Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.
Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.
The substantia gelatinosa (SG, lamina II of the spinal dorsal horn) is an indisputably important relay center for transmitting and regulating sensory information. It is composed of excitatory and inhibitory interneurons, which receive inputs from the primary afferent fibers, local interneurons, and the endogenous descending inhibitory system1. In recent decades, the development of acute spinal cord slice preparation and the advent of whole-cell patch-clamp recording have enabled various studies on the intrinsic electrophysiological and morphological properties of SG neurons2,3,4 as well as studies of the local circuitry in SG5,6. In addition, by using the in vitro spinal cord slice preparation, researchers can interpret the changes in neuronal excitabilities7,8, the function of ion channels9,10, and synaptic activities11,12 under various pathological conditions. These studies have deepened our understanding of the role that SG neurons play in the development and maintenance of chronic pain and neuropathic itch.
Essentially, the key prerequisite to achieve a clear visualization of neuronal soma and ideal whole-cell patching using acute spinal cord slices is to ensure the excellent quality of slices so healthy and patchable neurons can be obtained. However, preparing spinal cord slices involves several steps, such as performing a ventral laminectomy and removing the pia-arachnoid membrane, which may be obstacles in obtaining healthy slices. Although it is not easy to prepare spinal cord slices, performing recordings in vitro on spinal cord slices has several advantages. Compared to cell culture preparations, spinal cord slices can partially preserve inherent synaptic connections that are in a physiologically relevant condition. In addition, whole-cell patch-clamp recording using spinal cord slices could be combined with other techniques, such as double patch clamp13,14, morphological studies15,16 and single-cell RT-PCR17. Therefore, this technique provides more information on characterizing the anatomical and genetic diversities within a specific region and allows for investigation of the composition of local circuitry.
Here, we provide a basic and detailed description of our method for preparing acute spinal cord slices and acquiring whole-cell patch-clamp recordings from SG neurons.
All experimental protocols described were approved by the Animal Ethics Committee of Nanchang University (Nanchang, PR China, Ethical No.2017-010). All efforts were made to minimize the stress and pain of the experimental animals. The electrophysiological recordings performed here were carried out at room temperature (RT, 22–25 °C).
1. Animals
2. Preparation of Solutions and Materials
3. Acute Spinal Cord Slice Preparation
Note: Transverse or parasagittal spinal cord slices are prepared as previously described18,19,20.
4. Whole-cell Patch-clamp Recordings
5. Morphological study
Acute spinal cord slices were prepared according to the diagram shown in Figure 1. After slicing and recovery, a spinal cord slice was transferred to the recording chamber. Healthy neurons were identified based on soma appearance using IR-DIC microscopy. Next, the action potentials of SG neurons were elicited by a series of depolarizing current pulses (1 s duration) when neurons were held at RMP. As shown in Figure 2, the firing patterns observed in SG neurons included tonic-firing, delayed-firing, gap-firing, initial-burst, phasic-bursting, single-spike and reluctant-firing, which have been described and categorized by previous studies.
Implementing this preparation, we also recorded subthreshold currents and spontaneously appearing currents in voltage clamp. Representative traces of subthreshold currents, including hyperpolarization-activated current (Ih), T-type calcium current (IT) and A-type potassium current (IA), are given in Figure 3A. These currents were obtained by holding cells at -50 mV and gradually stepping in 10-mV decrements from -60 to -120 mV. Ih was activated by hyperpolarizing voltage steps. However, IT and IA were activated by hyperpolarizing prepulses to release from inactivation followed with a depolarized voltage. Figure 3B, 3C show representative spontaneous EPSCs (sEPSCs) and IPSCs (sIPSCs) recorded from SG neurons, respectively. The amplitude and frequency of these synaptic events could be analyzed using the Mini-analysis software offline.
