions for each current were made to overcome the high variability because of spontaneous synaptic activity. Figure 4B depicts the distribution of the sag amplitude in response to injection of 500 pA ( 3 ± 0.2 mV, n = 49 cells), and Figure 4C depicts the distribution of the sag ratio (SR; see definition in Experimental Procedures). The average sag ratio value was 12% of the membrane voltage change (sag ratio, 12% ± 1%; n = 49). To confirm that the sag evoked by negative current injections was due to Ih, we used the Ih blocker ZD7288 (Magee, 1998). HCN channels are densely expressed in the apical tuft, located close to the surface of the brain, and in many L5PCs, the apical tuft could span a large region (up to 500 mm laterally and 350 mm in depth) (Lo¨rincz et al., 2002). We reasoned that the most effective way to apply ZD7288 would be by using topical application. After application of 0.5 mM ZD7288 (Experimental Procedures), we performed an additional series of hyperpolarization current injections from the same neuron and measured the sag. An example of the results of this protocol is shown in Figure 4D, clearly showing a reduction in sag amplitude. Also evident is hyperpolarization of the resting membrane potential and an increase in input resistance. Figure 4D compares the average response before and after the application of ZD7288. In addition, the voltage response after application of ZD7288 is shown shifted and normalized to the response of control, illustrating the reduction in sag ratio. An increase in input resistance is also evident in the population (Figure 4E; Rinctl = 83 ± 7 megaohm [MU] versus RinZD = 102 ± 12 MU, n = 15, p < 0.001, paired t test), combined with hyperpolarization of the membrane potential (Vmctl = 74 ± 4 mV versus VmZD = 89 ± 6 mV, n = 10, p < 0.01, paired t test). Analysis of the population data showed that the sag ratio was significantly reduced by application of ZD7288 (Figure 4F; SRctl = 13% ± 2% versus SRZD = 5% ± 1%, n = 17, p < 0.001). Following the validation that application of ZD7288 reduces Ih under our experimental conditions, we applied the critical frequency protocol before and after application of ZD. Importantly, blocking Ih resulted in a significant drop in the critical frequency (Figure 4G; CFbefore = 97 ± 9 Hz versus CFZD = 69 ± 6 Hz, n = 8, p < 0.05, paired t test). To further confirm that blocking Ih affects Ca2+ activity in the apical tuft, we performed two-photon imaging of Ca2+ from the apical tuft of L5PCs in awake head-fixed mice before and after application of ZD7288. Example Ca2+ traces for two ROIs as well as raster plots for 17 ROIs from the same experiment are presented in Figures 4H and 4I and show that application of ZD7288 results in a significant increase in the Ca2+ activity in the tuft, similar to the results obtained for guanfacine (Figure 1). Analysis of the fluorescent values shows that the rightward tail of the distribution increased significantly under these conditions as well (Figures 4J and 4K; n = 2 mice, 3 experiments, 55 ROIs; before application, 0.05% ± 0.01%; after application, 0.17% ± 0.02%; p < 0.0001, paired t test). Moreover, in two additional experiments, we added guanfacine following application of ZD7288. In the dendrites that increased their activity following ZD7288, we did not observe a significant increase following additional guanfacine application (2 mice, 46 ROIs; tail integral values: control, 0.05 ± 0.009; after ZD, 0.13 ± 0.016; ZD7288 [ZD] + guanfacine [guan], 0.14 ± 0.02, p > 0.1, paired t test between ZD and Guan). 1038 Cell Reports 23, 1034–1044, April 24, 2018
The similarity between the effects observed for the application of guanfacine and for ZD7288 on the drop in critical frequency and on the increase in Ca2+ activity in the apical tuft is consistent with the hypothesis that the effects of guanfacine are at least partially due to modulation of Ih in the apical tuft of L5PCs; however, these two effects could possibly represent two independent mechanisms. Application of Guanfacine Shifts the Activation Curve of Ih toward Hyperpolarized Potential The effects of guanfacine and blocking of Ih on dendritic excitability are similar. Does guanfacine modulate Ih? To test this, we performed another set of recordings (in awake mice) and measured the sag before and after application of guanfacine in a similar manner to that used in the experiment presented above with ZD7288 (Figure 4). We first applied repeated hyperpolarization current injections of various amplitudes and measured the sag under control conditions (Figure 5A, control). We repeated this protocol after application of 5 mM guanfacine. For a current of 300 pA, the membrane potential response resulted in a very small sag (Figure 5A, top purple trace). However, for larger-amplitude current injections ( 400 pA and 500 pA), the sag amplitude was measurable (Figure 5A, bottom two purple traces). Analysis at the population level of the sag obtained for the larger current amplitude ( 500 pA) measured before and after application of guanfacine