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.
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 nothing to disclose.
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.).
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 |