To characterize neuronal morphological features, parasagittal slices were applied because most of the SG neurons have significantly rostrocaudal spread of dendritic trees, and neurobiotin 488 was added to intracellular solutions. The size of neuronal soma and the extent and dimensions of their dendritic processes were evaluated after confocal microscopy imaging. As reported previously, SG neurons show morphological distinctions and could be categorized into central cells, radial cells, vertical cells, islet cells and unclassified cells. Representative micrographs of these cells are shown in Figure 4.
Figure 1: Diagram for acute spinal cord slice preparation. After being deeply anesthetized with urethan (i.p.), rats are transcardially perfused with ice-cold carbogenated sucrose-ACSF. The spinal column is then quickly dissected, and a ventral laminectomy is performed. The meninges, pia-arachnoid membrane and attached spinal nerve roots are removed. Then, the spinal cord specimen is mounted on an agarose block. Transverse or parasagittal slices are cut with a vibratome as needed. Please click here to view a larger version of this figure.
Figure 2: Firing patterns of SG neurons. Firing patterns are determined by injecting a series of 1 s depolarizing current pulses into an SG neuron at RMP. The firing patterns may be classified as tonic-firing, delayed-firing, gap-firing, initial-burst, phasic-bursting, single-spike, and reluctant-firing. Please click here to view a larger version of this figure.
Figure 3: Voltage-clamp recordings in SG neurons. (A) Representative traces showing the response to hyperpolarizing current injection classified as Ih, IA and IT. The lower panel shows the evoking protocol for sub-threshold currents in voltage-clamp. (B) Representative traces of sEPSCs recorded from SG neurons at -70 mV in the absence and presence of 50 μM APV and 20 μM CNQX. Lower consecutive traces, which are shown in an expanded time scale before (left) and under (right) the action of APV and CNQX, correspond to a period indicated by a bar shown below the chart recording. (C) Representative traces of sIPSCs recorded from SG neurons in the absence and presence of 10 μM bicuculline and 1 μM strychnine at 0 mV. Please click here to view a larger version of this figure.
Figure 4: Representative morphology of rat SG neurons. According to soma sizes and dendrite properties shown in confocal microscopy images, SG neurons may be classified as the central cell (A), radial cell (B), vertical cell (C), islet cell (D) and unclassified cell (E). V: ventral; D: dorsal; R: rostral; C: caudal. Scale bar = 50 µm. Please click here to view a larger version of this figure.
Component | Molecular Weight | Concentration (mM) | g/L |
NaCl | 58.5 | 117 | 6.84 |
KCl | 74.5 | 3.6 | 0.27 |
NaH2PO4·2H2O | 156 | 1.2 | 0.19 |
CaCl2·2H2O | 147 | 2.5 | 0.37 |
MgCl2·6H2O | 203 | 1.2 | 0.24 |
NaHCO3 | 84 | 25 | 2.1 |
D-Glucose | 180 | 11 | 1.98 |
Ascorbic acid | 198.11 | 0.4 | 0.08 |
Sodium pyruvate | 110 | 2 | 0.22 |
Table 1: Recipe for ACSF.
Component | Molecular Weight | Concentration (mM) | g/L |
NaCl | 58.5 | 117 | 6.84 |
KCl | 74.5 | 3.6 | 0.27 |
NaH2PO4·2H2O | 156 | 1.2 | 0.19 |
CaCl2·2H2O | 147 | 2.5 | 0.37 |
MgCl2·6H2O | 203 | 1.2 | 0.24 |
NaHCO3 | 84 | 25 | 2.1 |
D-Glucose | 180 | 11 | 1.98 |
Ascorbic acid | 198.11 | 0.4 | 0.08 |
Sodium pyruvate | 110 | 2 | 0.22 |
Table 2: Recipe for sucrose-ACSF.