revealed that guanfacine application reduced the Ih sag ratio (Figure 5B; SRctl 500pA = 11% ± 1% versus SRguan 500pA = 8% ± 1%, n = 19, p < 0.05, paired t test). This reduction of sag ratio suggests lower availability of Ih after application of guanfacine. In addition to the effect on sag ratio, a change in time course of the sag was evident; the sag time course after application of guanfacine was slower. Analysis of the rise and decay times of the sag response showed a clear increase in both parameters after application of guanfacine (Figure 5C; Risectl = 34 ± 2 ms versus Riseguan = 43 ± 3 ms; Decayctl = 31 ± 2 ms versus Decayguan = 40 ± 3 ms, n = 17, p < 0.01 in both). The application of guanfacine also caused a small but significant increase in input resistance (difference, 7 ± 2.5 MU, n = 25, p = 0.008, paired t test). These results are in line with the hypothesis that both guanfacine and ZD7288 affect Ih, but although ZD7288 application blocks Ih (by blocking the HCN channels), guanfacine appears to modulate the current. For small hyperpolarization current injections, Ih appears to be blocked, but further hyperpolarization can open the current, although to a lesser degree than in the control. These results are consistent with a shift of the activation curve of Ih toward more hyperpolarized membrane potential values. We used the Zap protocol to further explore the effect of guanfacine on the properties of Ih (Lampl and Yarom, 1997; Hutcheon and Yarom, 2000; Narayanan and Johnston, 2008; Sheets et al., 2011). The method is based on a current clamp protocol in which a chirp sinusoidal current (with instantaneously increasing frequency) is injected into the neuron. The input impedance is then estimated by dividing the amplitude frequency spectrum of the voltage response by that of the current. This method has been used to demonstrate that Ih reduces the neuron’s impedance at low frequencies, and, therefore, the resulting impedance
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Figure 5. a2A-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
curve has a resonance frequency (with a peak amplitude obtained for sinusoidal input currents in the range of 4–8 Hz). Figure 5D shows the injected current and an example of the average voltage response from a recorded neuron before and after topical application of 5 mM guanfacine. Only neurons that have demonstrated clear sag under control conditions were selected for this experiment. In response to the injected current, the amplitude of the membrane potential increases and then decreases as a function of frequency. This effect is reduced by application of guanfacine (Figure 5D). Figure 5E plots the impedance as a function of frequency, calculated for one example cell (Experimental Procedures) and clearly shows an impedance curve with a resonance frequency under control conditions and, to a much lesser degree, after application of guanfacine. This result is consistent with previous reports obtained in vitro of the effect of Ih on the impedance curve in a subtype of projection pyramidal neurons in the prefrontal and motor cortex (Dembrow et al., 2010; Sheets et al., 2011). As shown in Figures 5E and 5F, the presence of a resonant frequency under control conditions and its decrease after application of guanfacine were consistent and significant for the population (p < 0.05, t test; n = 14 control, n = 6 guanfacine; 6 cells were recorded under both conditions). The data from Figure 5E are useful to obtain a rough estimate of the degree by which guanfacine shifts the activation curve of Ih toward more hyperpolarized values. To obtain this estimate, we used a model of a single isopotential cell. The model is based on two measured variables from the experiment: the change in the direct current (DC) input impedance Zin(0) and the slope of the impedance curve at high frequencies. These two measures are sufficient to uniquely define a simplified isopotential model and determine the shift of the activation curve (Supplemental Experimental Procedures). Figures 5G and 5H show traces obtained from the model using the same current injected in the experiment. However, comparison of the impedance curve from the experiment with that of the model (Figure 5I) shows a significant discrepancy so that the model could not account for the relative amplitude of the observed resonance (compare solid and dashed black curves). We therefore used a full compartmental model containing only a passive membrane and Ih (Experimental Procedures). The full model accounted better for the observed resonance (Figure 5J); based on this model, we estimate that a shift of 15 mV is required to achieve agreement between the models and the data. Together, the results presented in Figures 4 and 5 are consistent with the hypothesis that a2A-adrenoceptor activation modulates Ih in the apical tuft of L5PCs by shifting the activation
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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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.
