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Research Articles: Systems/Circuits
Dendritic A-current in Rhythmically Active PreBötzinger ComplexNeurons in Organotypic Cultures from Newborn Mice
Wiktor S. Phillips1,2, Christopher A. Del Negro2 and Jens C. Rekling1
1Department of Neuroscience, University of Copenhagen, Panum Building 33.3.84, Blegdamsvej 3, DK-2200Copenhagen N, Denmark.2Department of Applied Science, Integrated Science Center, 540 Landrum Dr., The College of William and Mary,Williamsburg, Virginia, United States.
DOI: 10.1523/JNEUROSCI.3342-17.2018
Received: 26 November 2017
Revised: 22 January 2018
Accepted: 9 February 2018
Published: 19 February 2018
Author contributions: W.P., C.A.D.N., and J.R. designed research; W.P. and J.R. performed research; W.P.,C.A.D.N., and J.R. analyzed data; W.P., C.A.D.N., and J.R. wrote the paper.
Conflict of Interest: The authors declare no competing financial interests.
This work was supported by The Danish Research Council, The Lundbeck Foundation, Mindefonden for AliceBrenaa, Den Owensenke Fond, Lægeforeningens forskningsfond, Agnes og Pouls Friis Fond, Fonden tilLægevidenskabens Fremme, Brødrene Hartmans fond, as well as the U.S. National Institutes of Health (R01HL104127 PI: Del Negro). We thank Morten Bjerre Nielsen for technical assistance.
Corresponding author: Jens C. Rekling, Department of Neuroscience, University of Copenhagen, PanumBuilding 33.3.84, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. Email: [email protected]
Cite as: J. Neurosci ; 10.1523/JNEUROSCI.3342-17.2018
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Dendritic A-current in Rhythmically Active PreBötzinger Complex Neurons in Organotypic 1
Cultures from Newborn Mice 2
Abbreviated title: Dendritic A-current in preBötzinger neurons 3
Authors: Wiktor S. Phillips1,2, Christopher A. Del Negro2, and Jens C. Rekling1* 4
Affiliations: 5
1: Department of Neuroscience, University of Copenhagen, Panum Building 33.3.84, 6
Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. 7
2: Department of Applied Science, Integrated Science Center, 540 Landrum Dr., The College 8
of William and Mary, Williamsburg, Virginia, United States. 9
Corresponding author: Jens C. Rekling 10
Department of Neuroscience, University of Copenhagen, Panum Building 33.3.84, 11
Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. 12
Email: [email protected] 13
Number of pages: 37 14
Number of figures: 6 15
Number of words for Abstract, Introductions, and Discussion: 236, 665, 1489. 16
Conflict of Interest: The authors declare no competing financial interests. 17
Acknowledgements: This work was supported by The Danish Research Council, The Lundbeck 18
Foundation, Mindefonden for Alice Brenaa, Den Owensenke Fond, Lægeforeningens 19
forskningsfond, Agnes og Pouls Friis Fond, Fonden til Lægevidenskabens Fremme, Brødrene 20
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Hartmans fond, as well as the U.S. National Institutes of Health (R01 HL104127 PI: Del Negro). 21
We thank Morten Bjerre Nielsen for technical assistance. 22
Abstract 23
The brainstem preBötzinger complex (preBötC) generates the inspiratory rhythm for breathing. 24
The onset of neural activity that precipitates the inspiratory phase of the respiratory cycle may 25
depend on the activity of type-1 preBötC neurons, which exhibit a transient outward K+ current, 26
IA. Inspiratory rhythm generation can be studied ex vivo since the preBötC remains rhythmically 27
active in vitro, both in acute brainstem slices and organotypic cultures. Advantageous optical 28
conditions in organotypic slice cultures from newborn mice of either sex allowed us to 29
investigate how IA impacts Ca2+ transients occurring in the dendrites of rhythmically active type-30
1 preBötC neurons. The amplitude of dendritic Ca2+ transients evoked via voltage increases 31
originating from the soma significantly increased after an IA antagonist, 4-aminopyridine (4-AP), 32
was applied to the perfusion bath or to local dendritic regions. Similarly, glutamate-evoked 33
post-synaptic depolarizations recorded at the soma increased in amplitude when 4-AP was co-34
applied with glutamate at distal dendritic locations. We conclude that IA is expressed on type-1 35
preBötC neuron dendrites. We propose that IA filters synaptic input, shunting sparse excitation, 36
while enabling temporally-summated events to pass more readily as a result of IA inactivation. 37
Dendritic IA in rhythmically active preBötC neurons could thus ensure that inspiratory motor 38
activity does not occur until excitatory synaptic drive is synchronized and well-coordinated 39
among cellular constituents of the preBötC during inspiratory rhythmogenesis. The biophysical 40
properties of dendritic IA might thus promote robustness and regularity of breathing rhythms. 41
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Significance Statement. Brainstem neurons in the preBötC generate the oscillatory activity that 42
underlies breathing. PreBötC neurons express voltage-dependent currents that can influence 43
inspiratory activity, among which is a transient potassium current (IA) previously identified in a 44
rhythmogenic excitatory subset of type-1 preBötC neurons. We sought to determine whether IA 45
is expressed in the dendrites of preBötC. We found that dendrites of type-1 preBötC neurons 46
indeed express IA, which may aid in shunting sparse non-summating synaptic inputs, while 47
enabling strong summating excitatory inputs to readily pass and thus influence somatic 48
membrane potential trajectory. The subcellular distribution of IA in rhythmically active neurons 49
of the preBötC may thus be critical for producing well-coordinated ensemble activity during 50
inspiratory burst formation. 51
Introduction 52
Bilaterally distributed in the ventrolateral medulla, the preBötC contains a network of 53
excitatory interneurons that generate the rhythm for inspiratory breathing movements (Smith 54
et al., 1991; Feldman and Del Negro, 2006; Feldman et al., 2013). PreBötC neurons can be 55
subdivided into two classes, which differ by their electroresponsive properties and membrane 56
potential trajectory during the respiratory cycle (Rekling et al., 1996; Picardo et al., 2013). 57
During rhythmic activity, type-1 preBötC neurons integrate synaptic drive and exhibit a ramp of 58
preinspiratory depolarization ~400 ms prior to inspiratory bursts. Type-2 neurons exhibit 59
preinspiratory depolarization as well, but it occurs approximately ~200 ms later than type-1 60
neurons (Rekling et al., 1996). Earlier preinspiratory activity in type-1 neurons suggests that 61
they may be more important for initiating the cascade of recurrent excitation that leads to 62
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synchronized network bursts (Smith et al., 1990; Rekling et al., 1996; Carroll and Ramirez, 2013; 63
Kam et al., 2013; Feldman and Kam, 2015). Type-1 neurons are thus putatively rhythmogenic. 64
The biophysical basis by which type-1 neurons synchronize and coordinate the onset of activity 65
across the rhythmic neuronal population has yet to be fully explained, but may be related to 66
the presence of a transient outward IA-like current (Rekling et al., 1996). Pharmacological 67
inhibition of IA in acute slices containing the preBötC results in spurious burst generation at 68
both the cellular and network levels. Such changes in rhythmic activity have been attributed to 69
disorderly recruitment of rhythmogenic preBötC neurons during recurrent excitation, which 70
precedes the inspiratory burst in each cycle of the rhythm (Hayes et al., 2008). 71
IA can influence synaptic integration, as shown in principal neurons outside of the preBötC. For 72
example, IA is expressed at a high density on the dendrites of hippocampal pyramidal cells and 73
cerebellar Purkinje cells, where it acts locally to inhibit Ca2+ spikes and limits the effect of sparse 74
or transient excitatory inputs (Hoffman et al., 1997; Kampa and Stuart, 2006; Otsu et al., 2014). 75
Conversely, IA on dendrites permits robust post-synaptic responses to temporally-summated 76
excitatory input, which may be long-lasting enough to inactivate IA and thereby depolarize the 77
membrane (Magee et al., 1998). 78
Dendrites of rhythmically active preBötC neurons feature active integrative properties, but the 79
dendritic expression of inward and outward currents in subclasses of preBötC neurons is 80
