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.).

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&#39;s Animal Care and Ethics Committee (protocols A-2013-312, and A-2020-002).
1. In vitro electrophysiology
<ol>
	<li>Preparation of solutions for spinal cord slice preparation and recording
	<ol>
		<li>Artificial cerebrospinal fluid 
		&#8203;NOTE: Artificial cerebrospinal fluid (aCSF) is used in an interface incu...

<ol>
	<li>Smith, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calretinin+positive+neurons+form+an+excitatory+amplifier+network+in+the+spinal+cord+dorsal+horn.">Calretinin positive neurons form an excitatory amplifier network in the spinal cord dorsal horn.</a> eLife. 8, 49190 (2019).</li><li>Smith, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+heterogeneity+of+calretinin-expressing+neurons+in+the+mouse+superficial+dorsal+horn:+implications+for+spinal+pain+processing.">Functional heterogeneity of calretinin-expressing neurons in the mouse superficial dorsal horn: implications for spinal pain processing.</a> The Journal of physiology. 593 (19), 4319-4339 (2015).</li><li>Boyle, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+a+spinal+microcircuit+that+gates+myelinated+afferent+input:+Implications+for+tactile+allodynia.">Defining a spinal microcircuit that gates myelinated afferent input: Implications for tactile allodynia.</a> Cell Reports. 28 (2), 526-540 (2019).</li><li>Browne, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+cross-referencing+of+inhibitory+and+excitatory+interneuron+populations+to+dissect+neuronal+heterogeneity+in+the+dorsal+horn.">Transgenic cross-referencing of inhibitory and excitatory interneuron populations to dissect neuronal heterogeneity in the dorsal horn.</a> Frontiers in Molecular Neuroscience. 13, 32 (2020).</li><li>Graham, B. A., Hughes, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rewards,+perils+and+pitfalls+of+untangling+spinal+pain+circuits.">Rewards, perils and pitfalls of untangling spinal pain circuits.</a> Current Opinion in Physiology. 11, 35-41 (2019).</li><li>Todd, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+circuitry+for+pain+processing+in+the+dorsal+horn.">Neuronal circuitry for pain processing in the dorsal horn.</a> Nature Reviews Neuroscience. 11 (12), 823-836 (2010).</li><li>Hughes, D. I., Todd, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+nervous+system+targets:+inhibitory+interneurons+in+the+spinal+cord.">Central nervous system targets: inhibitory interneurons in the spinal cord.</a> Neurotherapeutics. 17 (3), 874-885 (2020).</li><li>Duan, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+spinal+circuits+transmitting+and+gating+mechanical+pain.">Identification of spinal circuits transmitting and gating mechanical pain.</a> Cell. 159 (6), 1417-1432 (2014).</li><li>Hachisuka, J., Chiang, M. C., Ross, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Itch+and+neuropathis+itch.">Itch and neuropathis itch.</a> Pain. 159 (3), 603 (2018).</li><li>Foster, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+ablation,+silencing,+and+activation+establish+glycinergic+dorsal+horn+neurons+as+key+components+of+a+spinal+gate+for+pain+and+itch.">Targeted ablation, silencing, and activation establish glycinergic dorsal horn neurons as key components of a spinal gate for pain and itch.</a> Neuron. 85 (6), 1289-1304 (2015).</li><li>Bourane, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+spinal+circuit+for+light+touch+and+fine+motor+control.">Identification of a spinal circuit for light touch and fine motor control.</a> Cell. 160 (3), 503-515 (2015).</li><li>Cheng, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+spinal+circuits+involved+in+touch-evoked+dynamic+mechanical+pain.">Identification of spinal circuits involved in touch-evoked dynamic mechanical pain.</a> Nature neuroscience. 20 (6), 804-814 (2017).</li><li>Peirs, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+allodynia+circuitry+in+the+dorsal+horn+is+defined+by+the+nature+of+the+injury.">Mechanical allodynia circuitry in the dorsal horn is defined by the nature of the injury.</a> Neuron. 109 (1), 73-90 (2021).</li><li>Huang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circuit+dissection+of+the+role+of+somatostatin+in+itch+and+pain.">Circuit dissection of the role of somatostatin in itch and pain.