curve toward a more hyperpolarized potential (Biel et al., 2009). This shift of the activation curve reduces the effective available Ih in the tuft, resulting in an increase in the excitability of the apical tuft. Functional Implications for Neuromodulation of Apical Tuft Excitability in L5PCs What is the quantitative relationship between the modulation of Ih and the excitability of apical tufts? Pharmacological application of ZD7288 or guanfacine provides only a few reference points and, thus, makes it difficult to evaluate this relationship. An important question is whether neuromodulation by NE of Ih acts as an on-off switch for the excitability of the apical tuft or whether it allows a gradual change. To answer this question, we used a solid compartmental model that replicates most of 1040 Cell Reports 23, 1034–1044, April 24, 2018
the known electrical behaviors of L5PCs (Hay et al., 2011; Shai et al., 2015; Figure 6A). Minimal modifications to the model were made so that it is consistent with recently published data regarding the relationship between Ih and tuft excitability (Harnett et al., 2015). To evaluate the excitability of the tuft, we used a 10-ms 1-nA current injection pulse in the nexus of the apical tree. This perturbation resulted in a small regenerative voltage response (it does not decay passively when the current is terminated; Figure 6B, black curve). When we completely removed Ih from the model (simulating application of ZD7288), it resulted in hyperpolarization of the membrane potential but also in a full Ca2+ spike in response to an identical current, consistent with recently published data (Harnett et al., 2015). A negative shift of the activation curve of Ih by 15 mV (simulating guanfacine application) has an intermediate effect. The resting membrane potential is slightly hyperpolarized, and the peak amplitude of the membrane potential response to the current injection is between the control and the no Ih conditions. Figure 6C shows the peak amplitude of the nexus response for various values of shift in the activation curve of Ih. It shows that even a very small shift of Ih can create a strong effect on the excitability of the tuft. A further shift in the activation curve results in a gradual shift of the peak amplitude in the nexus. Thus, the modulation of Ih has both a non-linear component (for small shifts) and a gradual linear effect for larger shifts. DISCUSSION Layer 5 pyramidal neurons are ideally suited to be the integrators of information in the cortical column because their dendritic tree collects synaptic inputs throughout the whole range of cortical layers, and their apical tuft receives inputs from axons carrying brain-wide contextual information (Stuart and Spruston, 2015). The attenuation associated with these remote electrotonic locations were thought to limit the contribution of tuft synapses to synaptic integration (Rall, 1959; Zador et al., 1995); however, electrical and optical dendritic recordings have shown that the local integration in the apical tuft involves excitable mechanisms (e.g., branch-specific N-Methyl-D-aspartic acid (NMDA) spikes and global Ca2+ spikes) that may boost the efficacy of these inputs (Amitai et al., 1993; Schiller et al., 1997; Larkum et al., 1999b, 2009). Recently it has been demonstrated that tuft excitability affects behavioral performance (Xu et al., 2012; Manita et al., 2015; Yang et al., 2016; Palmer et al., 2016; Takahashi et al., 2016). These studies suggest that L5PCs are composed of at least two distinct compartments (Figure 6D): the somatic compartment, which integrates feedforward and recurrent input in the deep layers, and the apical tuft compartment, which integrates top-down feedback input arriving from many sources to the upper layers. The coupling between these two compartments enables the interaction between feedforward (e.g., sensory) information and feedback information (e.g., expectations), which is essential for cognitive processes. This interaction is achieved through the back propagation of the somatic action potential and of the massive depolarization created in the tuft by Ca2+ spikes. It is unclear, however, to what extent the degree of coupling is a static property of the neuron (Schaefer et al., 2003) or might be modulated on relatively short