presently unclear. The amplitude of the synaptic drive potential underlying inspiratory bursts in 81
rhythmically active preBötC neurons depends on a Ca2+-activated non-specific cation current 82
(ICAN), recruited by concurrent Ca2+ influx through voltage-gated channels or intracellular Ca2+ 83
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release linked to group I metabotropic glutamate receptors (mGluRs) (Pace et al., 2007; 84
Mironov, 2008; Pace and Del Negro, 2008; Mironov and Skorova, 2011). Excitatory potentials 85
recorded at the soma increase when group I mGluR agonists are co-applied with AMPA on 86
dendrites (Pace and Del Negro, 2008), suggesting that intrinsic dendritic conductances can 87
locally amplify synaptic input. Spontaneous synaptic activity during the interval between 88
inspiratory bursts could cause spurious burst generation if that activity were to evoke ICAN on its 89
own. Therefore, a biophysical mechanism that selectively inhibits sparse synaptic inputs could 90
prevent aberrant dendritic amplification, thus avoiding spurious bursts prior to a synchronized 91
build-up in network activity. 92
Fast-activating voltage-dependent outward currents on dendrites can shunt sparse excitatory 93
input, and might explain the disordered behavior seen during network-wide blockade of IA in 94
rhythmically active slices. We therefore hypothesize that the blockade of IA modifies active 95
synaptic integration occurring in the dendrites in type-1 neurons. Here, we utilize an 96
organotypic slice culture containing the preBötC to investigate how IA might influence transient 97
depolarization in the dendrites of rhythmically active preBötC neurons. We show that loss of 98
dendritically-localized IA significantly increases the amplitude of voltage-sensitive dendritic Ca2+ 99
transients as well as glutamate-evoked postsynaptic depolarizations recorded at the soma. 100
These phenomena demonstrate a possible mechanism by which dendritic IA might filter inputs 101
and thus modulate the postsynaptic output of type-1 neurons, blocking sparse synaptic 102
excitation while permitting temporally- and spatially-summated inputs during the 103
preinspiratory phase of the respiratory cycle, which may enhance the regularity of inspiratory 104
rhythms. 105
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Materials and Methods 106
Ethical approval 107
The Department of Experimental Medicine at the Faculty of Health and Medical Sciences, 108
University of Copenhagen, approved all experiments and procedures according to protocols laid 109
out by Danish Ministry of Justice and the Danish National Committee for Ethics in Animal 110
Research. 111
Organotypic slice cultures 112
US Naval Medical Research Institute (NMRI) mice post-natal ages P3.5 to P6.5 days of either sex 113
were anesthetized with isoflurane (Baxter, Soeborg, Denmark) and immediately dissected in 114
sterile-filtered chilled artificial cerebrospinal fluid (ACSF) containing (in mM): 184 glycerol, 2.5 115
KCl, 1.2 NaH2PO4, 30 NaHCO3, 5 HEPES acid, 15 HEPES base, 25 D-glucose, 5 sodium ascorbate, 116
2 thiourea, 3 sodium pyruvate, 10 MgSO4, 0.5 CaCl2; pH 7.3, equilibrated by bubbling with 95 % 117
O2/5 % CO2. A single transverse slice of the brainstem, 400 μm in thickness, were taken at the 118
level of the preBötC using a vibrating microtome (Thermo Fisher Scientific Microm 650V, 119
RRID:SCR_008452). Anatomical markers, such as the principle loop of the inferior olive and 120
obex of the fourth ventricle (Ruangkittisakul et al., 2011; Ruangkittisakul et al., 2014), were 121
used to verify the rostral-caudal location of the preBötC and thus properly calibrate the slice. 122
Cultures were prepared as previously described (Phillips et al., 2016) via the Stoppini interface 123
method, placing 1-4 transverse brainstem slices onto semi-porous culture well inserts (Millipore 124
Cat# PIC03050, RRID:SCR_008983). Mounted preparations were maintained in sterile-filtered 125
organotypic culture media containing: 50 % Eagle’s MEM with Earle’s Salts, 25 % Hank’s 126
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balanced salt solution, 25 % horse serum, 2 mM GlutaMAX (Gibco), 200 U/mL penicillin, 5 127
μg/mL streptomycin, 25 mM HEPES and an additional 3.6 mM D-glucose. The osmolarity of the 128
culture medium measured 320-340 mOsm with a pH of 7.25. The cultures were treated with 10 129
μM MK-801 for the first three days in vitro to prevent ischemia-related cell death (Newell et al., 130
1990). Fresh culture media was supplied every 48 hours thereafter until experimentation. These 131
slices were then kept in a sterile, humidified incubator at 35° C and atmospheric CO2 132
concentrations during incubation. All compounds and media were from Sigma (Sigma-Aldrich, 133
RRID:SCR_008988). 134
Electrophysiology 135
Somatic whole-cell patch-clamp recordings were performed in current clamp and discontinuous 136
single-electrode voltage clamp (dSEVC; sampling rate 1-2 kHz) using an AxoClamp 2B amplifier 137
(Molecular Devices, Sunnyvale, CA, USA). Data were digitally acquired at a sampling rate of 10 138
kHz, with a low-pass filter of 2 kHz from the output of the AxoClamp 2B. Glass micropipettes 139
were pulled from filamented capillary glass (O.D. 1.5 mm, I.D. 0.86 mm, Harvard Apparatus, 140
Holliston, MA, USA) using a PUL-100 micropipette puller (World Precision Instruments, 141
RRID:SCR_008593) to a tip resistance of 4-6 MΩ. Patch pipettes were filled with a solution 142
containing (in mM): 130 HCH3SO3, 130 KOH, 10 HEPES, 0.4 NaGTP, 4 Na2ATP, 5 Na2-143
phosphocreatine, 4 MgCl2 (all from Sigma-Aldrich, RRID:SCR_008988), 0.05 Alexa 594 hydrazide 144
(Thermo Fisher Scientific, RRID:SCR_008452), 0.1 Fluo-8L (AAT Bioquest , Sunnyvale, CA, USA). 145
The osmolarity of the patch pipette solution measured 310 mOsm with a pH of 7.3. Patch 146
pipettes were visually guided to target neurons under visual control using MPC-200 147
micromanipulator system (Sutter Instruments, Novato, CA, USA) on a fixed-stage upright 148
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microscope (modified Olympus BX51, Olympus Corporation, Tokyo, Japan) under 40x 149
magnification (NA = 0.8,WD = 3.3 mm). 150
Iontophoresis was performed using a IP-X5 instrument (Neuro Data Instruments Corp., New 151
York, NY, USA). Single-barrel glass micropipettes were pulled as described above to a tip 152
resistance of 8-10 MΩ for single-drug trials. For experiments involving 4-aminopyridine (4-AP) 153
pipettes were filled with an aqueous solution containing: 165 mM NaCl, 10 mM HEPES, and 0.2 154
% tetramethylrhodamine-dextran. For inhibition of IA localized to dendrites, we added 45 mM 155
4-AP (pH of 7.5). For experiments involving local iontophoretic application of Cd2+, an aqueous 156
solution containing exclusively 200 mM CdSO4 was used. ACSF in Cd2+experiments was free of 157
phosphates (omitting NaH2PO4) to avoid precipitation. Pulses were delivered at 1 Hz (500 ms 158
pulse duration) with the ejection currents: +20-30 nA to eject 4-AP, +5-10 nA to eject Cd2+. 159
Filamented theta capillary glass (O.D. 1.5 mm, I.D. 1.17, septum 0.17 mm, Harvard Apparatus, 160
Holliston, MA, USA) was used in some experiments to apply glutamate (150 mM in water, pH=8, 161
+20 nA holding, -150 nA ejection current, 0.2 Hz cycle time, 100 ms pulse duration) from one 162
barrel and 4-AP (50 mM with 10mM HEPES in 165 mM NaCl, pH 7.5, -10 nA holding, +90 nA 163
ejection current, 10-30 s per application bout) from the other. 164
During voltage-clamp recordings and dendritic glutamate iontophoresis, 1 μM TTX (Tocris 165
Bioscience, RRID:SCR_003689) was added to the bath. A-current was blocked with bath-applied 166
2 mM 4-AP when not applied locally. During spike train response experiments, 10 μM NBQX 167
disodium salt (Tocris Bioscience,RRID:SCR_003689) was added to the bath to preclude Ca2+ 168
transients that might arise due to spontaneous excitatory synaptic transmission. 169
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Rhythmic activity in slices was recorded in ACSF containing (in mM): 124 NaCl, 3 KCl, 5 KH2PO4, 170
25 NaHCO3, 25 D-Glucose, 1 ascorbic acid, 1 MgCl2, 1.5 CaCl2 (all from Sigma-Aldrich, 171
RRID:SCR_008988), with a pH of 7.4, equilibrated by bubbling with 95 % O2/5 % CO2. The final 172
concentration of K+ was [K+]o = 8 mM, which elevates baseline membrane potentials and 173
