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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: jrekling@sund.ku.dk

Cite as: J. Neurosci ; 10.1523/JNEUROSCI.3342-17.2018

Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formattedversion of this article is published.

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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: jrekling@sund.ku.dk 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

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0

0.5

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-80

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-40

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