</a> Nature Neuroscience. 21 (5), 707-716 (2018).</li><li>Obien, M. E. J., Deligkaris, K., Bullmann, T., Bakkum, D. J., Frey, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+neuronal+function+through+microelectrode+array+recordings.">Revealing neuronal function through microelectrode array recordings.</a> Frontiers in Neuroscience. 8, 423 (2015).</li><li>Nam, Y., Wheeler, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+microelectrode+array+technology+and+neural+recordings.">In vitro microelectrode array technology and neural recordings.</a> Critical Reviews in Biomedical Engineering. 39 (1), 45-61 (2011).</li><li>Johnstone, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microelectrode+arrays:+a+physiologically+based+neurotoxicity+testing+platform+for+the+21st+century.">Microelectrode arrays: a physiologically based neurotoxicity testing platform for the 21st century.</a> Neurotoxicology. 31 (4), 331-350 (2010).</li><li>Stett, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+application+of+microelectrode+arrays+in+drug+discovery+and+basic+research.">Biological application of microelectrode arrays in drug discovery and basic research.</a> Analytical and Bioanalytical Chemistry. 377 (3), 486-495 (2003).</li><li>Xu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+and+recent+development+of+the+microelectrode+arrays+(MEAs).">Trends and recent development of the microelectrode arrays (MEAs).</a> Biosensors and Bioelectronics. 175 (1), 112854 (2020).</li><li>Chapman, R. J., Cilia La Corte, P. F., Asghar, A. U. R., King, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network-based+activity+induced+by+4-aminopyridine+in+rat+dorsal+horn+in+vitro+is+mediated+by+both+chemical+and+electrical+synapses.">Network-based activity induced by 4-aminopyridine in rat dorsal horn in vitro is mediated by both chemical and electrical synapses.</a> The Journal of Physiology. 587, 2499-2510 (2009).</li><li>Ruscheweyh, R., Sandk&#252;hler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epileptiform+activity+in+rat+spinal+dorsal+horn+in+vitro+has+common+features+with+neuropathic+pain.">Epileptiform activity in rat spinal dorsal horn in vitro has common features with neuropathic pain.</a> Pain. 105 (1-2), 327-338 (2003).</li><li>Kay, C. W., Ursu, D., Sher, E., King, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Cx36+and+Cx43+in+4-aminopyridine-induced+rhythmic+activity+in+the+spinal+nociceptive+dorsal+horn:+an+electrophysiological+study+in+vitro.">The role of Cx36 and Cx43 in 4-aminopyridine-induced rhythmic activity in the spinal nociceptive dorsal horn: an electrophysiological study in vitro.</a> Physiological Reports. 4 (14), 12852 (2016).</li><li>Jankowska, E., Lundberg, A., Rudomin, P., Sykova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+4-aminopyridine+on+synaptic+transmission+in+the+cat+spinal+cord.">Effects of 4-aminopyridine on synaptic transmission in the cat spinal cord.</a> Brain Research. 240 (1), 117-129 (1982).</li><li>Semba, K., Geller, H. M., Egger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4-Aminopyridine+induces+expansion+of+cutaneous+receptive+fields+of+dorsal+horn+cells.">4-Aminopyridine induces expansion of cutaneous receptive fields of dorsal horn cells.</a> Brain Research. 343 (2), 398-402 (1985).</li><li>Ruscheweyh, R., Sandk&#252;hler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-range+oscillatory+Ca2++waves+in+rat+spinal+dorsal+horn.">Long-range oscillatory Ca2+ waves in rat spinal dorsal horn.</a> European Journal of Neuroscience. 22 (8), 1967-1976 (2005).</li><li>Egert, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+organotypic+long-term+culture+of+the+rat+hippocampus+on+substrate-integrated+multielectrode+arrays.">A novel organotypic long-term culture of the rat hippocampus on substrate-integrated multielectrode arrays.</a> Brain Research Protocols. 2 (4), 229-242 (1998).</li><li>Thiebaud, P., De Rooij, N., Koudelka-Hep, M., Stoppini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microelectrode+arrays+for+electrophysiological+monitoring+of+hippocampal+organotypic+slice+cultures.">Microelectrode arrays for electrophysiological monitoring of hippocampal organotypic slice cultures.</a> IEEE Transactions on Biomedical Engineering. 44 (11), 1159-1163 (1997).</li><li>Rey, H. G., Pedreira, C., Quiroga, R. