Volume 44, Issue 2, Pages 351-364 (October 2004) Unique Roles of SK and Kv4.2 Potassium Channels in Dendritic Integration  Xiang Cai, Conrad W. Liang, Sukuman Muralidharan, Joseph P.Y. Kao, Cha-Min Tang, Scott M. Thompson  Neuron  Volume 44, Issue 2, Pages 351-364 (October 2004) DOI: 10.1016/j.neuron.2004.09.026

Figure 1 Dendritic Plateau Potentials (A) Montage of an Alexa 568-filled CA1 pyramidal cell from a hippocampal slice culture illustrating the sites of photolysis used in this study (white circle in this and subsequent figures). Families of responses elicited with UV pulses of increasing duration (1, 2, 4, 8, 16, and 32 ms) directed at a terminal apical dendrite are shown at left. Note that photolytic EPSPs are elicited from the terminal dendrite with the briefest UV pulses, but that all-or-none plateau-like potentials are elicited with suprathreshold UV pulse durations. Saline contained TTX. (B) In a terminal apical dendrite of another cell, a train of three subthreshold photolytic EPSPs at 20 Hz, elicited with 2 ms laser pulses, summated to elicit a plateau potential that has the same amplitude and duration as a plateau potential elicited with a single 32 ms UV pulse. (C) Responses of distal apical dendrites of CA1 pyramidal cells in an acute hippocampal slice to photolysis of caged glutamate using UV pulses of increasing duration (left) or trains of five stimuli at 10 Hz of increasing intensity from a stimulating electrode placed close to the dendrite (right). In both conditions, EPSPs are elicited with weak stimulation until the threshold for triggering a plateau potential is reached. Subsequent stimuli elicit responses of roughly the same amplitude. These plateau potentials are essentially identical to those elicited with photostimulation from CA1 cell dendrites in cultured hippocampal slices (e.g., [A]). (D) Pairs of plateau potentials elicited from a single terminal apical dendrite with a pair of UV pulses separated by 50 ms (middle) or 100 ms (right) did not show evidence of amplitude summation, unlike the pair of subthreshold photolytic EPSPs shown in (B). Scale bar, 3 mV and 100 ms. (E) Plateau potentials were elicited from two adjacent terminal dendrites using a UV light pulse at one and a brief pressure application of 5 mM glutamate from a second patch pipette at the other. In contrast to the pair of potentials elicited from the same dendrite shown in (D), pairs of plateau potentials at different dendrites did display amplitude summation. Scale bar, 3 mV and 100 ms. Neuron 2004 44, 351-364DOI: (10.1016/j.neuron.2004.09.026)

Figure 2 A Calcium-Activated, TEA-Sensitive Process Terminates Dendritic Plateau Potentials (A) Responses of a terminal apical dendrite to sub- and suprathreshold UV pulses in control saline (left) and after addition of 10 mM TEA (middle). Note that TEA prolonged the duration of the plateau potential but did not affect its amplitude, as shown in the superimposed traces at right. TEA also had no effect on the amplitude or duration of subthreshold photolytic EPSPs. (B) Summary graph of the effects of TEA at various concentrations on the amplitude, duration, and decay time of dendritic plateau potentials (n = 9, 11, and 9 cells at 0.1, 1, and 10 mM, respectively). (C) Dialysis of 5 mM BAPTA from the pipette solution resulted in a progressive increase in the duration of plateau potentials if cells were impaled in control saline (left). In contrast, if cells were impaled in saline containing 10 mM TEA, then the plateau potentials were already prolonged and were not further increased in duration by BAPTA (right). (D) Plot of plateau potential duration as a function of time after impalement with pipettes containing 5 mM BAPTA in control saline or saline containing 10 mM TEA (n = 6 and 11 cells, respectively). Duration is normalized to the duration 5 min after impalement. We conclude that BAPTA prolongs plateau potentials by decreasing their rate of repolarization and that this process is occluded by prior application of TEA. Neuron 2004 44, 351-364DOI: (10.1016/j.neuron.2004.09.026)