timescales. Several studies have suggested that dendritic ionic channels such as HCN and voltage dependent potassium channels (Kv) control the magnitude and likelihood of such excitable tuft events (Berger et al., 2003; Kole et al., 2007; Tsay et al., 2007; Barth et al., 2008; Harnett et al., 2013, 2015), but the expression level of these ionic currents is implicitly considered as a static property. Dendritic inhibition is currently the only mechanism proposed for dynamic control of coupling and has been shown to be effective at short timescales (Larkum et al., 1999b; Gidon and Segev, 2012; Palmer et al., 2012; Pe´rez-Garci et al., 2013). Here, we propose that neuromodulation provides another level of control, resulting in varying degrees of coupling for different brain states (Figures 6D–6F). Using two-photon imaging, we find that application of guanfacine (an a2A-adrenoceptors agonist) in awake mice leads to a significant increase in the Ca2+ transients observed in the tuft. Using whole-cell recordings, we measured the critical frequency in the awake state (corresponding to the Ca2+ spike threshold in the apical tuft) and showed that it is sensitive to the application of ZD7288 and guanfacine. We hypothesized that the mechanism by which the a2A-adrenoceptor affects dendritic excitability is through its effect on HCN channels. To test this, we blocked Ih and saw a very similar effect on the tuft’s excitability as that of guanfacine. We then showed that guanfacine application reduces the amplitude and increases the duration of the sag; it increases input resistance and nearly eliminates the resonance in membrane potential response to Zap current injection. All of these results are consistent with the hypothesis that guanfacine affects Ih in vivo in L5PCs. Last, using compartmental modeling, we show that a significant shift in the activation curve of Ih is required to replicate our results and predict that a gradual change in NE levels can result in either a sharp or gradual change in the excitability of the tuft, depending on the initial availability of Ih. Our results are consistent with previous in vitro studies in the PFC and in the motor cortex of rodents, which have shown that adrenergic modulation affects Ih (Sheets et al., 2011; Zhang et al., 2013; Dembrow and Johnston, 2014), Ih affects dendritic excitability (Berger et al., 2003; Harnett et al., 2015), and adrenergic modulation affects dendritic excitability (Carr et al., 2007; Barth et al., 2008). In vivo, NE has been shown to affect synaptic integration (Constantinople and Bruno, 2011; Polack et al., 2013; Schiemann et al., 2015), and optogenetic silencing of locus coeruleus (LC) has shown cognitive impairment (Janitzky et al., 2015). There is only one in vivo study of the effect of NE modulation on Ih (Wang et al., 2007) reported a behavioral effect, but it did not focus on dendritic excitability and could not conclusively measure the effect of guanfacine on Ih because of methodological limitations. The ambient concentration of NE in the cortex is difficult to determine. An important advantage of our in vivo approach is that the baseline level of NE is likely to be in the physiological range when recording in the awake state (before the manipulation). Although it is difficult to be certain of the effective concentration near dendrites because of our perturbation (topical application of the drug), our results at least demonstrate that the system is not saturated in the quiet resting state of the brain, while the animal is awake but not performing a task. We show that, in that regime, there is a place of further activation
of adrenoceptors, which, in turn, may have an effect on dendritic excitability. Future experiments are required to confirm that physiological release of endogenous norepinephrine results in increased dendritic excitability and has behavioral implications. It is important to note that some of our results might be explained by additional mechanisms, such as off-targets effects of guanfacine on excitatory and inhibitory neurons in layer 2/3 of the cortex. Our whole-cell recordings from L5PCs demonstrate that many of the properties of these neurons in the somatosensory cortex, which have previously been described in vitro and in vivo in anesthetized animals, are similarly expressed in the awake state (Margrie et al., 2002; Haider et al., 2013; Yang et al., 2016). In particular, we have shown that Ih is a key ionic current in these neurons, affecting basic properties such as resting membrane potential and input resistance, which are critical for synaptic integration. Our measurements of the sag ratio are consistent with reported values in the motor cortex (Schiemann et al., 2015) and are typically lower than reported values in vitro. Since its discovery in the sinus node of the heart, Ih has been a known target for adrenergic modulation (Brown et al., 1979). Subsequently, HCN channels have been identified as the carriers of Ih, and some of the molecular targets for modulation have also been identified (Gauss et al., 1998; Ludwig et al., 1998). Our results suggest that Ih can be modulated by a negative shift of its activation curve of as much