Component | Molecular Weight | Concentration (mM) | mg/100 mL |
K-gluconate | 234.2 | 130 | 3044.6 |
KCl | 74.5 | 5 | 37.28 |
Na2-Phosphocreatine | 453.38 | 10 | 453.38 |
EGTA | 380.35 | 0.5 | 19.02 |
HEPES | 238.31 | 10 | 238.3 |
Mg-ATP | 507.18 | 4 | 202.9 |
Li-GTP | 523.18 | 0.3 | 15.7 |
Table 3: Recipe for K+-based intracellular solution.
Component | Molecular Weight | Concentration (mM) | mg/100 mL |
CsMeSO4 | 228 | 92 | 2097.6 |
CsCl | 168.36 | 43 | 723.95 |
Na2-Phosphocreatine | 453.38 | 10 | 453.38 |
TEA-Cl | 165.71 | 5 | 82.86 |
EGTA | 380.35 | 0.5 | 19.02 |
HEPES | 238.31 | 10 | 238.3 |
Mg-ATP | 507.18 | 4 | 202.9 |
Li-GTP | 523.18 | 0.3 | 15.7 |
Table 4: Recipe for Cs+-based intracellular solution.
This protocol details the steps for preparing spinal cord slices, which we have used successfully when performing whole-cell patch-clamp experiments on SG neurons18,19,20,21. By implementing this method, we recently reported that minocycline, a second generation of tetracycline, could markedly enhance inhibitory synaptic transmission through a presynaptic mechanism in SG neurons19. In addition, this agent could decrease the amplitude of Ih and further inhibit the excitability of SG neurons21. In support of these published data and the representative results that we show here, the currently described method is suitable for use in a wide range of electrophysiological studies.
As we noted previously, transcardial perfusion is a crucial element for obtaining healthy specimens. First, we use ice-cold solution for perfusion so the spinal cord can be rapidly cooled and the neuronal metabolism can be slowed22. Second, sucrose-substituted ACSF, a 'protective cutting' solution with low Na+ concentration, can ameliorate passive Na+ influx and thus decrease neuronal edema through water entry23. Third, it is beneficial to obtain and analyze neuronal morphology because perfusion could minimize the background caused by biocytin22. For successful preparations, it is also important to use some antioxidants to reduce oxidative damage, which allows neuronal preservation24. Hence, in our protocol, we supplement ascorbic acid and sodium pyruvate, which are powerful antioxidants and can ameliorate edema in spinal cord slices effectively, in both ACSF and sucrose-ACSF. Also, in our experience, we can obtain healthy spinal cord slices successfully from neonatal as well as 3–10 weeks old SD rats. Thus, for this protocol to be successful, we recommend using SD rats that are less than 10 weeks old.
While performing 'ventral' laminectomy and removing the meninges and spinal nerves, one should be patient and careful to avoid cutting, stretching or splitting the spinal cord. In some studies, spinal cord slices with attached dorsal roots have been used to evaluate the synaptic transmission SG neurons received peripherally25,26. The procedure of removing pia-arachnoid membrane is of technical difficulty in this case, and it requires a lot of patience.
This slice preparing technique also has some limitations. One clear drawback is that although acute slices preserve abundant synaptic connections, it could not reflect the real state and address what exactly happens in vivo. Thus, some studies have implemented in vivo recordings that are normally performed 'blind'27,28,29. However, this in vivo approach is technically challenging, and it is difficult to tell whether a recording is performed from the soma or dendrite without sufficient experience. Another limitation of our current method is that sucrose-ACSF may not be sufficient for neuronal preservation when preparing slices from aging rodents. An updated approach using N-methyl-D-glucamine as a Na+ substitute has been proposed, and this optimized methodology could markedly improve morphological and functional preservation of neurons in acute slices30,31,32,33. Finally, SG neurons show different morphological and electrophysiological properties3. It seems difficult to interpret data obtained from whole-cell recordings while overlooking the heterogeneity. This limitation may be sidestepped by further verifying the morphological details of recorded neurons5 or using transgenic mice, which could help researchers identify specific neurons20,34. Furthermore, optogenetics, a novel tool allowing control of a sub-population of cells35, could be combined with whole-cell patch-clamp recording to study the role of specific ion channels or proteins and to investigate specific neuronal circuitry.