Figure 3 Apamin-Sensitive Ca2+-Activated K+ Channels in the Distal Dendrites Are Responsible for Repolarization of Plateau Potentials (A) Responses of a terminal apical dendrite to sub- and suprathreshold UV pulses in control saline (left) and after addition of 100 nM apamin (middle). Note that apamin prolonged the duration of the plateau potential but did not affect its amplitude, as seen in the superimposed traces at right. Apamin also had no effect on the amplitude or duration of subthreshold photolytic EPSPs. The effects of apamin were thus identical to the effects of TEA described above. (B) Apamin also prolonged the duration of plateau potentials elicited in a terminal apical dendrite by a train of three brief, subthreshold UV pulses at 10 Hz. The effects of apamin are thus not due to the prolonged photorelease of glutamate. (C) Summary graph illustrating the effects of apamin alone (black bars) and after subsequent application of 10 mM TEA (white bars) on the decay time and duration of plateau potentials (n = 6 cells) normalized to the response before apamin application. The effects of TEA and apamin were mutually occlusive, thus indicating that all of the effects of TEA on plateau potentials, described above, were mediated by actions at apamin-sensitive Ca2+-activated K+ channels. (D) False color fluorescence image showing the stimulated terminal apical dendrite (green) and the focal application of apamin (1 μM, visualized with the red dye Alexa 568). Focal apamin application produced a prolongation of plateau potentials that was identical to that produced by bath-applied apamin, indicating that the apamin-sensitive channels responsible for its effect are located in the distal dendrites. Scale bar, 20 μm. (E) Summary graph illustrating the effects of focal apamin application (striped bar) and bath-applied blockers of SK-type Ca2+-activated K+ channels (apamin, 100 nM; scyllatoxin, 20 nM; bicuculline methiodide, 10 μM) (black bars), another GABAA receptor antagonist (GABAzine, 10 μM, 1:1000 DMSO) (stippled bar), a BK-type Ca2+-activated K+ channel blocker (charybdotoxin, 30 nM) (white bar), and blockers of the slow AHP (forskolin, 40 μM; 8-Br-cAMP, 10 μM; carbachol, 300 μM; Sp-cAMP, 200 μM) (gray bars) on the duration of plateau potentials in terminal apical dendrites. The pharmacology of the channels responsible for repolarization of the plateau potential are consistent with the properties of recombinant SK2 and SK3 channels (n = 8, 14, 4, 6, 5, 11, 5, 5, 3, and 3 cells, respectively). Neuron 2004 44, 351-364DOI: (10.1016/j.neuron.2004.09.026)

Figure 4 Apamin Increases Ca2+ Influx during Plateau Potentials in the Stimulated Branch but Does Not Affect Compartmentalization (A) Image of Alexa in CA1 cell dendrites and the site of photostimulation. Fluorescence measurements for the stimulated (a) and unstimulated (b) branches were made between the two open arrowheads. Scale bar, 10 μm. The arrowhead that is labeled “s” indicates the direction of the soma. (B) Image of the maximal change in fluo-4 fluorescence after eliciting a plateau potential in the left branch in control saline. The resulting plateau potential and the change in fluo-4 fluorescence over time in the stimulated (a) and unstimulated (b) branches are shown below. (C) Images of the maximal change in fluo-4 fluorescence after eliciting a plateau potential in the left branch after bath application of apamin (same dendrites as in [B]). The resulting plateau potential and the change in fluo-4 fluorescence are shown below. Note that more Ca2+ entered the stimulated branch after apamin, because the plateau potential is prolonged, but that no Ca2+ entered the unstimulated branch before or after apamin. Fluorescence intensity scale in arbitrary units is shown below image. Neuron 2004 44, 351-364DOI: (10.1016/j.neuron.2004.09.026)

Figure 5 Block of SK Channels in Distal Dendrites Facilitates the Triggering of Action Potentials with Dendritic Glutamate Application (A) Plateau potentials were elicited in a terminal dendritic branch and at an apical trunk dendrite before and after bath application of apamin in saline lacking TTX. Action potentials were reliably elicited from the trunk dendrite in control saline, but not in the terminal branch. After apamin, in contrast, an identical UV pulse elicited trains of action potentials from both sites. Fast action potentials elicited from terminal branches had a mean amplitude of 93 ± 6 mV and a duration of 5.3 ± 1.0 ms (n = 5 cells). (B) False color image (left) showing the stimulated terminal apical dendrite (green) and the cloud of Alexa 568 (red) ejected locally from the apamin-containing puffer pipette. Corresponding plateau potentials before (left) and after (right) ejection of apamin are shown at right. Local application of apamin to distal dendrites in the absence of TTX thus facilitated the ability of dendritic excitation to elicit action potential discharge, as did bath application of apamin. Scale bar, 10 μm. Neuron 2004 44, 351-364DOI: (10.1016/j.neuron.2004.09.026)