as 15 mV. We note that there is controversy regarding whether this modulation is carried by direct action of cAMP on HCN channels (Wang et al., 2007) or through other pathways, such as PLC-PKC (Carr et al., 2007; Kole et al., 2006; Wahl-Schott and Biel, 2009). In a wider context, other neuromodulators can affect dendritic excitability through Ih or through other mechanisms. An opposite effect, enhancement of Ih by neuromodulation, has been attributed to Serotonin in pyramidal neurons of the cingulate cortex, affecting neuropathic pain (Santello and Nevian, 2015). Dopaminergic neuromodulation has also been demonstrated to enhance Ih in L5PCs of the entorhinal cortex (Rosenkranz and Johnston, 2006). These findings suggest a bidirectional effect of the modulation of Ih and possible ‘‘competition’’ between the effects of different neuromodulators (e.g., NE and Serotonin both innervate sensory areas; Foote and Morrison, 1987). The multiple sources of modulation and their fast time course (Aston-Jones and Cohen, 2005) suggest that the coupling between the perisomatic region and the apical tuft is subject to fast and complex interaction between different types of neuromodulators, receptor sub-types, their spatial distribution on the cell membrane, their efficacy, and their temporal dynamics. Alternatively, neuromodulation could change the gain of a whole population of neurons, as we might expect to happen with a shift in attention (Figure 6F). The past few decades have seen an increase in the understanding of dendritic properties and function. Because some of the major targets of neuromodulation are located on dendrites, we expect that many of the dramatic effects of neuromodulation stem from their effect on specific dendritic computations. Here we have demonstrated this using the adrenergic system (neuromodulation), HCN channels (targets), and Ca2+ spikes in the tuft of layer 5 pyramidal neurons (dendritic mechanism). Cell Reports 23, 1034–1044, April 24, 2018 1041
EXPERIMENTAL PROCEDURES Animals We used C57BL/6 and Rbp4-Cre transgene (GENSAT #RP24-285K21) male mice (8–13 weeks old). The Hebrew University Animal Care and Use Committee approved all experiments. Surgery and Viral Vector Injections Viral vectors were delivered using standard stereotactic injections to L5 in the right somatosensory cortex (2 mm caudal, 3 mm lateral) under isoflurane anesthesia. For sparse labeling of L5 neurons with GCaMP6s (Chen et al., 2013), AAV9.CAG.Flex.GCaMP6s.WPRE.SV40 (Penn Vector Core) was co-injected with diluted (1:100) AAV9.CamKII.Cre (Xu et al., 2012). For the data in Figures S1E and S1F, Rbp4-Cre mice were used, and only AAV9-DIO-GCaMP6s was injected. Two weeks after the virus injection, a 3-mm imaging window (with a laser-drilled hole) and a headpost were installed. For electrophysiology, a 1-mm-diameter craniotomy was made, and the dura was removed under light isoflurane anesthesia (1%–2%). The mouse was habituated on a custom-made walking wheel for 45 min prior to the initiation of recordings. Two-Photon Calcium Imaging from L5 Dendrites Imaging from awake animals was performed with a low-power temporal oversampling (LOTOS) two-photon microscope (LotosScan2015, Suzhou Institute of Biomedical Engineering and Technology; http://english.sibet.cas.cn/) at 920 nm with a Ti:Sapphire laser (Vision II, Coherent, CA) and imaged through a 253, 1.05 numerical aperture (NA) water immersion objective (Olympus, Japan). Full-frame images (600 3 600 pixels) were acquired from apical dendrites of L5PCs expressing GCaMP6s at depths of 150–350 mm below the pia at 40 frames per second (fps). Imaging data for Figure 2 were obtained from a galvo-galvo Sutter movable objective microscope (MOM) two-photon system with MScan software (Sutter Instrument, CA). Images (460 3 240 pixels) were acquired at 7.25 Hz through a 163, 0.8 NA objective (Nikon, Japan). Electrophysiology Whole-cell recordings were performed using ‘‘blind-patching’’ (Margrie et al., 2002). The selection criteria for L5PC were as follows: distance from the pia on the approach axis (at a 60 angle): 946 ± 137 mm, n = 51; firing properties, Ih sag, and input resistance > 30 milliohm (mU) average (avg): 85 ± 34 MU, n = 51. Some neurons were recovered using neurobiotin 488 staining (Vector Laboratories, CA) and two-photon imaging. All recordings were obtained using a Multiclamp 700B amplifier (Molecular Devices, CA) and sampled at 10 kHz (low pass-filtered at 3 kHz), digitized using ITC-18 (HEKA, Germany), and analyzed using Igor Pro (http://wavemetrics.com), Neuromatics/Nclamp (http:// neuromatic.thinkrandom.com), and MATLAB (https://www,mathworks.com). For details of extracellular recordings in Figure S3, see the Supplemental Experimental Procedures.