increases the frequency of spontaneous respiratory rhythm. 174
After recording more than 15 minutes of rhythmic activity, the standard ACSF was always 175
exchanged with a low-excitability ACSF in which external K+ concentration was reduced from 8 176
to 3 mM and external Ca2+ was raised from 1.5 mM to 2 mM. Thus, all subsequent 177
electrophysiological measurements and subcellular imaging were performed at physiological K+ 178
concentrations. These modifications reduced or stopped network rhythmic activity by 179
hyperpolarizing neuronal membrane potentials and increasing the threshold for Na+ channel 180
activation (Panaitescu et al., 2009; Ruangkittisakul et al., 2011). Low-excitability ACSF contained 181
(in mM): 124 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 25 D-glucose, 1 ascorbic acid, 1 MgCl2, 2 182
CaCl2, with a pH of 7.4, equilibrated by bubbling with 95 % O2/5 % CO2. 183
Ca2+ imaging 184
The region of the organotypic culture containing the preBötC was targeted via anatomical 185
landmarks and visualization of synchronized rhythmic network activity during whole-slice Ca2+ 186
imaging (Phillips et al., 2016). A membrane-permeable fluorescent Ca2+ indicator, either Fura-2 187
AM or Fluo-8, AM (AAT Bioquest , Sunnyvale, CA, USA), was bath loaded prior to each 188
experiment. Cultures were submerged in 1.5 mL of loading solution containing 35 μM of Fura-2 189
AM or Fluo-8, AM in DMSO along with 0.05 % pluronic acid dissolved in low excitability ACSF. 190
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Slices were incubated in loading solution at room temperature for 30-40 minutes, and then 191
perfused with ACSF in the recording chamber for 15 minutes of recovery and additional de-192
esterification prior to experimentation. Preheated and oxygenated ACSF was perfused into the 193
recording chamber during recording at 2 ml/min and 29° C. 194
Fluorescent Ca2+ activity was recorded in wide-field on a fixed-stage upright microscope 195
(modified Olympus BX51), illuminated by a metal halide light source PhotoFluor II (89North, 196
Burlington, USA) or a blue (470 nm) LED light source, M470L2 (Thorlabs, Newton, NJ, USA). Red 197
and green channel fluorescence was visualized using a dual-bandpass filter set (Chroma 59022: 198
excitation dual band-pass 450-490 nm/555-590 nm, emission dual band-pass 500-543 nm/603-199
665 nm). Red and green channels (for Alexa 594 hydrazide, Fluo-8, AM, Fluo-8L) were separated 200
during acquisition by manually exchanging an additional excitation filter in the light path 201
(Semrock FF01: band-pass 565-605 nm; Semrock FF02: band-pass 457-487 nm, Rochester, NY, 202
USA). Imaging of Fura-2 AM loaded cultures was done using a U-MNU filter (Olympus, UV 203
Narrow, Excitation: 360-370 nm, dichroic mirror: 400 nm (long-pass), emission: 420 nm (long 204
pass). Time series acquisition was performed with a sCMOS camera (Neo DC-152Q, Andor 205
Technology, Belfast, UK) controlled by SOLIS software (Andor Technology, Belfast, UK). Imaging 206
protocols employed 10x (NA 0.3) and 40x (NA 0.8) water immersion objectives. Time series 207
were acquired at 10-50 Hz. At least 30 minutes of perfusion was allowed after break-in during 208
whole-cell patch clamp to allow equilibration of intracellular Ca2+ dye. Dark current noise - the 209
background sensor noise in the absence of illumination - was sampled prior to the start of each 210
acquisition and subtracted from all imaging sweeps. In some experiments 40 nM Pandinotoxin-211
Kα (Sigma-Aldrich, RRID:SCR_008988), 500 nM Phrixotoxin-1 or 100 nM AmmTX3 (Alomone 212
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Labs, RRID:SCR_013570)—which are IA-blockers that act selectively on Kv4.x channel 213
subtypes—were applied to the perfusion bath. 214
Experimental design and statistical analysis 215
Optical data were analyzed offline using ImageJ 1.51p (ImageJ, RRID:SCR_003070), and Igor Pro 216
7 (IGOR Pro, RRID:SCR_000325). Dendritic Ca2+ transients were calculated as the percent 217
change in fluorescence relative to baseline values (ΔF/F0). Background was subtracted frame-218
by-frame, taken as the equal-sized mean background fluorescence immediately adjacent to the 219
dendritic ROI. Dendritic ROIs were hand drawn along neural processes, extending for no more 220
than 10 μm in length. The mean pre-stimulus fluorescence in the dendritic ROI was defined as 221
F0. For each condition, 4-6 sweeps were acquired at 10-30 second intervals, and subsequently 222
averaged after calculation of ΔF/F0. 223
Subcellular differences in the concentration of the Ca2+ indicator, surface area to volume ratio, 224
and Ca2+ channel density can alter the amplitude of ΔF/F0 in response to a given voltage 225
increase (Yasuda et al., 2004). These experiments seek to measure relative changes in 226
membrane excitability caused by manipulations of IA, but not the absolute change in Ca2+ 227
concentration. The changes to Ca2+ transients before and after 4-AP application were therefore 228
also computed at each measurement location to control for such differences between cellular 229
compartments or between experiments that might bias the weight of fluorescence 230
measurements. The formula for this metric, which we term normalized 4-AP effect, was the 231
difference (i.e., before and after blockade 4-AP) in Ca2+ transient amplitude in response to 232
stimuli divided by the sum of the responses: 233
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Electrophysiological data were acquired using pClamp 10.0 (pClamp, RRID:SCR_011323) and 234
subsequently analyzed using custom scripts written in Igor Pro 7 (IGOR Pro, RRID:SCR_000325). 235
Statistical values are reported as mean ± SD, unless otherwise stated. Student’s t-test was used 236
for statistical comparisons of one- and two-sample populations. Fisher’s exact test was used to 237
assess the distribution of neuronal membrane currents. All statistical tests were performed 238
using OriginPro 2017 (Microcal Origin, RRID:SCR_002815) or Prism 7 (Graphpad Prism, 239
RRID:SCR_002798). 240
Results 241
Effects of low-dose 4-AP on rhythmic bursting in organotypic cultures of the preBötC 242
Brainstem organotypic slice cultures containing the preBötC generate spontaneous bilaterally-243
synchronized rhythmic bursts of ensemble neuronal activity resembling inspiratory rhythms 244
analogous generated by acutely-prepared slices (Phillips et al., 2016), which can be recorded 245
via population fluorescent Ca2+ activity. If IA moderates post-synaptic amplification of excitatory 246
input, then partial inhibition of IA ought to lower the threshold for post-synaptic burst 247
production, thereby advancing inspiratory phase onset, which we surmise would measurably 248
increase rhythmic frequency. We imaged rhythmic cultures and found that bath-applied low 249
doses of the IA-blocker 4-AP (50 μM) increased the oscillatory burst frequency observed in the 250
preBötC from 13.6 ± 2.7 to 24.3 ± 5.1 bursts/min (Figure 1A, n = 7 cultures, t(6) = 4.68, p = 251
0.0034, paired two-tailed t-test). 252
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In addition to its affinity for potassium channels of the Kv4 family (associated with IA-like 253
currents), 4-AP can also block delayed rectifier Kv1.1 and Kv1.2 channels, which are typically 254
expressed in axons. We thus attempted to pharmacologically isolate IA-related effects via bath 255
application of three scorpion and spider toxin IA-blockers that have greater, albeit 256
heterogenous selectivity for the Kv4 channel subfamily, i.e. Pandinotoxin-Kα, Phrixotoxin-1, or 257
AmmTX3 (Diochot et al., 1999; Klenk et al., 2000; Maffie et al., 2013). However, none of these 258
selective blockers changed burst frequency (Figure 1A, n = 3 cultures in each group, F(1,2) = 259
2.62, p = 0.24, repeated-measures one-way ANOVA). 260
Low doses of 4-AP also affected the relative amplitude of synchronized Ca2+ activity in regions 261
of the slice putatively corresponding to downstream motor and sensory nuclei (i.e., hypoglossal 262
motor nucleus and the nucleus of the solitary tract). The amplitude of synchronized Ca2+ 263
transients located dorsomedial to the preBötC was increased to 190 ± 55 % in response to low-264
doses of 4-AP (Figure 1B, calculated as the percentage change in the ratio of dorsomedial 265
region: preBötC Ca2+ transient amplitudes for equal-sized areas, n = 8 cultures, t(7) = 5.83, p = 266
0.0006, two-tailed paired t-test). 267