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Past,+present+and+future+of+spike+sorting+techniques.">Past, present and future of spike sorting techniques.</a> Brain Research Bulletin. 119, 106-117 (2015).</li><li>Satuvuori, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measures+of+spike+train+synchrony+for+data+with+multiple+time+scales.">Measures of spike train synchrony for data with multiple time scales.</a> Journal of Neuroscience Methods. 287, 25-38 (2017).</li><li>Mendis, G. D. C., Morrisroe, E., Reid, C. A., Halgamuge, S. K., Petrou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+local+field+potentials+of+dissociated+cultures+grown+on+multi-electrode+arrays+for+pharmacological+assays.">Use of local field potentials of dissociated cultures grown on multi-electrode arrays for pharmacological assays.</a> 38th Annual International Conference of the IEEE Engineering in Medicine and Biology Society. , 952-956 (2016).</li><li>Hughes, D. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological,+neurochemical+and+electrophysiological+features+of+parvalbumin-expressing+cells:+a+likely+source+of+axo-axonic+inputs+in+the+mouse+spinal+dorsal+horn.">Morphological, neurochemical and electrophysiological features of parvalbumin-expressing cells: a likely source of axo-axonic inputs in the mouse spinal dorsal horn.</a> The Journal of Physiology. 590 (16), 3927-3951 (2012).</li><li>Peirs, C., Seal, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+circuits+for+pain:+recent+advances+and+current+views.">Neural circuits for pain: recent advances and current views.</a> Science. 354 (6312), 578-584 (2016).</li><li>Li, J., Baccei, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+regulation+of+membrane+excitability+in+rat+spinal+lamina+I+projection+neurons.">Developmental regulation of membrane excitability in rat spinal lamina I projection neurons.</a> Journal of Neurophysiology. 107 (10), 2604-2614 (2012).</li><li>Li, J., Baccei, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pacemaker+neurons+within+newborn+spinal+pain+circuits.">Pacemaker neurons within newborn spinal pain circuits.</a> Journal of Neuroscience. 31 (24), 9010-9022 (2011).</li><li>Sandk&#252;hler, J., Eblen-Zajjur, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+rhythmic+nociceptive+and+non-nociceptive+spinal+dorsal+horn+neurons+in+the+rat.">Identification and characterization of rhythmic nociceptive and non-nociceptive spinal dorsal horn neurons in the rat.</a> Neuroscience. 61 (4), 991-1006 (1994).</li><li>Lucas-Romero, J., Rivera-Arconada, I., Roza, C., Lopez-Garcia, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+classification+of+spontaneous+discharges+in+mouse+superficial+dorsal+horn+neurons.">Origin and classification of spontaneous discharges in mouse superficial dorsal horn neurons.</a> Scientific Reports. 8 (1), 9735-9735 (2018).</li><li>Antonio, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L.+al.+In+vitro+seizure+like+events+and+changes+in+ionic+concentration.">L. al. In vitro seizure like events and changes in ionic concentration.</a> Journal of Neuroscience Methods. 260, 33-44 (2016).</li><li>Avoli, M., Jefferys, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+drug-induced+epileptiform+synchronization+in+vitro.">Models of drug-induced epileptiform synchronization in vitro.</a> Journal of Neuroscience Methods. 260, 26-32 (2016).</li><li>Taccola, G., Nistri, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+micromolar+concentrations+of+4-aminopyridine+facilitate+fictive+locomotion+expressed+by+the+rat+spinal+cord+in+vitro.">Low micromolar concentrations of 4-aminopyridine facilitate fictive locomotion expressed by the rat spinal cord in vitro.</a> Neuroscience. 126 (2), 511-520 (2004).</li><li>Mitra, P., Brownstone, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+spinal+cord+slice+preparation+for+recording+from+lumbar+motoneurons+of+the+adult+mouse.">An in vitro spinal cord slice preparation for recording from lumbar motoneurons of the adult mouse.</a> Journal of Neurophysiology. 107 (2), 728-741 (2012).</li><li>Egert, U., Heck, D., Aertsen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+monitoring+of+spiking+networks+in+acute+brain+slices.">Two-dimensional monitoring of spiking networks in acute brain slices.</a> Experimental Brain Research. 142 (2), 268-274 (2002).</li></ol>

