Name: recording synaptic plasticity in acute hippocampal slices maintained in a small-volume recycling-, perfusion-, and submersion-type chamber system
Uploaded: 2018-01-01T13:00:00
Description: Watch this Scientific Journal Video about Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System at JoVE.com

W.W. conducted, analyzed, and designed the experiments and wrote the manuscript. D.X. and C.P. assisted in figure preparation and conducted the experiments. This work was supported by NSFC (31320103906) and 111 Project (B16013) to T.B.

The animals were maintained in accordance with the established standards of animal care and procedures of the Institutes of Brain Science and State Key Laboratory of Medical Neurobiology of Fudan University, Shanghai, China.
1. Solution Preparation
NOTE: See the Table of Materials.
<ol>
	<li>Prepare the slicing buffer (modified Gey&#39;s solution): 92 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 25 mM glucose, 20 mM HEPES, 3 mM Na+-pyruva...

<ol>
	<li>McIlwain, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+response+in+vitro+to+electrical+stimulation+of+sections+of+mammalian+brain.">Metabolic response in vitro to electrical stimulation of sections of mammalian brain.</a> Biochem J. 48 (4), (1951).</li><li>McIlwain, H., Richards, C. D., Somerville, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+in+vitro+from+the+piriform+cortex+of+the+rat,+and+their+susceptibility+to+centrally-acting+drugs.">Responses in vitro from the piriform cortex of the rat, and their susceptibility to centrally-acting drugs.</a> J Neurochem. 14 (9), 937-938 (1967).</li><li>Yamamoto, C., McIlwain, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+activities+in+thin+sections+from+the+mammalian+brain+maintained+in+chemically-defined+media+in+vitro.">Electrical activities in thin sections from the mammalian brain maintained in chemically-defined media in vitro.</a> J Neurochem. 13 (12), 1333-1343 (1966).</li><li>Andersen, P., Bliss, T. V., Lomo, T., Olsen, L. I., Skrede, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lamellar+organization+of+hippocampal+excitatory+pathways.">Lamellar organization of hippocampal excitatory pathways.</a> Acta Physiol Scand. 76 (1), 4-5 (1969).</li><li>Skrede, K. K., Westgaard, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transverse+hippocampal+slice:+a+well-defined+cortical+structure+maintained+in+vitro.">The transverse hippocampal slice: a well-defined cortical structure maintained in vitro.</a> Brain Res. 35 (2), 589-593 (1971).</li><li>Kirov, S. A., Sorra, K. E., Harris, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slices+have+more+synapses+than+perfusion-fixed+hippocampus+from+both+young+and+mature+rats.">Slices have more synapses than perfusion-fixed hippocampus from both young and mature rats.</a> J Neurosci. 19 (8), 2876-2886 (1999).</li><li>Schwartzkroin, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+CA1+neurons+recorded+intracellularly+in+the+hippocampal+in+vitro+slice+preparation.">Characteristics of CA1 neurons recorded intracellularly in the hippocampal in vitro slice preparation.</a> Brain Res. 85 (3), 423-436 (1975).</li><li>Schwartzkroin, P. A., Wester, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-lasting+facilitation+of+a+synaptic+potential+following+tetanization+in+the+in+vitro+hippocampal+slice.">Long-lasting facilitation of a synaptic potential following tetanization in the in vitro hippocampal slice.</a> Brain Res. 89 (1), 107-119 (1975).</li><li>Reymann, K. G., Frey, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+late+maintenance+of+hippocampal+LTP:+requirements,+phases,+'synaptic+tagging',+'late-associativity'+and+implications.">The late maintenance of hippocampal LTP: requirements, phases, 'synaptic tagging', 'late-associativity' and implications.</a> Neuropharm. 52 (1), 24-40 (2007).</li><li>Bliss, T. V., Collingridge, G. L., Morris, R. 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Although interface slice chambers exhibit more robust synaptic responses25,26,27,28, submersion chambers provide additional convenience for patch-clamp recording and fluorescence imaging. Thus, we have described several aspects of field potential recordings in acute hippocampal slices using a commercial submersion slice chamber that can easily be extended to the imaging of fluorescence probes i...