We took advantage of the improved optical conditions in the cultures and sampled 10 somatic 268
ROIs within the preBötC, as well as a pooled sample of dendritic ROIs in the neighboring 269
neuropil (area: 4000-5000 μm2) imaged in a single field of view, before and after bath 270
application of 4-AP (Figure 1C,D). Baseline rhythmic Ca2+ transients measured in dendrites of 271
the neuropil had a shorter duration (half-amplitude width: 672 ± 188 ms) than those arising in 272
somatic compartments (955 ± 262 ms, n = 7 cultures, t(6) = 7.53, p = 0.0003, two-tailed paired 273
t-test). In the presence of 4-AP the half-amplitude width and decay time constant of the Ca2+ 274
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transients increased in both somatic ROIs (half-amplitude width: 955 ± 262 ms vs. 1181 ± 416 275
ms, decay time constant: 0.96 ± 0.19 s vs. 1.73 ± 0.77 s, n = 7 cultures, t(6) = 3.30, p = 0.0163, 276
t(6) = 2.82, p = 0.030, two-tailed paired t-test ) and neuropil dendritic ROIs (half-amplitude 277
width: 672 ± 188 ms vs. 929 ± 316 ms, decay time constant: 0.84 ± 0.85 s vs. 1.72 ± 1.07 s, n = 278
7 cultures, t(6) = 4.34, p = 0.0049, t(6) = 2.68, p = 0.037, two-tailed paired t-test) (Figure 1E). 279
The mean rise-time did not change significantly (somata: 338 ± 68 vs. 344 ± 103, n = 7 cultures, 280
t(6) = 0.29, p = 0.7769, neuropil dendrites: 248 ± 53 vs. 278 ± 121, n = 7 cultures, t(6) = 0.54, p = 281
0.6092, two-tailed paired t-test). These data suggest that 4-AP increases network excitability. 282
To determine whether this increased excitability might be attributable to cellular-level changes 283
in synaptic integration, we investigated the subcellular expression of IA in a class of neurons 284
thought to be most critical to rhythm generation. 285
Two rhythmic neuron classes in the preBötC 286
Organotypic slice cultures containing the preBötC generate respiratory rhythm analogous to 287
acute slice preparations and en bloc brainstem-spinal cord preparations (Funk and Greer, 2013; 288
Forsberg et al., 2016; Phillips et al., 2016). However, it is not yet known whether rhythmically 289
active preBötC neurons in culture retain the dichotomous membrane properties classified as 290
type-1 and type-2 first characterized in acute slice recordings (Rekling et al., 1996). Type-1 and 291
type-2 neurons are distinguished by voltage trajectory (e.g., pre-inspiratory activity) and the 292
expression of either a transient outward current, IA, or a hyperpolarization-activated inward 293
current, Ih. Type-1 neurons express IA, lack Ih, and display a ramp-like increase in membrane 294
potential, known as pre-inspiratory activity, beginning ~400 ms prior to the inspiratory burst. 295
Conversely, type-2 neurons lack IA, express Ih, and display more latent pre-inspiratory activity 296
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~200 ms prior to the inspiratory burst (Rekling et al., 1996). Motor nerve rootlets are not 297
retained in slice cultures, so here type-1 and type-2 neurons were differentiated based on the 298
presence of either IA or Ih, but not preinspiratory latency. 299
We recorded rhythmically active neurons in organotypic cultures and tested for the expression 300
of both IA and Ih in current clamp upon break-in in the whole-cell configuration (Figure 2, n = 301
42). Rhythmic activity was first recorded using the minimum amount of negative holding 302
current to inhibit spontaneous action potentials between rhythmic bursts (Figure 2A, 0 to -0.15 303
nA). Among all rhythmically active neurons, the mean interburst interval was 5.4 ± 3.2 s, and 304
the mean burst duration was 405 ± 134 ms (n = 42). 305
The presence of IA was determined by first hyperpolarizing neurons with negative holding 306
current to a baseline membrane potential (Vm) lower than -65 mV, which deinactivates IA (Hayes 307
et al., 2008). Square-wave positive current pulses of 400 ms duration were then delivered to 308
evoke repetitive firing of action potentials. Neurons with IA display a delay in membrane 309
depolarization lasting 100-200 ms before firing repetitively, whereas neurons without IA 310
discharge action potentials throughout the duration of the current pulse without a delay 311
exceeding the membrane time constant (Figure 2B shows cycle-triggered averages of 5-10 312
sweeps to demonstrate the repeatability of delayed excitation or the lack thereof in type-1 and 313
type-2 preBötC neurons). 314
The presence of Ih was determined by setting baseline Vm between -40 and -50 mV and 315
delivering 400 ms negative current pulses of sufficient amplitude to hyperpolarize the neuron 316
to -70 to -90 mV, which is sufficient to evoke Ih if it is expressed by the neuron (Mironov et al., 317
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2000; Thoby-Brisson et al., 2000). Neurons with Ih exhibit a ‘sag’ depolarization of ~10 mV after 318
being transiently hyperpolarized as well as a post-inhibitory rebound after the negative current 319
pulse terminates (Figure 1B shows cycle-triggered averages of 5-10 sweeps to demonstrate the 320
repeatability of ‘sag’ or the lack thereof in type-1 and type-2 preBötC neurons). 321
Among the rhythmic neurons recorded in our preBötC cultures, 57 % (n = 24) were type-1 322
neurons and 31 % (n = 13) were type-2 neurons. However, 7 % (n = 3) of the neurons showed 323
both sag potential and delayed excitation and 5 % (n = 2) displayed neither property, so these 324
neurons (n = 5) could not be categorized as either type-1 or -2. These results are in line with the 325
distribution of respiratory neuron classes in acute slices (Rekling et al., 1996; Picardo et al., 326
2013). If we consider the null hypothesis to be that there is no relationship governing the 327
expression of IA and Ih in respiratory neurons, then the allotment of recorded neurons into type-328
1 and type-2 phenotypes is unlikely to have occurred by random chance (p = 0.0001, Fisher’s 329
exact test), suggesting that the dichotomous electrophysiological properties of rhythmically 330
active type-1 and type-2 neurons observed in the preBötC from acute slices, also persists in 331
culture. 332
Voltage ramps during blockade of IA increase electrotonic compactness of type-1 neurons 333
To determine whether IA might actively inhibit the spread of voltage transients along dendrites 334
in type-1 neurons, we next performed simultaneous single-electrode voltage clamp (SEVC) and 335
fluorescent Ca2+-imaging of rhythmically active neurons (Figure 3). This allowed us to track the 336
relative amplitude of voltage changes at distal dendritic compartments using voltage-sensitive 337
Ca2+ influx as a surrogate for direct measurements of membrane potential. 338
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Since voltage clamping inherently suffers from a lack of space clamp as a function of distance 339
from the recording pipette, changes in membrane potential enforced at the soma via SEVC are 340
attenuated at distal dendritic locations. The exact amount of that attenuation depends on 341
passive cable properties (e.g. length, diameter, branch order) and density of voltage-gated 342
conductances (Bar-Yehuda and Korngreen, 2008), but space-clamp error must be factored into 343
data analysis and interpretation. 344
In type-1 neurons, IA activates at subthreshold membrane potentials and fully inactivates at 345
membrane potentials less negative than -40 mV (Hayes et al., 2008). Therefore, neurons were 346
first held at a command potential (VC) of -75 to -80 mV, where IA is steady-state deinactivated in 347
the presence of TTX (Hayes et al., 2008). We then evoked IA by 400 ms step commands to 348
+20mV, which inactivated after approximately 100-200 ms (Figure 3A). 349
Dendritic Ca2+ transients were then imaged during delivery of fast (150 ms duration) positive-350
going voltage ramps, starting at VC and increasing to a final membrane potential capable of 351
activating voltage-gated Ca2+ currents (mean = 7.8 ± 10.2 mV, n = 9). These short-duration, 352
rapidly-increasing voltage ramps mimic the onset of endogenous inspiratory bursts and elicit 353
supra-threshold Ca2+ responses within the transient phase of IA activation (Figure 3B). We then 354
added 2 mM 4-AP to the bath, confirmed blockade of IA with voltage-clamp step commands 355
(Figure 3A), and then repeated the voltage-ramp command to re-measure the dendritic Ca2+ 356
transients (Figure 3B). 357