The authors have no conflicts of interest to declare.

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 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 &#39;pain-like&#39; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-aminopyridine</td>
			<td>Sigma-Aldrich</td>
			<td>275875-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>Thermo Fisher</td>
			<td>AJA214-2.5LPL</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 1M</td>
			<td>Banksia Scientific</td>
			<td>0430/1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonox (Carbogen - 95% O2, 5% CO2)</td>
			<td>Coregas</td>
			<td>219122</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved long handle spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15015-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom made air interface incubation chamber</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal bovine serum</td>
			<td>Thermo Fisher</td>
			<td>10091130</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Dumont #5</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Thermo Fisher</td>
			<td>AJA783-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Thermo Fisher</td>
			<td>16050130</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Zeiss</td>
			<td>Axiovert10</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Thermo Fisher</td>
			<td>AJA383-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Ceva</td>
			<td>KETALAB04</td>
			<td></td>
		</tr>
		<tr>
			<td>Large surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14007-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Loctite 454 Instant Adhesive</td>
			<td>Bolts and Industrial Supplies</td>
			<td>L4543G</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td></td>
			<td>R2018b</td>
		</tr>
		<tr>
			<td>MEAs, 3-Dimensional</td>
			<td>Multichannel Systems</td>
			<td>60-3DMEA100/12/40iR-Ti, 60-3DMEA200/12/50iR-Ti</td>
			<td>60 titanium nitride (TiN) electrodes with 1 internal reference electrode, organised in an 8x8 square grid. Electrodes are 12 &#181;m in diameter, 40 &#181;m (100/12/40) or 50 &#181;m (200/12/50) high and equidistantly spaced 100 &#181;m (100/12/40) or 200 &#181;m (200/12/50) apart.</td>
		</tr>
		<tr>
			<td>MEA headstage</td>
			<td>Multichannel Systems</td>
			<td>MEA2100-HS60</td>
			<td></td>
		</tr>
		<tr>
			<td>MEA interface board</td>
			<td>Multichannel Systems</td>
			<td>MCS-IFB 3.0 Multiboot</td>
			<td></td>
		</tr>
		<tr>
			<td>MEA net</td>
			<td>Multichannel Systems</td>
			<td>ALA HSG-MEA-5BD</td>
			<td></td>
		</tr>
		<tr>
			<td>MEA perfusion system</td>
			<td>Multichannel Systems</td>
			<td>PPS2</td>
			<td></td>
		</tr>
		<tr>
			<td>MEAs, Planar</td>
			<td>Multichannel Systems</td>
			<td>60MEA200/30iR-Ti, 60MEA500/30iR-Ti</td>
			<td>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 &#181;m in diameter and equidistantly spaced 200 &#181;m (200/30) or 500 &#181;m (500/30) apart.</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Thermo Fisher</td>
			<td>AJA296-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Motic</td>
			<td>Moticam X Wi-Fi</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi Channel Analyser software</td>
			<td>Multichannel Systems</td>
			<td></td>
			<td>V 2.17.4</td>
		</tr>
		<tr>
			<td>Multi Channel Experimenter software</td>
			<td>Multichannel Systems</td>
			<td></td>
			<td>V 2.17.4</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Thermo Fisher</td>
			<td>AJA465-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Thermo Fisher</td>
			<td>AJA475-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Thermo Fisher</td>
			<td>ACR207805000</td>
			<td></td>
		</tr>
		<tr>
			<td>Rongeurs</td>
			<td>Fine Science Tools</td>
			<td>16021-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Small spring scissors</td>
			<td>Fine Science Tools</td>
			<td>91500-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Small surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Thermo Fisher</td>
			<td>AJA530-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Superglue</td>
			<td></td>
			<td></td>
			<td>cyanoacrylate adhesive</td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Abcam</td>
			<td>AB120055</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibration isolation table</td>
			<td>Newport</td>
			<td>VH3048W-OPT</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrating microtome</td>
			<td>Leica</td>
			<td>VT1200 S</td>
			<td></td>
		</tr>
	</tbody>
</table>