pass-filtered at 3 Hz. The fluorescence change, DF/F0 (percent) was calculated as (F F0) / F0, where F0 is the mode defined from the histogram of F. In very active traces, F0 was defined as the fifth percentile of F. Spontaneous Ca2+ activity in the dendrites was defined as DF/F0 > 25%. Electrophysiology To assess the degree of Ih, we used the sag ratio: SR = (Vs.s Vmin) / (Vrest Vmin) 3 100 (in percent), where Vmin is the minimal potential achieved at the beginning of the current step, Vrest is the resting membrane potential, and Vs.s is the steady-state potential estimated by the average over the last 100 ms of the pulse (Van Welie et al., 2006). The sag ratio was calculated for an average of 50 repetitions. Decay and rise time of the Ih sag were calculated by fitting an exponential from baseline to trough (decay) and trough to Vs.s of the hyperpolarized injected current (rise time). Impedance measurements using the Zap protocol were performed as described previously (Puil et al., 1986; Gutfreund et al., 1995; Lampl and Yarom, 1997). To improve resolution at low frequencies, the injected Zap current frequency increased logarithmically with time: i_zap(t) = sin(0.196 exp(0.324t / 1,000)). The impedance was computed as Z(f) = O PSD(v(f) / PSD(i(f))), where PSD means power spectrum density (Supplemental Experimental Procedures). Statistical Analysis Statistical significance (p < 0.05) was assessed using a single-tailed Student’s t test, and all data are reported as mean ± SEM unless otherwise specified. A paired-sample t test was used for electrophysiology results comparing before and after drug application in the same cell. To test for a change in spontaneous Ca2+ activities between different conditions, a paired-sample t test composed of all ROIs under both conditions was used. ROIs showing strong correlation (above 0.5 correlation coefficient) before or after application of the drug were treated as redundant, and only one of them was used for the statistical test. Model All simulations were done using the Neuron simulation environment (Hines and Carnevale, 1997). Ih was simulated using the deterministic model by Kole et al. (2006), with a positive shift of 14 mV (to accommodate our uncorrected liquid junction potential values). To explore the effect of shifting of Vhalf, we introduced a variable Vshift for both functions, alpha(V) and beta(V), which resulted in the correct shift in both activation curve and activation time constant. For Figure 6, we modified the published (Shai et al., 2015) L5PC model to fit experimental results of Ih (Harnett et al., 2015; Supplemental Experimental Procedures). SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and three figures and can be found with this article online at https://doi.org/ 10.1016/j.celrep.2018.03.103. ACKNOWLEDGMENTS
Pharmacology In all intracellular electrophysiology experiments, one cell per mouse was used for analysis because application of the pharmacological agents compromised further recordings. 20 mL of 0.5 mM of the Ih blocker ZD7288 (Tocris) and 5 mM guanfacine (Z3777-5MG, G1043-10MG, Sigma-Aldrich) diluted in external solution were applied topically onto the craniotomy for blocking and modulation. For Figure 2, 10 mM guanfacine diluted in external solution was applied via a pipette. Addition of 5 mM and 10 mM guanfacine increased the osmolality of the solution by 16 ± 2.6 and 24.3 ± 4.0 mOsm, respectively). We compared the effect of a high-osmolality (+24 mOsm) external solution with our standard external solution and found no significant difference in Ca2+ activity in the dendrites (3 mice, 3 experiments, 52 ROIs, p > 0.1).
This research was supported by a grant from the Israeli Science Foundation (845/12), the Einstein Foundation, the National Institute for Psychobiology, and the Gatsby Charitable Foundation. C.L. was supported by a fellowship of the Edmond and Lily Safra Center for Brain Science. M.L. is a Sachs Family Lecturer in Brain Science. We thank N. Yayon for assistance with imaging analysis; A. Libster, Y. Nir, Y. Yarom, and I. Segev for fruitful discussions; and I. Goshen, A. Citri, I. Duguid, S. Goodman, and M. Ha¨usser for comments on the manuscript.
Data Analysis Ca2+ Imaging All analyses were performed using ImageJ software (Schneider et al., 2012) and custom-written codes in MATLAB. Movements were corrected using the moco plugin (Dubbs et al., 2016). ROIs in the apical dendrites of L5 pyramidal neurons were manually selected, and fluorescence traces were low
M.L. conceived the project and designed the experiments. C.L. performed all electrophysiology experiments and analysis. Y.D. performed all imaging experiments and analysis. A.D. and Y.D. performed the experiments shown in Figure 2. B.W. performed the extracellular recordings shown in Figure S3. A.K.A. and N.Z. contributed to the experiments shown in Figure S1. M.L. wrote the manuscript with comments from all authors.
1042 Cell Reports 23, 1034–1044, April 24, 2018
AUTHOR CONTRIBUTIONS
DECLARATION OF INTERESTS
Dubbs, A., Guevara, J., and Yuste, R. (2016). moco: Fast Motion Correction for Calcium Imaging. Front. Neuroinform. 10, 6.
The authors declare no competing interests.
Foote, S.L., and Morrison, J.H. (1987). Extrathalamic modulation of cortical function. Annu. Rev. Neurosci. 10, 67–95.
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