The command stimulus was constant between control and 4-AP sweeps for each recorded 358
neuron, but the peak voltage amplitude achieved by the stimulus waveform (which is 359
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measurable in SEVC) nevertheless increased across all neurons after addition of 4-AP because 360
of increased effectiveness of the SEVC (type-1: 8.5 ± 2.2 %, n = 5; type-2: 2.0 ± 1.5 %, n = 4). 361
Changes in peak amplitude of the stimulus waveform were significantly greater in type-1 362
neurons than those in type-2 neurons (n = 5 type-1, n = 4 type-2 neurons, t(7) = 4.93, p = 363
0.0017, two-tailed unpaired t-test). Input resistance in each cell was measured by taking the 364
slope of the IV curve from the first 20 mV of increase above command potential—a range in 365
which no apparent active conductances were activated. Mean input resistance was 221 ± 97 366
MΩ in type-1 neurons and 181 ± 77 MΩ in type-2 neurons, which was a negligible difference (n 367
= 5 type-1, n = 4 type-2 neurons, t(7) = 0.68, p = 0.52, two-tailed unpaired t-test). 368
In order to visualize how voltage propagation through dendritic compartments is affected by a 369
blockade of IA, we measured the relative increase in Ca2+ indicator fluorescence (ΔF/F0) evoked 370
by voltage ramps before and after exposure to 4-AP (Figure 3C,D). Proximal dendritic 371
compartments (less than or equal to 33 μm from the soma) and distal dendritic compartments 372
(greater than or equal to 69 μm from the soma) were sampled with a minimum distance 373
between measurement sites of 54 μm. The mean proximal ROI distance from the soma was 14 374
± 9 μm (n = 9) and the mean distal ROI distance from the soma was 101 ± 21 μm (n = 9). Among 375
type-1 neurons, the amplitude of fluorescent Ca2+ transients elicited by voltage ramps increased 376
significantly by 228 ± 98 % at proximal regions after addition of 4-AP (Figure 3D, ΔF/F0 before 377
vs. ΔF/F0 after 4-AP, n = 5 type-1 neurons, t(4) = 2.40, p = 0.037, one-tailed paired t-test). The 378
amplitude of transients measured at distal dendritic compartments also increased significantly 379
by 382 ± 123 % (ΔF/F0 before vs. ΔF/F0 after 4-AP, n = 5 type-1 neurons, t(4) = 2.87, p = 0.022, 380
one-tailed paired t-test). The observed increase in response amplitude at distal regions in type-381
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1 neurons was significantly greater than proximal regions (normalized 4-AP effect proximal vs. 382
normalized 4-AP effect distal, n = 5 type-1 neurons, t(8) = 2.56, p = 0.034, two-tailed unpaired t-383
test), suggesting a non-linear increase in the amplitude of Ca2+ influx between peri-somatic and 384
distal dendritic compartments. 385
Among type-2 neurons, the amplitude of fluorescent Ca2+ transients did not increase 386
significantly at proximal regions (ΔF/F0 before vs. ΔF/F0 after 4-AP, n = 4 type-2 neurons, t(3) = 387
0.46, p = 0.34, one-tailed paired t-test), nor did the amplitude at distal regions (ΔF/F0 before vs. 388
ΔF/F0 after 4-AP, n = 4 type-2 neurons, t(3) = 1.72, p = 0.091, one-tailed paired t-test). This 389
suggests that IA does not play a significant role in governing dendritic depolarization of type-2 390
preBötC neurons, particularly compared to their counterparts the type-1 preBötC neurons 391
(compare columns 1,2 to 3,4 in Figure 3D). 392
These results demonstrate that a large increase in the voltage ramp-evoked Ca2+ transient 393
occurs globally in type-1 neurons when 4-AP-sensitive currents are blocked (Figure 3C, depicting 394
raw ΔF/F0 values above 100 %), and that this increase is greatest at distal dendritic regions. The 395
data imply either: 1) blocking IA increases the length constant by removing IA-mediated shunting 396
of the dendritic plasma membrane, which enhances the ability of somatic command potentials 397
to depolarize the dendrite, or 2) blocking IA augments Ca2+ channel activation at distal dendritic 398
sites. In either scenario, our data cannot distinguish whether changes in the measured Ca2+ 399
transient arose predominantly from diminution of somatic or dendritic IA (or both). Thus, we 400
sought to determine whether ionic membrane currents could be blocked on distal dendritic 401
sites while minimally affecting the soma. 402
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Ion channels can be blocked in a spatially restricted manner via iontophoresis 403
To demonstrate that ion channels can be blocked in morphologically selective regions of 404
preBötC neurons in culture, we conducted a positive control experiment in which Cd2+ was 405
applied focally via iontophoresis to block Ca2+ channels in distal dendrites (Figure 4). 406
Simultaneous whole-cell patch-clamp and Ca2+-imaging recordings were acquired from preBötC 407
neurons in slice cultures. Spontaneous network activity was suppressed by reducing 408
extracellular K+ concentration from 8 to 2.5 mM and increasing extracellular Ca2+ concentration 409
from 1.5 to 2 mM. Membrane potential was held between -55 and -60 mV to prevent 410
spontaneous spiking. A pipette containing an aqueous solution of 200 mM CdSO4 was 411
positioned at distal dendritic regions. Positive current through the patch-recording pipette was 412
then injected at the soma in either square-wave pulses (400 ms, 0.1-0.3 nA, 1 Hz, causing 413
repetitive spiking, Figure 4A) or short repetitive current pulses (3 ms, 0.8-1.2 nA, 100 Hz; 414
evoking brief trains of action potentials. The stimulus was maintained between control and Cd2+ 415
application in each cell. After acquiring control sweeps, Cd2+ was applied to the dendrite using 416
iontophoresis (400-500 ms, 1 Hz, +5-10 nA), 30-60 seconds were allowed to pass for 417
equilibration of drug ejection, and imaging sweeps were repeated. Peak ΔF/F0 values were 418
sampled from two locations in each cell, separated by a minimum of 50 μm (n = 7 neurons): 419
proximal and distal dendritic regions as before, as well as the soma. The decrease in Ca2+ 420
transient amplitude after application of Cd2+ was normalized to control sweeps. 421
Distance between the tip of the iontophoresis pipette and center of each ROI was measured in 422
the xy-plane. Since both the iontophoresis pipette and imaged cellular compartments occupy 423
approximately the same focal plane, the difference in their positions estimated the distance in 424
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three-dimensional space between the point of drug application and the ROI. Regression analysis 425
revealed a linear relationship between the degree of Ca2+ signal attenuation and straight-line 426
distance from the pipette (Figure 4C, y-intercept = 1.0, R2 = 0.85; ANOVA F-value = 64.45, p < 427
0.001, n = 14). This model suggests that less than 50 % of the somatically evoked, dendritic Ca2+ 428
transient is attenuated when the drug (Cd2+ in this case) is applied 79 μm away from the ROI. 429
Although the mobility in the extracellular environment due to applied electric field undoubtedly 430
differs between Cd2+ and 4-AP, both drugs are extracellular ion channel blockers that are not 431
taken up by cellular processes. Thus, to ensure that recorded somata remained unaffected by 432
iontophoretic drug application, we maintained a minimum of 96 μm (and average of 121 ± 20 433
μm, n = 12 experiments) between the point of drug application and the nearest edge of somatic 434
compartments in all subsequent local drug application experiments. 435
Dendritic IA blockade increases Ca2+ response to somatically evoked stimuli in type-1 neurons 436
To determine whether 4-AP increases the Ca2+ response of type-1 neurons by blocking dendritic 437
IA, we applied 4-AP via iontophoresis to distal dendrite sites (Figure 5, mean dendritic length 438
from soma: 118 ± 21 μm, n = 12) in rhythmically active preBötC neurons and measured the 439
amplitude of Ca2+ transients in response to trains of 10 action potentials triggered by somatic 440
current injection. Whole-cell patch-clamp recordings of rhythmically active preBötC neurons 441
were acquired and their membrane properties were tested in current clamp recording mode to 442
test for the presence of IA and Ih, and thus determine whether they were type-1 or type-2. At 443
least 40 minutes of whole-cell recording was allowed to elapse to allow for dye diffusion at 444
distal compartments. All imaging sweeps were performed in the presence of 2.5 mM K+ and 2 445