In 1951, the first-reported acute brain slice experiments were conducted1. In 1971, after successful in vitro recordings from the piriform cortex2,3 and the discovery that hippocampal neurons are interconnected transversely along the septotemporal axis of the hippocampus4, one of the first in vitro recordings of hippocampal neuronal activity was achieved5. The similarity of the neurophysiological or neurostructural parameters of neurons under in vivo and in vitro conditions are still the subject of some debate6, but in 1975, Schwartzkroin7 indicated that the basal properties of neurons are maintained in vitro and that high-frequency stimulation (i.e., tetanization) of afferents in the hippocampal formation induces a long-lasting facilitation of synaptic potentials8. Electrophysiological recording of neuronal activity in vitro greatly expanded the study of the cellular mechanisms of activity-dependent synaptic plasticity9,10, which had been discovered in 1973 by Bliss et al.11 in in vivo experiments with rabbits.
The study of neuronal activity or signaling pathways in brain slices, and especially in acute hippocampal slices, is now a standard tool. However, surprisingly, in vitro experiments have yet to be standardized, as evidenced by the multiple approaches that still exist for the preparation and maintenance of acute hippocampal slices. Reid et al. (1988)12 reviewed the methodological challenges for the maintenance of acute brain slices in different types of slice chambers and the choices of bathing medium, pH, temperature, and oxygen level. These parameters are still difficult to control in the recording chamber due to the custom-made elements of in vitro slice-recording setups. Publications can be found that might help to overcome some of the methodological challenges and that describe new types of submersion slice chambers, such as an interstitial 3D microperfusion system13, a chamber with enhanced laminar flow and oxygen supply14, a system with computerized temperature control15, and a multi-chamber recording system16. Since these chambers are not easy to build, most scientists rely on commercially available slice chambers. These chambers can be mounted on a microscope system, thus allowing for the combination of electrophysiology and fluorescence imaging17,18,19. Since these chambers keep the brain slices submerged in artificial cerebrospinal fluid (aCSF), a high flow rate of the buffer solution needs to be maintained, increasing the expense of drug application. To this end, we have incorporated a recycling perfusion system with outflow-carbogenation that provides sufficient stability for the long-term recording of field potentials in a submersion slice chamber using a relatively small aCSF volume. In addition, we summarized how the use of this experimental carbogenation/perfusion system affects the outcome of activity-dependent synaptic plasticity10 and how inhibition of eukaryotic elongation factor-2 kinase (eEF2K) modulates synaptic transmission20.