recording network activity in spinal nociceptive circuits using microelectrode arrays

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 na&#239;ve 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.

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 ...

Watch this Scientific Journal Video about Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays at JoVE.com

Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays

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 ...

The MEA 4-AP spinal slice preparation described to you provides a platform to study the connectivity of dorsal horn circuits and how these networks interact during spinal sensory processing. The methodology presented enables dorsal horn circuit activity to be investigated at a macroscopic regional level of resolution. This preparation can be used as a rapid screening tool to assess the ability of compounds to disrupt signaling in spinal sensory circuits.This method helps bridge the gap in our understanding of how the activity of specific cell types and small micro circuits influence large populations of neurons to subsequently shape the output of the dorsal horn behavior responses and ultimately the pain experience. To begin, fill the MEA well with horse serum for 30 minutes. After removing the serum, thoroughly rinse the MEA five times with distilled water until the distilled water becomes non-foamy.Then, fill the well with artificial CSF. After decapitating the anesthetized mouse, remove the skin over the abdominal region by making a small cut in the skin at the level of the hips. Then, pull the skin on either side of the cut rostrally until all the skin is removed from the top of the rib cage to the top of the pelvis, both ventrally and dorsally.After placing the body on ice, use a ventral approach to expose the vertebral column by removing the viscera and cutting through the ribs laterally to the sternum. Remove the ventral rib cage, both scapulae, and the lower limbs and pelvis. Transfer the vertebral column and rib preparation to a dissecting bath containing ice cold sucrose artificial CSF.Pin all four corners of the preparation by placing pins through the lower back muscles and the attached upper ribs. Remove all muscle and connective tissue overlying the ventral surface of the vertebrae with the rongeurs and identify the vertebral region over the lumbo-sacral enlargement, which lies approximately beneath the T12-L2 vertebral bodies. Remove a vertebral body caudal to the lumbo-sacral enlargement region to access the spinal cord sitting on the vertebral canal.Using curved spring scissors, cut through the vertebral pedicles bilaterally while lifting and pulling the vertebral body rostrally to separate the ventral and dorsal aspects of the vertebrae and expose the spinal cord. Once the vertebral bodies are removed to reveal the lumbo-sacral enlargement, carefully clear the remaining roots that anchor the spinal cord with spring scissors until the cord floats free. Isolate the spinal cord with rostral and caudal cuts above and below the lumbo-sacral enlargement to make the target region of the cord float free.For transverse slices, lift and place the lumbo-sacral segment by an attached route on a precut polystyrene block with a shallow channel cut in the center. Then, use cyanoacrylate adhesive to attach the block and cord to the sectioning platform and place it in the cutting bath containing ice cold sucrose artificial CSF slurry. Once the 300 micrometers thick slices are obtained, transfer the slices to an air interface incubation chamber containing oxygenated artificial CSF and allow the slices to equilibrate for one hour at room temperature.To begin recording dorsal horn activity, transfer the slice from the incubator to the MEA well using a large tip pasteur pipette filled with artificial CSF and add additional artificial CSF. Use a fine short hair paint brush to position the slice over the 60 electrode recording array. Then, place a weighted neck over the tissue to hold it in place and promote good contact with MEA electrodes.Place the MEA in the recording head stage. Check the position of the tissue over the electrodes using an inverted microscope to confirm that as many electrodes as possible are under the superficial DH.Ensure that at least