mM Ca2+, which suppresses respiratory rhythm. 446
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We further added the ionotropic excitatory amino acid receptor antagonist NBQX (10-20 μM) to 447
the perfusate to suppress excitatory synaptic activity. In both control trials and local 4-AP 448
application trials, ROIs were sampled from distal dendritic regions within 30 μm of the 449
iontophoresis pipette. To verify that the effect of 4-AP observed near the site of iontophoresis 450
was caused by local ion channel blockade, we also sampled the relative change in fluorescence 451
from a presumably unaffected compartment (i.e., other dendritic branches, proximal sites on 452
the branch of drug application, and the soma) located at least 100 μm away in the xy plane 453
from the site of iontophoresis (mean distance: 139 ± 32 μm, n = 12). Pipettes containing 45 mM 454
4-AP dissolved in saline (165 mM NaCl, 0.2 % Dextran-TMR, pH of 7.5) were positioned at distal 455
dendritic sites. During drug application trials, we applied continuous ejection pulses as before 456
(400-500 ms, 1 Hz, +20-45 nA). In type-1 neurons, spike train-evoked Ca2+ transients at distal 457
dendritic sites (near the point of drug application) increased substantially to 155 ± 29 % (Figure 458
5A-C, ΔF/F0 before vs. ΔF/F0 after 4-AP, n = 9 type-1 neurons, t(8) = 5.22, p = 0.0008, two-tailed 459
paired t-test) compared to control. Regions farther more than 100 μm away from the site of 460
iontophoresis did not show an increase in ΔF/F0 compared to the control response (ΔF/F0 461
before vs. ΔF/F0 after 4-AP, n = 9 type-1 neurons, t(8) = 2.08, p = 0.071, two-tailed paired t-test). 462
The increase at sites near the point of iontophoresis was significantly greater than on other 463
dendritic branches or the soma (normalized 4-AP effect proximal vs. normalized 4-AP effect 464
distal, n = 9 type-1 neurons, t(16) = 5.65, p = 0.0001, two-tailed unpaired t-test). 465
Type-2 neurons did not show an increase in ΔF/F0 evoked by current pulses at the site of 466
iontophoresis (Figure 5C, ΔF/F0 before vs. ΔF/F0 after 4-AP, n = 3 type-2 neurons, t(2) = 0.26, p = 467
0.82, two-tailed paired t-test) or at sites >100 μm from the site of iontophoresis (ΔF/F0 before 468
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vs. ΔF/F0 after 4-AP, n = 3 type-2 neurons, t(2) = 0.18, p = 0.87, two-tailed paired t-test). These 469
results demonstrate that blockade of IA on dendrites has a substantial effect on the size of distal 470
dendritic Ca2+ transients evoked by somatic current pulses in type-1 preBötC neurons, but that 471
IA blockade has a negligible effect on distal dendritic Ca2+ transients evoked at the soma in type-472
2 neurons. 473
IA blockade increases dendritic depolarizing response to glutamate application in type-1 474
neurons 475
To better understand how the dendritic IA might influence the synaptic integration in type-1 476
neurons, we applied glutamate to distal dendritic sites of type-1 neurons and recorded the 477
resulting somatic depolarization before and after iontophoresis of 4-AP at the same site. These 478
experiments were performed in 1 μM TTX to preclude action-potential driven synaptic 479
transmission not under our control. We used a dual-barrel theta pipette containing glutamate 480
(150 mM) in one barrel and 4-AP (50 mM) in the other. The tip of the theta pipette was placed 481
at dendritic sites that were 116-138 μm from the soma (n = 4 neurons in 4 different cultures). 482
Short repetitive (100 ms; 0.2 Hz) puffs of glutamate at dendritic sites gave rise to evoked 483
depolarizations recorded at the soma (total duration ~500 ms or less, half-amplitude width 90-484
244 ms; maximum amplitude 5-15 mV, Figure 6B, C). We inhibited A-current on dendrites at the 485
site of glutamate application via 10-20 s of 4-AP iontophoresis. Upon resuming pulsed 486
glutamate delivery, we recorded a 149 ± 14 % increase in amplitude and 160 ± 42 % increase in 487
half-amplitude width of the glutamate responses recorded at the soma (Figure 6C, n = 4 type-1 488
neurons, t(3) = 2.74, p = 0.036, and t(3) = 4.22, p = 0.012, one-tailed paired t-test). 489
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Discussion 490
The subcellular distribution of active membrane currents in preBötC neurons is largely 491
unknown. Here we show that an IA-like current is expressed on the dendrites of type-1 preBötC 492
neurons, which can influence synaptic integration and thus inspiratory rhythm- and burst 493
generation. 494
IA in type-1 preBötC neurons has previously been posited to affect the onset of the inspiratory 495
burst phase during rhythmic network activity. A complete network-wide blockade of IA causes 496
disordered inspiratory rhythms (Hayes et al., 2008). These authors measured IA in somatic 497
whole-cell and outside-out patches, which cannot provide information about its subcellular 498
distribution. However, the distribution of IA could influence the integrative properties of 499
rhythmically active neurons. Dendrites expressing IA can selectively promote and amplify 500
responses to network activity that is synchronized, while inhibiting postsynaptic responsiveness 501
to asynchronous and sparse spontaneous input. 502
Minute inhibition of 4-AP sensitive currents has a strong effect on network excitability. Network 503
activity responds even to doses of 4-AP (50 μM) that are 40-fold less than the reported IC50 for 504
IA (~ 2mM) in acute slices containing the preBötC (Hayes et al., 2008). Attenuation of IA appears 505
to advance the phase of the inspiratory cycle, nearly doubling the rhythm frequency. 506
Attenuation of IA also increases the amplitude of neural bursts in regions dorsomedial to the 507
preBötC, which likely arises from inspiratory-related hypoglossal motoneurons. Lastly, 508
attenuation of IA lengthens the duration of somatic and dendritic Ca2+ transients in preBötC 509
neurons. Although 4-AP can inhibit delayed rectifier current, conceivably accounting for an 510
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overall increase in excitability, we suspect that these off-target effects are negligible at such low 511
(50 μM) doses of 4-AP. Therefore, a more parsimonious explanation for these data is that 512
partial blockade of IA diminishes its ability to gate burst amplifying currents during the 513
preinspiratory phase. 514
Increased activity in dorsomedial areas outside the preBötC suggests three possibilities: 1) 515
activity in the preBötC is augmented, 2) preBötC activity propagates more readily to 516
downstream areas, or 3) that excitability is increased in dorsomedial pre-motor or motor pools. 517
Ca2+ activity measured in neurons within the preBötC suggests local network activity may 518
indeed be augmented. Ca2+ transients in both somata and the neuropil (dominated by 519
dendrites) lasted longer and decayed more slowly following a 50 μM dose of 4-AP, 520
demonstrating that burst activity at the systems level is prolonged when IA is partially blocked. 521
We failed to pharmacologically characterize the IA-like current present in the preBötC after 522
using three different channel-specific Kv4 inhibitors—the compounds did not affect rhythmic 523
output. Thus, pharmacological specificity and molecular identification of the channel types 524
giving rise to IA in the preBötC remains an issue. Transcriptomic data show that preBötC 525
neurons sharing a common lineage with type-1 neurons (i.e. Dbx1-derived cells) differentially 526
and robustly express Kv4.2 with KChIP2 (Hayes et al., 2017), forming IA-like channels that favor 527
non-inactivated states with relatively fast deinactivation kinetics (Amadi et al., 2007). The 528
transcriptome study catalogs all K+ channels in rhythmogenic and non-rhythmogenic preBötC 529
neurons (Hayes et al., 2017). Specific targeting of these channel types (using genetic rather than 530
pharmacological tools alone) will be necessary to assess the rhythmogenic role of IA. 531
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To determine what IA might be doing at the cellular level, we needed a means of interrogating 532
activity in the dendrites of rhythmically active preBötC neurons. Organotypic slice cultures 533
containing the preBötC flatten out and become transparent over several days, which improves 534
the signal-to-noise ratio of Ca2+ fluorescence dendritic imaging. To better understand how type-535
