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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.
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.
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.
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
Chemical | aCSF (mM) | aCSF (g/100 mL) | Sucrose-substituted aCSF (mM) | Sucrose-substituted aCSF (g/100 mL) | High-potassium aCSF (mM) | High-potassium aCSF (g/100 mL) |
Sodium chloride (NaCl) | 118 | 0.690 | - | - | 118 | 0.690 |
Sodium hydrogen carbonate (NaHCO3) | 25 | 0.210 | 25 | 0.210 | 25 | 0.210 |
Glucose | 10 | 0.180 | 10 | 0.180 | 10 | 0.180 |
Potasium chloride (KCl) | 2.5 | 0.019 | 2.5 | 0.019 | 4.5 | 0.034 |
Sodium dihydrogen phosphate (NaH2PO4) | 1 | 0.012 | 1 | 0.012 | 1 | 0.012 |
Magnesium cloride (MgCl2) | 1 | 0.01 | 1 | 0.01 | 1 | 0.01 |
Calcium chloride (CaCl2) | 2.5 | 0.028 | 2.5 | 0.028 | 2.5 | 0.028 |
Sucrose | - | - | 250 | 8.558 | - | - |
Table 1: Artificial Cerebrospinal Fluid compositions. Abbreviation: aCSF = artificial cerebrospinal fluid.
Figure 1: Spinal cord slice orientations, mounting and cutting methods. (A) Transverse slices require a Styrofoam cutting block with a supporting groove cut into it. The spinal cord is rested against the block in the support groove, the dorsal side of the cord facing away from the block. The block and cord are glued onto a cutting stage with cyanoacrylate adhesive. (B) Sagittal slices are prepared by placing a thin line of cyanoacrylate adhesive on the cutting stage and then positioning the spinal cord on its side on the glue. (C) Horizontal slices are prepared by placing a thin line of cyanoacrylate adhesive on the cutting stage and then positioning the spinal cord ventral side down on the glue. Please click here to view a larger version of this figure.
Microelectrode Array Layouts | ||||
Microelectrode Array Model | 60MEA 200/30iR-Ti | 60-3DMEA 100/12/40iR-Ti | 60-3DMEA 200/12/50iR-Ti | 60MEA 500/30iR-Ti |
Planar or 3-Dimensional (3D) | Planar | 3D | 3D | Planar |
Electrode Grid | 8 x 8 | 8 x 8 | 8 x 8 | 6 x 10 |
Electrode Spacing | 200 µm | 100 µm | 200 µm | 500 µm |
Electrode Diameter | 30 µm | 12 µm | 12 µm | 30 µm |
Electrode Height (3D) | N/A | 40 µm | 50 µm | N/A |
Experiments | Transverse slice | Transverse slice | Sagittal + Horizontal | Sagittal + Horizontal |
Table 2: Microelectrode array layouts.
Figure 2: Tissue positioning on the microelectrode array. (A) Image shows an open MEA headstage with an MEA placed in position. (B) Same as A with MEA headstage closed for recordings and tissue perfusion system in place. (C) Image shows an MEA as supplied by the manufacturer. Contact pads, which interface with the gold springs of the headstage, and the MEA tissue bath that holds the tissue bathing solution and tissue slice are shown. The area highlighted by the red square in the center is the location of the electrode array. (D) Schematics show the two MEA electrode configurations used in this study, with further details presented in Table 2. The reference electrode is denoted by the blue trapezoid. The left MEA electrode layout shows a 60-electrode square configuration, used most in the presented work-models 60MEA200/30iR-Ti with 30 μm diameter electrodes spaced 200 μm apart, or 200 μm spaced and 100 μm spaced 3-dimensional MEAs (60MEA200/12/50iR-Ti and 60MEA100/12/40iR-Ti) with electrodes 12 μm in diameter and either 50 μm or 40 μm high, respectively. The left MEA electrode layout shows a 6 x 10 electrode rectangular layout-60MEA500/30iR-Ti. (E) High-magnification image of a 60MEA100/12/40iR-Ti square MEA with transverse spinal cord slice positioned for recording. The slice sits on electrode rows 3-8. The top row of electrodes, which do not contact any tissue, serve as reference electrodes. The SDH area appears as a semitransparent band. In this case, the SDH overlies electrodes in rows 4, 5, and 6 and columns 2, 3, 4, 5, and 7 of the MEA. Scale bar = 200 µm. Abbreviations: MEA = microelectrode array; SDH = superficial dorsal horn. Please click here to view a larger version of this figure.
2. Data processing and analysis
NOTE: The following steps detail how to use the analysis software for MEA experiments on spinal cord slices. One of the 60 electrodes serves as an internal reference (marked by a trapezoid in Figure 2 C,D), while between four and twenty-five of the remaining 59 are positioned under the SDH in an adult mouse spinal cord slice. Subsequent analysis detects extracellular action potential (EAP) and local field potential (LFP) waveforms (see Figure 3B for examples) from the raw signal in this region.
Figure 3: Data recording and analysis tool layouts and example microelectrode array recordings showing extracellular action potential and local field potential waveforms. (A) Schematic shows preconfigured recording templateused for the acquisition of MEA data. Linking the MEA2100 and the recording (headstage/amplifier) tool enables the data to be named and saved. Four example traces of raw data (right, 5-min epochs) were collected by one MEA channel showing activity at baseline, 12 min after 4-AP application, a further 15 min after established 4-AP activity, and following bath application of TTX (1 µM). Note, the addition of 4-AP (second trace) produces a clear increase in background noise and EAP/LFP activity. Importantly, the activity remains relatively stable for at least 15 min after 4-AP-induced activity is established (third trace). Addition of TTX (1 µM) abolishes all activity (bottom trace). (B) Schematic (left) shows analyzer software configuration for data analysis. The raw data explorer tool is used to import recordings collected by recording software. These data are then run through a cross-channel filter tool that subtracts the selected reference electrode(s) signal(s) from other electrodes to remove background noise. Data pass through the EAP filter and the LFP filter tools to optimize signal-to-noise relationships for each waveform. Following this step, the EAP path data enter the EAP detector tool, where thresholds are set. EAPs are detected and then sent to the EAP analyzer tool where the latencies of each event are recorded and exported as a txt. file. An identical workflow occurs for LFP data using a corresponding LFP toolkit. Right traces show data from a single MEA channel containing various extracellular waveforms. Location of EAP and LFP signals are highlighted in the above 'count rasters.' Lower traces are epochs from upper recording (denoted by red bars) showing waveforms on an expanded timescale, including various LFP signals (note the variety of appearances) and individual extracellular EAPs (red circles). Note, LFP/EAP waveform and polarity vary relative to the number of neurons producing these signals, their proximity to the recording electrode, and their location in relation to the nearby electrode(s). Abbreviations: MEA = microelectrode array; EAP = extracellular action potential; LFP = local field potential; 4-AP = 4-aminopyridine; TTX = tetrodotoxin. Please click here to view a larger version of this figure.
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 ...
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...
The authors have no conflicts of interest to declare.
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.).
Name | Company | Catalog Number | Comments |
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 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 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 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. |
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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