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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The combined use of microelectrode array technology and 4-aminopyridine-induced chemical stimulation for investigating network-level nociceptive activity in the spinal cord dorsal horn is outlined.

Abstract

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an increasingly detailed view of the circuits underpinning spinal pain processing. However, the effects of these connections for broader network activity in the DH remain less well understood because most studies focus on the activity of single neurons and small microcircuits. Alternatively, the use of microelectrode arrays (MEAs), which can monitor electrical activity across many cells, provides high spatial and temporal resolution of neural activity. Here, the use of MEAs with mouse spinal cord slices to study DH activity induced by chemically stimulating DH circuits with 4-aminopyridine (4-AP) is described. The resulting rhythmic activity is restricted to the superficial DH, stable over time, blocked by tetrodotoxin, and can be investigated in different slice orientations. Together, this preparation provides a platform to investigate DH circuit activity in tissue from naïve animals, animal models of chronic pain, and mice with genetically altered nociceptive function. Furthermore, MEA recordings in 4-AP-stimulated spinal cord slices can be used as a rapid screening tool to assess the capacity of novel antinociceptive compounds to disrupt activity in the spinal cord DH.

Introduction

The roles of specific types of inhibitory and excitatory interneurons within the spinal cord DH are being uncovered at a rapid rate1,2,3,4. Together, interneurons make up over 95% of the neurons in the DH and are involved in sensory processing, including nociception. Furthermore, these interneuron circuits are important for determining whether peripheral signals ascend the neuroaxis to reach the brain and contribute to the perception of pain5,6,7. T....

Protocol

Studies were carried out on male and female c57Bl/6 mice aged 3-12 months. All experimental procedures were performed in accordance with the University of Newcastle's Animal Care and Ethics Committee (protocols A-2013-312, and A-2020-002).

1. In vitro electrophysiology

  1. Preparation of solutions for spinal cord slice preparation and recording
    1. Artificial cerebrospinal fluid
      ​NOTE: Artificial cerebrospinal fluid (aCSF) is used in an interface incu.......

Representative Results

Model of network activity in the spinal cord dorsal horn
Application of 4-AP reliably induces synchronous rhythmic activity in the spinal cord DH. Such activity presents as increased EAPs and LFPs. The later signal is a low-frequency waveform, which has previously been described in MEA recordings30. Changes in EAP and/or LFP activity following drug application reflect altered neural activity. Examples of EAPs and LFPs are shown in Figure 3B and .......

Discussion

Despite the importance of the spinal DH in nociceptive signaling, processing, and the resulting behavioral and emotional responses that characterize pain, the circuits within this region remain poorly understood. A key challenge in investigating this issue has been the diversity of neuron populations that comprise these circuits6,31,32. Recent advances in transgenic technologies, led by optogenetics and chemogenetics, are beginn.......

Acknowledgements

This work was funded by the National Health and Medical Research Council (NHMRC) of Australia (grants 631000, 1043933, 1144638, and 1184974 to B.A.G. and R.J.C.) and the Hunter Medical Research Institute (grant to B.A.G. and R.J.C.).

....

Materials

NameCompanyCatalog NumberComments
4-aminopyridineSigma-Aldrich275875-5G
100% ethanolThermo FisherAJA214-2.5LPL
CaCl2 1MBanksia Scientific0430/1L
Carbonox (Carbogen - 95% O2, 5% CO2)Coregas219122
Curved long handle spring scissorsFine Science Tools15015-11
Custom made air interface incubation chamber
Foetal bovine serumThermo Fisher10091130
Forceps Dumont #5Fine Science Tools11251-30
GlucoseThermo FisherAJA783-500G
Horse serumThermo Fisher16050130
Inverted microscopeZeissAxiovert10
KClThermo FisherAJA383-500G
KetamineCevaKETALAB04
Large surgical scissorsFine Science Tools14007-14
Loctite 454 Instant AdhesiveBolts and Industrial SuppliesL4543G
MATLABMathWorksR2018b
MEAs, 3-DimensionalMultichannel Systems60-3DMEA100/12/40iR-Ti, 60-3DMEA200/12/50iR-Ti60 titanium nitride (TiN) electrodes with 1 internal reference electrode, organised in an 8x8 square grid. Electrodes are 12 µm in diameter, 40 µm (100/12/40) or 50 µm (200/12/50) high and equidistantly spaced 100 µm (100/12/40) or 200 µm (200/12/50) apart.
MEA headstageMultichannel SystemsMEA2100-HS60
MEA interface boardMultichannel SystemsMCS-IFB 3.0 Multiboot
MEA netMultichannel SystemsALA HSG-MEA-5BD
MEA perfusion systemMultichannel SystemsPPS2
MEAs, PlanarMultichannel Systems60MEA200/30iR-Ti, 60MEA500/30iR-Ti60 titanium nitride (TiN) electrodes with 1 internal reference electrode, organised in either a 8x8 square grid (200/30) or a 6x10 rectangular grid (500/30). Electrodes are 30 µm in diameter and equidistantly spaced 200 µm (200/30) or 500 µm (500/30) apart.
MgCl2Thermo FisherAJA296-500G
Microscope cameraMoticMoticam X Wi-Fi
Multi Channel Analyser softwareMultichannel SystemsV 2.17.4
Multi Channel Experimenter softwareMultichannel SystemsV 2.17.4
NaClThermo FisherAJA465-500G
NaHCO3Thermo FisherAJA475-500G
NaH2PO4Thermo FisherACR207805000
RongeursFine Science Tools16021-14
Small spring scissorsFine Science Tools91500-09
Small surgical scissorsFine Science Tools14060-09
SucroseThermo FisherAJA530-500G
Supergluecyanoacrylate adhesive
TetrodotoxinAbcamAB120055
Vibration isolation tableNewportVH3048W-OPT
Vibrating microtomeLeicaVT1200 S

References

  1. Smith, K. M., et al. Calretinin positive neurons form an excitatory amplifier network in the spinal cord dorsal horn. eLife. 8, 49190 (2019).
  2. Smith, K. M., et al.

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