two to six electrodes do not contact the slice. After connecting the camera to the device, take a reference image of the slice relative to the MEA for use during the analysis.Then, press start DAQ in the recording software and confirm that all electrodes receive a clear signal. Next, attach the perfusion inlet and outlet lines to the MEA well filled with artificial CSF and turn the perfusion system on. Check the flow rate and ensure that the outflow is sufficient to prevent overflow of the superfusate.After equilibrating the tissue for five minutes, record the raw baseline data for five minutes. Move the perfusion inlet line from artificial CSF to a 4-aminopyridine solution and wait for 12 minutes for the 4-aminopyridine induced rhythmic activity to reach steady state. Then, record five minutes of 4-aminopyridine induced activity and be prepared for the subsequent recordings to test the drugs or check the stability of 4-aminopyridine.Following each recording session, rinse the lines with artificial CSF. After removing the MEA from the head stage and removing the net and the tissue from the MEA well, rinse the MEA and net with artificial CSF and repeat the whole process with a new slice. Open the analysis software and load the pre-made analysis layout.Open the file of interest and deselect the reference electrode and any electrodes deemed to be excessively noisy. Set the time window for analysis. Then, move to the cross channel filter tab.Select the complex reference and reference electrodes based on the image taken and notes made during the experiment and press Explore before continuing. Move to the EAP filter tab and apply a second order high-pass Butterworth filter to remove LFP activity. Move to the LFP filter tab and apply a second order band pass Butterworth filter to remove EAP activity.In the EAP detector tab, ensure to select auto threshold, tick the rising and falling edge boxes, and set the dead time to three milliseconds. To set positive and negative thresholds, inspect the data by returning to the raw data analyzer screen, moving the time marker, returning to the EAP detector tab, and pressing explore. Repeat the process until the set detection threshold captures EAPs without capturing noise or non-physiological activity.In the LFP detector tab, ensure to select the manual threshold, tick the rising and falling edge boxes, and set the dead time to three milliseconds. Set threshold for one electrode and, once satisfied, select apply to all and then press start analysis. The bath application of the voltage gated sodium channel antagonist tetrodotoxin abolished both EAP and LFP activity, confirming the spike dependency of these signals.The stability of 4-aminopyridine induced activity parameters was characterized for EAP or LFP. All activity characteristics plus the coincidence of activity for LFPs were stable based on the similarity of 4-aminopyridine induced activity at 12 minutes after 4-aminopyridine application and 15 minutes later. The feature's EAPs or LFPs were characterized by an increase in the number of coincident events detected across multiple electrodes.The number of linked adjacent electrodes, and the strength of linkages between adjacent electrodes and LFPs following 4-aminopyridine stimulation and then relative stability. The slice orientation is an important consideration for in vitro preparations. 4-aminopyridine stimulation induced a similar rhythmic activity in the SDH regardless of slice orientation.Finally, to shed further light on the relevance of 4-aminopyridine activation of the DH networks, an elevated potassium artificial CSF solution was bath applied and shown to evoke a similar DH response to 4-aminopyridine stimulation. Key steps in the protocol are in the tissue preparation and placement of sections, ensuring the tissue is healthy through careful, but timely, dissection, as well as delicate optimal positioning of the tissue on the MEA will aid in obtaining quality results.

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JoVE Journal

Neuroscience

2.7K 조회수.  University of Newcastle. 귀하에게 설명된 MEA 4-AP 척추 슬라이스 준비는 등쪽 경적 회로의 연결성과 척추 감각 처리 중에 이러한 네트워크가 어떻게 상호 작용하는지 연구하는 플랫폼을 제공합니다. 제시된 방법론은 등쪽 경적 회로 활동을 거시적 인 지역 해상도 수준에서 조사 할 수있게합니다. 이 준비는 척추 감각 회로에서 신호 전달을 방해하는 화합물의 능력을 평가하는 신속한 스크리닝 도구로...