1 and type-2 neurons integrate synaptic input, we then asked whether IA in rhythmically active 536
neurons exists on dendrites and whether it has a significant impact on the ability for voltage to 537
spread between somatic and distal dendritic compartments. 538
Blockade of IA in type-1 neurons results in a ~130 % increase in the amplitude of ramp-evoked 539
Ca2+ transients at proximal dendritic regions and a ~280 % increase at distal dendritic regions. In 540
contrast, type-2 neurons show no significant increase at proximal or distal dendritic regions 541
after bath application of 4-AP. Global increases in the Ca2+ transients of type-1 neurons may 542
reflect a change in the electrotonic compactness of the neuron: more of the somatically-543
triggered depolarization propagates from the soma to the distal dendrite. Alternatively, 544
depolarization triggered at the soma might be less counteracted by IA before passively 545
propagating along dendrites. 546
Bath application of 4-AP mediated a ~2-8 % rise in the maximum amplitude of delivered voltage 547
ramps in both type-1 and type-2 neurons, signifying a change in the strength of the SEVC. By 548
some immeasurable degree, the consistent increase in ramp amplitudes definitely contributes 549
to the observed change in evoked transients on both proximal and distal dendrites. The 550
strength of the SEVC, and by extension the ability to increase the membrane potential of the 551
neuron, is thus correlated with the presence or absence of IA, supporting the hypothesis that it 552
affects either electrotonic compactness or resists membrane depolarization. The data from 553
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these experiments is unable to distinguish whether the apparent increase in electrotonic 554
compactness, or reduced inhibition of membrane depolarization, is the result of solely somatic 555
IA or additionally includes dendritic IA. To definitively determine whether the increases in the 556
amplitude of ramp-evoked Ca2+ transients were caused by dendritic IA in conjunction with 557
somatic IA, we attempted to locally block IA on dendrites of rhythmically active neurons. 558
Spike train-evoked Ca2+ fluorescence increased by ~155 % at local sites of 4-AP iontophoresis, 559
with no change in fluorescence in other compartments located either proximally on the same 560
parent branch, at the soma, or on other dendritic branches occupying the same focal plane. 561
Thus, these results indeed suggest that the density of ion channels giving rise to IA in type-1 562
preBötC neurons extends well beyond the soma into distal dendritic compartments. It is 563
important to distinguish that increased dendritic Ca2+ influx in the presence of 4-AP does not 564
necessarily map one-to-one with a rise in voltage, but is generally indicative of changes in 565
voltage. The non-linear increase in evoked Ca2+ transients at distal dendritic sites can be equally 566
interpreted as a reduction in voltage decay between the soma and dendrites or as the 567
recruitment of previously inhibited inward currents. Considering the overall global increase in 568
Ca2+response, IA at minimum alters electrotonic compactness since all cellular compartments 569
appear to charge in response to voltage stimuli more effectively. 570
Finally, we show that dendritic IA blockade increases the amplitude of glutamate-evoked 571
postsynaptic depolarizations recorded at the soma of type-1 neurons (Figure 6). Small-572
amplitude glutamate responses (5-15 mV) triggered at distal dendritic sites became augmented 573
when nearby dendritic IA was blocked. Thus, IA demonstrably dampens small-amplitude 574
dendritic input. 575
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The data suggest that IA on the dendrites can counteract sparse excitatory synaptic events in 576
preBötC neurons. Nevertheless, during oscillatory activity in the preBötC, IA would inactivate via 577
temporally-summating excitatory inputs lasting 200 ms (or more). Preinspiratory activity of 578
type-1 neurons exceeds 400 ms (Rekling et al., 1996), indicating that building recurrent 579
excitation ordinarily outlasts the transient phase of IA, which would allow steady-state 580
inactivation to occur during a typical inspiratory cycle in type-1 neurons. (Hayes et al., 2008) 581
estimated the contribution of IA to the currents flowing in preBötC neurons during an 582
inspiratory burst, and found that IA rapidly achieves maximal activation, diminished throughout 583
the burst, and carries very little current during the inter-inspiratory burst interval. With IA 584
unavailable due to inactivation, type-1 neurons may transition from a relatively low-excitability 585
state—in which excitatory synaptic input is presumably inhibited by IA—to a high-excitability 586
state that is more electrotonically compact and where amplifying currents, such as ICAN are 587
more easily recruited. It would also explain why preinspiratory activity in type-1 neurons 588
becomes significantly shorter and rises more rapidly after network-wide block of IA in acute 589
slices (Hayes et al., 2008). This type of activity-dependent integration emphasizes why type-1 590
neurons expressing IA may be most critical in dictating the appropriately-timed onset of 591
inspiratory burst cycles. 592
In conclusion, IA is dendritically expressed in type-1 preBötC neurons, which ordinarily 593
diminishes excitatory synaptic input. However, the shunting role of IA can be relieved by steady-594
state inactivation. Apparent changes in electrotonic compactness after blockade of IA support 595
the notion that IA decreases the length constant of dendrites and shunts incoming synaptic 596
potentials, minimizing their influence on somatic voltage trajectory. During rhythmic network 597
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activity, steady-state inactivation of dendritic IA could be achieved via building recurrent 598
excitation during the pre-inspiratory phase of the inspiratory cycle. IA in the dendrites of type-1 599
neurons could thus limit the effects of synaptic inputs that arrive asynchronously or 600
sporadically. When activity in this recurrently connected network accumulates during each 601
respiratory cycle, IA likely inactivates, promoting depolarization and subsequent burst 602
generation. Being colocalized at both the site of excitatory input and postsynaptic drive 603
amplification (i.e. dendrites), IA is thus able to integrate the state of the network as it 604
stochastically builds in tonic excitability during recurrent excitation. A robust amplification of 605
synaptic drive will occur only when network activity reaches a degree of persistent coincident 606
activity capable of inactivating IA. 607
Legends 608
Figure 1 609
4-AP increases burst frequency, dorsomedial burst amplitude, and duration of soma and 610
dendritic Ca2+ transients. A: Left panel. Ca2+ transients in a Fluo-8, AM loaded slice culture 611
before (Control) and after bath applied 4-AP (50 μM). Right panel: Group data for bath applied 612
IA-blockers 4-AP (50 μM, n = 7), PiTX (Pandinotoxin-Kα, 40 nM, n = 3), PaTx1 (Phrixotoxin-1, 500 613
nM, n = 3), AmmTX3 (100 nM, n = 3). Note that only 4-AP showed an increase in burst. B: Cycle-614
triggered averages of F/F0 in a culture at low magnification before and after bath applied 4-AP 615
(50 μM). Right panels are 50 pixel averages of the left panel images showing grayscale-coded 616
normalized (min: black, max: white) averages. M: midline, v: ventral, d: dorsal. Note that 4-AP 617
induces a relative increased activity in dorsomedial regions (lower right), but that this type of 618
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non-ratiometric AM dye-based Ca2+ imaging cannot report on absolute amplitudes in a given 619
region before and after 4-AP. C: Left panel: Cycle-triggered averages of F/F0 in a slice culture 620
at medium magnification. Individual somas and Fluo-8, AM labeled neuropil in the PreBötC are 621
visible. Right panel: Region of interest over 10 somatas (circles) and the neuropil (black filled 622
areas). D: Left panel: Blow-up of region defined by the dotted line in C right panel, showing 623
individual somatas and dendritic profiles in the neuropil. Right panel: Amplitude-normalized 624
averages of the raw burst Ca2+ signal (F) from the 10 somas and neuropil, before (Cont.) and 625
after 4-AP (50 μM, 4-AP). Note the increase in duration of the Ca2+ signal in both soma and 626
