Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays

Published: February 09, 2022

doi:

Jacqueline A Iredale, Jeremy G. Stoddard, Hannah R. Drury, Tyler J. Browne, Augustus Elton, Jessica F. Madden, Robert J. Callister, James S. Welsh, Brett A. Graham

¹School of Biomedical Sciences and Pharmacy,University of Newcastle, ²School of Electrical Engineering,University of Newcastle

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. To date, most studies have investigated the role of DH neurons at either the single-cell or whole-organism level of analysis using combinations of in vitro intracellular electrophysiology, neuroanatomical labeling, and in vivo behavioral analysis¹^,³^,⁸^,⁹^,¹⁰^,¹¹^,¹²^,¹³^,¹⁴. These approaches have significantly advanced the understanding of the role of specific neuron populations in pain processing. However, a gap remains in understanding how specific cell types and small macro-circuits influence large populations of neurons at a microcircuit level to subsequently shape the output of the DH, behavioral responses, and the pain experience.

One technology that can investigate macro-circuit or multicellular-level function is the microelectrode array (MEA)¹⁵^,¹⁶. MEAs have been used to investigate nervous system function for several decades¹⁷^,¹⁸. In the brain, they have facilitated the study of neuronal development, synaptic plasticity, pharmacological screening, and toxicity testing¹⁷^,¹⁸. They can be used for both in vitro and in vivo applications, depending on the type of MEA. Furthermore, the development of MEAs has evolved rapidly, with different electrode numbers and configurations now available¹⁹. A key advantage of MEAs is their capacity to simultaneously assess electrical activity in many neurons with high spatial and temporal accuracy via multiple electrodes¹⁵^,¹⁶. This provides a broader readout of how neurons interact in circuits and networks, under control conditions and in the presence of locally applied compounds.

One challenge of in vitro DH preparations is that ongoing activity levels are typically low. Here, this challenge is addressed in spinal cord DH circuits using the voltage-gated K⁺ channel blocker, 4-aminopryidine (4-AP), to chemically stimulate DH circuits. This drug has previously been used to establish rhythmic synchronous electrical activity in the DH of acute spinal cord slices and under acute in vivo conditions²⁰^,²¹^,²²^,²³^,²⁴. These experiments have used single-cell patch and extracellular recording or calcium imaging to characterize 4-AP-induced activity²⁰^,²¹^,²²^,²³^,²⁴^,²⁵. Together, this work has demonstrated the requirement of excitatory and inhibitory synaptic transmission and electrical synapses for rhythmic 4-AP-induced activity. Thus, the 4-AP response has been viewed as an approach that unmasks native polysynaptic DH circuits with biological relevance rather than as a drug-induced epiphenomenon. Furthermore, 4-AP-induced activity exhibits a similar response profile to analgesic and antiepileptic drugs as neuropathic pain conditions and has been used to propose novel spinally-based analgesic drug targets such as connexins²⁰^,²¹^,²².

Here, a preparation that combines MEAs and chemical activation of the spinal DH with 4-AP to study this nociceptive circuitry at the macro-circuit, or network level of analysis, is described. This approach provides a stable and reproducible platform for investigating nociceptive circuits under naive and neuropathic 'pain-like' conditions. This preparation is also readily applicable to test the circuit-level action of known analgesics and to screen novel analgesics in the hyperactive spinal cord.

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 Preparation of solutions for spinal cord slice preparation and recording 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…

Disclosures

The authors have nothing to disclose.

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

4-aminopyridine	Sigma-Aldrich	275875-5G
100% ethanol	Thermo Fisher	AJA214-2.5LPL
CaCl₂ 1M	Banksia Scientific	0430/1L
Carbonox (Carbogen – 95% O₂, 5% CO₂)	Coregas	219122
Curved long handle spring scissors	Fine Science Tools	15015-11
Custom made air interface incubation chamber
Foetal bovine serum	Thermo Fisher	10091130
Forceps Dumont #5	Fine Science Tools	11251-30
Glucose	Thermo Fisher	AJA783-500G
Horse serum	Thermo Fisher	16050130
Inverted microscope	Zeiss	Axiovert10
KCl	Thermo Fisher	AJA383-500G
Ketamine	Ceva	KETALAB04
Large surgical scissors	Fine Science Tools	14007-14
Loctite 454 Instant Adhesive	Bolts and Industrial Supplies	L4543G
MATLAB	MathWorks		R2018b
MEAs, 3-Dimensional	Multichannel Systems	60-3DMEA100/12/40iR-Ti, 60-3DMEA200/12/50iR-Ti	60 titanium nitride (TiN) electrodes with 1 internal reference electrode, organised in an 8×8 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 headstage	Multichannel Systems	MEA2100-HS60
MEA interface board	Multichannel Systems	MCS-IFB 3.0 Multiboot
MEA net	Multichannel Systems	ALA HSG-MEA-5BD
MEA perfusion system	Multichannel Systems	PPS2
MEAs, Planar	Multichannel Systems	60MEA200/30iR-Ti, 60MEA500/30iR-Ti	60 titanium nitride (TiN) electrodes with 1 internal reference electrode, organised in either a 8×8 square grid (200/30) or a 6×10 rectangular grid (500/30). Electrodes are 30 µm in diameter and equidistantly spaced 200 µm (200/30) or 500 µm (500/30) apart.
MgCl₂	Thermo Fisher	AJA296-500G
Microscope camera	Motic	Moticam X Wi-Fi
Multi Channel Analyser software	Multichannel Systems		V 2.17.4
Multi Channel Experimenter software	Multichannel Systems		V 2.17.4
NaCl	Thermo Fisher	AJA465-500G
NaHCO₃	Thermo Fisher	AJA475-500G
NaH₂PO₄	Thermo Fisher	ACR207805000
Rongeurs	Fine Science Tools	16021-14
Small spring scissors	Fine Science Tools	91500-09
Small surgical scissors	Fine Science Tools	14060-09
Sucrose	Thermo Fisher	AJA530-500G
Superglue			cyanoacrylate adhesive
Tetrodotoxin	Abcam	AB120055
Vibration isolation table	Newport	VH3048W-OPT
Vibrating microtome	Leica	VT1200 S

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Iredale, J. A., Stoddard, J. G., Drury, H. R., Browne, T. J., Elton, A., Madden, J. F., Callister, R. J., Welsh, J. S., Graham, B. A. Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays. J. Vis. Exp. (180), e62920, doi:10.3791/62920 (2022).