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			<td height="21" style="width:311px;height:21px;">Reagents required</td>
			<td style="width:244px;"></td>
			<td style="width:179px;"></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">NaCl</td>
			<td style="width:244px;">Sinopharm Chemical Reagent, China</td>
			<td style="width:179px;">10019318</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">KCl</td>
			<td style="width:244px;">Sinopharm Chemical Reagent, China</td>
			<td style="width:179px;">10016318</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">KH2PO4</td>
			<td style="width:244px;">Sinopharm Chemical Reagent, China</td>
			<td style="width:179px;">10017618</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">MgCl2&#183;6H2O</td>
			<td style="width:244px;">Sinopharm Chemical Reagent, China</td>
			<td style="width:179px;">10012818</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">CaCl2</td>
			<td style="width:244px;">Sinopharm Chemical Reagent, China</td>
			<td style="width:179px;">10005861</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">NaHCO3</td>
			<td style="width:244px;">Sinopharm Chemical Reagent, China</td>
			<td style="width:179px;">10018960</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">Glucose</td>
			<td style="width:244px;">Sinopharm Chemical Reagent, China</td>
			<td style="width:179px;">10010518</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">NaH2PO4</td>
			<td style="width:244px;">Sinopharm Chemical Reagent, China</td>
			<td style="width:179px;">20040718</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">HEPES</td>
			<td style="width:244px;">Sigma</td>
			<td style="width:179px;">H3375</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">Sodium pyruvate</td>
			<td style="width:244px;">Sigma</td>
			<td style="width:179px;">A4043</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">MgSO4</td>
			<td style="width:244px;">Sinopharm Chemical Reagent, China</td>
			<td style="width:179px;">20025118</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="width:311px;height:27px;">NaOH</td>
			<td style="width:244px;">Sinopharm Chemical Reagent, China</td>
			<td style="width:179px;">10019718</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;"></td>
			<td style="width:244px;"></td>
			<td style="width:179px;"></td>
			<td></td>
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			<td height="21" style="width:311px;height:21px;">Tools and materials for dissection</td>
			<td style="width:244px;"></td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Decapitators</td>
			<td style="width:244px;">Harvard apparatus</td>
			<td style="width:179px;">55-0012</td>
			<td>for rat decapitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Bandage Scissors</td>
			<td style="width:244px;">SCHREIBER</td>
			<td style="width:179px;">12-4227</td>
			<td>for mouse decapitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">double-edge blade</td>
			<td style="width:244px;">Flying Eagle, China</td>
			<td style="width:179px;">74-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">IRIS Scissors</td>
			<td style="width:244px;">RWD, China</td>
			<td style="width:179px;">S12003-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Bone Rongeurs</td>
			<td style="width:244px;">RWD, China</td>
			<td style="width:179px;">S22002-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Spoon</td>
			<td style="width:244px;">Hammacher&#160;</td>
			<td style="width:179px;">HSN 152-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">dental cement spatula</td>
			<td style="width:244px;">Hammacher&#160;</td>
			<td style="width:179px;">HSN 016-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">dental double end excavator</td>
			<td style="width:244px;">Blacksmith Surgical, USA</td>
			<td style="width:179px;">BS-415-017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Vibrating Microtome</td>
			<td style="width:244px;">Leica, Germany</td>
			<td style="width:179px;">VT1200S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">surgical blade&#160;</td>
			<td style="width:244px;">RWD, China</td>
			<td style="width:179px;">S31023-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">surgical holder</td>
			<td style="width:244px;">RWD, China</td>
			<td style="width:179px;">S32007-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;"></td>
			<td style="width:244px;"></td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:311px;height:42px;">Electrophysiology equipment and materials</td>
			<td style="width:244px;"></td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Vertical Pipette Puller</td>
			<td style="width:244px;">Narishige, Japan</td>
			<td style="width:179px;">PC-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Vibration isolation table</td>
			<td style="width:244px;">Meirits, Japan</td>
			<td style="width:179px;">ADZ-A0806</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">submerged type recording chamber</td>
			<td style="width:244px;">Warner Instruments</td>
			<td style="width:179px;">RC-26GLP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">thermostatic water bath</td>
			<td style="width:244px;">Zhongcheng Yiqi&#65292;China</td>
			<td style="width:179px;">HH-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">4 Axis Micromanipulator</td>
			<td style="width:244px;">Sutter, USA</td>
			<td style="width:179px;">MP-285, MP-225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Platinum Wire</td>
			<td style="width:244px;">World Precision Instruments</td>
			<td style="width:179px;">PTP406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Amplifier</td>
			<td style="width:244px;">Molecular Devices, USA</td>
			<td style="width:179px;">Multiclamp 700B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Data Acquisition System</td>
			<td style="width:244px;">Molecular Devices, USA</td>
			<td style="width:179px;">Digidata 1440A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Anaysis software</td>
			<td style="width:244px;">Molecular Devices, USA</td>
			<td style="width:179px;">Clampex 10.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Fluorescence Microscope</td>
			<td style="width:244px;">Nikon, Japan</td>
			<td style="width:179px;">FN1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">LED light source</td>
			<td style="width:244px;">Lumen Dynamics Group, Canada</td>
			<td style="width:179px;">X-cite 120LED</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">micropipettes</td>
			<td style="width:244px;">Harvard apparatus</td>
			<td style="width:179px;">GC150TF</td>
			<td>extracelluar recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">borosilicate micropipettes</td>
			<td style="width:244px;">Sutter, USA</td>
			<td style="width:179px;">BF150-86</td>
			<td>patch clamp</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">tungsten electrode</td>
			<td style="width:244px;">A-M Systems, USA</td>
			<td style="width:179px;">575500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">peristaltic pump</td>
			<td style="width:244px;">Longer, China</td>
			<td style="width:179px;">BT00-300T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">tubes for peristaltic pump</td>
			<td style="width:244px;">ISMATEC, Wertheim, Germany</td>
			<td style="width:179px;">SC0309</td>
			<td>1x inflow, ID: 1.02mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">tubes for peristaltic pump</td>
			<td style="width:244px;">ISMATEC, Wertheim, Germany</td>
			<td style="width:179px;">SC0319</td>
			<td>2x tubes for outflow, ID: 2.79 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">CCD camera</td>
			<td style="width:244px;">PCO, Germany</td>
			<td style="width:179px;">pco.edge sCMOS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">lens cleaning paper</td>
			<td style="width:244px;">Kodak</td>
			<td style="width:179px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">50 ml conical centrifuge tube</td>
			<td style="width:244px;">Thermo scientific</td>
			<td style="width:179px;">339652</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Prechamber</td>
			<td style="width:244px;">Warner Instruments</td>
			<td style="width:179px;">BSC-PC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Inline heater</td>
			<td style="width:244px;">Warner Instruments</td>
			<td style="width:179px;">SF-28</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:311px;height:21px;">Temperature Controller</td>
			<td style="width:244px;">Warner Instruments</td>
			<td style="width:179px;">TC-324B</td>
			<td></td>
		</tr>
	</tbody>
</table>

recording synaptic plasticity in acute hippocampal slices maintained in a small-volume recycling-, perfusion-, and submersion-type chamber system

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

In the protocol section, we described the preparation of acute hippocampal slices from the ventral and intermediate part of the hippocampal formation (Figure 1) of male C57BL/6 mice and male Wistar rats (5-8 weeks). The position of the hemispheres on the slicer platform helps to keep them stable and removes the need of stabilization with agar or agarose. The perfusion system itself is based on a peristaltic pump operated on high rotation to give the required ...

Watch this Scientific Journal Video about Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System at JoVE.com

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Research

JoVE Journal

Neuroscience

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Preparation of Oligomeric ÃƒÅ½Ã‚Â²-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Pentylenetetrazole-Induced Kindling Mouse Model

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...