Figure 6 Block of A-Type K+ Channels with 4-Aminopyridine Potentiates Dendritic Glutamate Responses (A) Subthreshold photolytic EPSPs and plateau potentials are shown before (left) and after (middle) bath application of 5 mM 4-AP. In contrast to apamin or TEA, 4-AP potentiated both responses, as shown in the superimposed traces at right. (B) Summary graphs illustrating the dose dependence of the effects of 4-AP on the peak amplitude, duration, and decay time of the plateau potentials (n = 8, 8, and 19 cells at the three concentrations). (C) Responses of a distal dendrite to brief trains of 2 ms UV pulses at 20 Hz before (left) and after (right) bath application of 5 mM 4-AP. Temporal summation was enhanced by 4-AP, allowing plateau potentials to be elicited with fewer photolytic EPSPs. (D) AMPA receptor-mediated photolytic EPSPs elicited with 2 and 4 ms UV flashes (the smaller and larger responses under each condition) before (left) and after (right) application of 5 mM 4-AP in the presence of 80 μM D,L-AP5. (E) Summary graph illustrating the effects of 5 mM 4-AP on the amplitude and duration of isolated AMPA receptor-mediated photolytic EPSPs (n = 8 cells). Facilitation was not merely dependent on NMDA receptor activation. (F) Plateau potentials elicited before and during application of 5 mM 4-AP. Note the appearance of a second superimposed plateau potential during 4-AP wash-in. These observations suggest that, in the presence of 4-AP, photorelease of glutamate triggered plateau potentials in multiple branches. Neuron 2004 44, 351-364DOI: (10.1016/j.neuron.2004.09.026)

Figure 7 Block of A-Type K+ Channels with 4-Aminopyridine Eliminates Dendritic Compartmentalization (A) Image of Alexa 568 in CA1 cell dendrites and the site of photostimulation. Fluorescence measurements for the stimulated (a) and unstimulated (b) branches were made between the two open arrowheads. Scale bar, 20 μm. The arrowhead that is labeled “s” indicates the direction of the soma. (B) Image of the maximal change in fluo-4 fluorescence after eliciting a plateau potential in the left branch in control saline. The resulting plateau potential and the change in fluo-4 fluorescence over time in the stimulated (a) and unstimulated (b) branches are shown below. (C) Image of the maximal change in fluo-4 fluorescence after eliciting a plateau potential in the left branch after bath application of 5 mM 4-AP. The resulting plateau potential (left) and the changes in fluo-4 fluorescence are shown below. Note that Ca2+ was elevated in unstimulated branches after eliciting plateau potentials with A-type K+ channels blocked. Fluorescence intensity scale in arbitrary units shown below image. Neuron 2004 44, 351-364DOI: (10.1016/j.neuron.2004.09.026)

Figure 8 Focal Application of 4-Aminopyridine to Dendritic Branch Points Eliminates Dendritic Compartmentalization and Facilitates Action Potential Discharge (A) Focal application of 10 mM 4-AP at the site of photolysis in the middle of a terminal dendrite near the site of photostimulation (left) or at its branch point (right) exerted different actions on plateau potentials in saline that contained TTX. Only branch point application produced the significant increase in both amplitude and duration that indicates lack of compartmentalization, as summarized in the graphs below (n = 10 and 5 cells). (B) Focal application of 10 mM 4-AP at the branch point of the stimulated terminal dendrite facilitated the induction of fast action potentials in the absence of TTX. Neuron 2004 44, 351-364DOI: (10.1016/j.neuron.2004.09.026)

Figure 9 Kv4.2 Channels Mediate Dendritic A-Type K+ Current and Dendritic Compartmentalization (A) Glutamate responses elicited with UV pulses of increasing duration at terminal dendrites in a cell transfected with the dominant-negative A-type potassium channel Kv4.2W362F (left) and a cell transfected with wild-type Kv4.2 channels (right). Note that glutamate responses and plateau potentials are potentiated in the cell in which Kv4.2W362F was overexpressed, whereas overexpression of wild-type Kv4.2 channels decreased subthreshold photolytic EPSPs to almost undetectable amplitudes, increased the threshold UV pulse duration necessary to trigger a plateau potential, and decreased plateau potential amplitude. Plateau potential amplitude and duration were significantly greater in the Kv4.2W362F-transfected cells than in the cells overexpressing wild-type Kv4.2 (p < 0.001 and p < 0.05, for 32 ms UV pulses; n = 8 Kv4.2W362F and 6 wild-type cells). (B) DsRed image of the dendritic tree, demonstrating that the recording was made from a transfected cell, and site of photolysis (upper panels) with the corresponding maximal increase in fluo-4 fluorescence after photostimulation in CA1 cells transfected with dominant-negative Kv4.2W362F channels (left column) or wild-type Kv4.2 channels (right column) before and after application of 5 mM 4-AP. Scale bar, 20 μm. The arrowhead labeled “s” indicates the direction of the soma. Neuron 2004 44, 351-364DOI: (10.1016/j.neuron.2004.09.026)