dendrite regions. E: Group data of the half-amplitude width, decay time constant (TauM), and 627
risetime-10-90 % for raw burst Ca2+ signals in soma and dendritic regions before and after 4-AP 628
(50μM, n = 7). Note an increase in width, TauM, but not risetime in response to 4-AP. 629
Figure 2 630
Electroresponsive properties of oscillating type-1 and type-2 neurons. A: Spontaneous 631
oscillatory burst activity in two neurons recorded in current camp mode. Note that the burst in 632
the left-most neuron show afterhyperpolarizations following the bursts. B: Depolarizing and 633
hyperpolarizing square current pulses from a slightly hyperpolarized membrane potential, and 634
around resting Vm, give rise to two distinctive electroresponsive responses in the two neurons. 635
The type-1 neuron show delayed excitation (arrow, top left trace), and the type-2 neuron show 636
a ‘sag’ potential (arrow, lower right). Voltage traces are cycle-triggered averages of 5-10 637
sweeps, which truncates action potentials but retains the form of delayed excitation and ‘sag’ 638
potentials. 639
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31
Figure 3 640
Bath applied 4-AP increase dendritic Ca2+ transients in response to ramp depolarizations. A: 641
Voltage clamp traces, with TTX (1 μM) present in the ACSF, before and after adding 2 mM 4-AP 642
to the bath. Left-most neuron show a transient outward current, which is blocked by 4-AP 643
classifying the neuron as a type-1. Right-most neuron show no evidence out a transient 644
outward current, classifying the neuron as a type-2. B: Ca2+ transients in proximal and distal 645
dendritic compartments in the two neurons in response to a 150 ms voltage ramp (-80 to 20 646
mV), and associated voltage and current traces. Responses before and after adding 2 mM 4-AP 647
to the bath is overlaid. Blue lines: Proximal dendritic compartments. Green lines: Distal 648
dendritic compartments. C: Live morphology (Alexa 594 hydrazide present in the patch 649
solution) of the two neurons with thresholded dendritic Ca2+ transient amplitude overlaid in 650
red. Red indicates Ca2+ transient amplitudes ( F/F0) above 100 %. D: Group data (mean ± SEM) 651
expressing the relative Ca2+-transient amplitude after 4-AP- from a sample of type-1 (T-1, n = 6) 652
and type-2 (T-2, n = 5) neurons. Note that both proximal and distal dendritic regions of type-1 653
neurons show a larger Ca2+ transient amplitude after 4-AP (*), and that distal regions show a 654
larger increase than proximal regions (#). 655
Figure 4 656
Dendritic iontophoresis of Cd2+ reduce dendritic Ca2+-transients evoked by current pulses. A: 657
200 ms current pulses applied to a type-1 neuron evoking a spike train, and the resulting Ca2+ 658
transient in a dendritic compartment (red trace, ~130 μm from soma, at the Cd2+ application 659
site). Iontophoresis of Cd2+ (2 min, 500 ms pules, 1 Hz) reduced the dendritic Ca2+ transient 660
Page 32
32
(black trace). B: Same neuron as in A, showing the spatial distribution of the Cd2+ effect, 661
expressed as attenuation (Red: 0 to Blue: 0.8). Note that the site of application (pipette insert) 662
has the largest attenuation compared to more proximal sites along the dendrite. C: Group data 663
(n = 7) showing the attenuation of spike-train evoked Ca2+ transients in response to Cd2+ as a 664
function of distance along the dendrite towards the soma. Black line is a linear fit. 665
Figure 5 666
Dendritic iontophoresis of 4-AP increases dendritic Ca2+ transients evoked by current pulse 667
trains. A: A train of current pulses (10 pulses, 3 ms duration, 100 Hz) applied to a type-1 neuron 668
evoking 10 spikes. The resulting Ca2+ transients in a dendritic compartment (~110 μm from 669
soma, placed next to the 4-AP pipette) is shown above the spikes before, and after (overlaid 670
green traces) iontophoresis of 4-AP (7 min, 500 ms pulses, 1 Hz). Overlaid Ca2+ transients before 671
and after 4-AP is also shown for another more proximal dendritic site close to soma (blue 672
traces). B: Same neuron as in A, showing the color coded spatial distribution of the 4-AP effect, 673
expressed as Ca2+ transient amplitude after 4-AP (Blue: 105 % to Red: 120 %). Note that the site 674
of application (pipette insert) has the largest increase in the Ca2+ transient compared to more 675
proximal sites along the dendrite. C: Group data (mean ± SEM) showing the Ca2+ transient 676
amplitude after 4-AP application for proximal and distal (next to 4-AP pipette) sites in the two 677
types of neurons (n = 9 type-1 neurons, and n = 3 type-2 neurons). Black dotted line is 100 %, 678
i.e. no change. Note that distal dendritic sites in type-1 neuron show a large increase in Ca2+-679
transients after application of 4-AP (***). 680
Figure 6 681
Page 33
33
The amplitude and width of dendritic glutamate responses are increased by dendritic 682
iontophoresis of 4-AP in type-1 neurons. A: Morphology of a live type-1 neuron (Alexa 594 683
hydrazide present in the patch solution) with a somatic recording pipette (Rec.), and a 684
dendritically placed (138 μm from soma) theta-iontophoresis pipette containing glutamate in 685
one barrel and 4-AP in the other (Glu/4-AP). B: Repetitive 100 ms-duration iontophoretic pulses 686
of glutamate in the presence of bath-applied TTX before (Cont.) and after iontophoresis of 4-AP 687
(20 s). C: Overlaid traces of the glutamate response before (Cont.) and after 4-AP iontophoresis 688
(4-AP). Note the increase in amplitude and width of the glutamate response. 689
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776
A B
C D
1 s
Avg. 10 somas
Avg. neuropil
Norm.F
10 somasand neuropil
ControlControl
4-AP 4-AP
4-AP
4-AP
Cont.
Cont.
0
0.5
500 μm 50 μm
5 s0
5�F/F %0
1 mm
m
v
d
E
Contsoma
4-APsoma
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4-APneuropil
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4-APsoma
Contneuropil
4-APneuropil
0.0
1.0
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3.0
TauM
(s)
0
200
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(ms)
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500
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1500
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Half-a
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idth
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s) **
**
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4-AP PiTX PaTx1 AmmTX3
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% o
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100
200
300
A
BType-1
Type-2
Vm
(m
V)
200 ms
-40
-20
-90-80-70-60
-80
-60
-40
-20
5 s 10 s
Type-1 Type-2
20 mV
0.4 nA
C
D
B
A
Control
4- (bath)AP
Type-1
Type-1
�F/F >100%0
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(m
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I (n
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I (n
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Distal
4-AP
4-AP
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Control4- (bath)AP 4- (bath)AP
200 ms
100 ms
-80
0.4
0
-0.4
- 040
- 08
20
0
1
�F/F %0
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0
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Type-2
Control
4- (bath)AP
50 μm
50 μm
*
*#
Ca
-tra
nsie
nt am
plit
ude
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after
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.0
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400
600
A B
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0
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Cd2+
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0 40 80 120 1600.0
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tion
Distance from Cd2+
p μmipette ( )
25 μm
20 mV
0.2nA
100 ms
0
5�F/F %0
A B
4-APiont.
Ca -transient amplitude2+
after 4- (%)AP105
120
4-AP
Distal
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4-AP
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Control
0
1�F/F %0
40 mV
1nA
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200 msC
Ca
-tra
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100
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180
220
Type-1distal
***
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or otherdendrite
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A B
50 μm
Rec.
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Glu
30 s
10 mV
Glu Glu
4-AP
C
500 ms
10 mV
Glu
4-AP
Cont.