Overall, this preparation technique is an ideal way to investigate the electrophysiological, morphological, pharmacological, and biological characteristics of SG neurons, complemented by patch-clamp recording, immunofluorescent staining, specific agonists or antagonists, and the single-cell RT-PCR technique. Moreover, this approach can be applied together with paired patch-clamp recordings or optogenetics, and it is thus a valuable tool for illuminating the neuronal microcircuits.
The authors have nothing to disclose.
This work was supported by grants from the National Natural Science Foundation of China (No. 81560198, 31660289).
NaCl | Sigma | S7653 | Used for the preparation of ACSF and PBS |
KCl | Sigma | 60130 | Used for the preparation of ACSF, sucrose-ACSF, and K+-based intracellular solution |
NaH2PO4·2H2O | Sigma | 71500 | Used for the preparation of ACSF, sucrose-ACSF and PBS |
CaCl2·2H2O | Sigma | C5080 | Used for the preparation of ACSF and sucrose-ACSF |
MgCl2·6H2O | Sigma | M2670 | Used for the preparation of ACSF and sucrose-ACSF |
NaHCO3 | Sigma | S5761 | Used for the preparation of ACSF and sucrose-ACSF |
D-Glucose | Sigma | G7021 | Used for the preparation of ACSF |
Ascorbic acid | Sigma | P5280 | Used for the preparation of ACSF and sucrose-ACSF |
Sodium pyruvate | Sigma | A7631 | Used for the preparation of ACSF and sucrose-ACSF |
Sucrose | Sigma | S7903 | Used for the preparation of sucrose-ACSF |
K-gluconate | Wako | 169-11835 | Used for the preparation of K+-based intracellular solution |
Na2-Phosphocreatine | Sigma | P1937 | Used for the preparation of intracellular solution |
EGTA | Sigma | E3889 | Used for the preparation of intracellular solution |
HEPES | Sigma | H4034 | Used for the preparation of intracellular solution |
Mg-ATP | Sigma | A9187 | Used for the preparation of intracellular solution |
Li-GTP | Sigma | G5884 | Used for the preparation of intracellular solution |
CsMeSO4 | Sigma | C1426 | Used for the preparation of Cs+-based intracellular solution |
CsCl | Sigma | C3011 | Used for the preparation of Cs+-based intracellular solution |
TEA-Cl | Sigma | T2265 | Used for the preparation of Cs+-based intracellular solution |
Neurobiotin 488 | Vector | SP-1145 | 0.05% neurobiotin 488 could be used for morphological studies |
Agar | Sigma | A7002 | 3% agar block was used in our protocol |
Paraformaldehyde | Sigma | P6148 | 4% paraformaldehyde was used for immunohistochemical processing |
Na2HPO4 | Hengxing Chemical Reagents | Used for the preparation of PBS | |
Mount Coverslipping Medium | Polyscience | 18606 | |
Urethan | National Institute for Food and Drug Control | 30191228 | 1.5 g/kg, i.p. |
Borosilicate glass capillaries | World Precision Instruments | TW150F-4 | 1.5 mm OD, 1.12 mm ID |
Micropipette puller | Sutter Instrument | P-97 | Used for the preparation of micropipettes |
Vibratome | Leica | VT1000S | |
Vibration isolation table | Technical Manufacturing Corporation | 63544 | |
Infrared CCD camera | Dage-MIT | IR-1000 | |
Patch-clamp amplifier | HEKA | EPC-10 | |
Micromanipulator | Sutter Instrument | MP-285 | |
X-Y stage | Burleigh | GIBRALTAR X-Y | |
Upright microscope | Olympus | BX51WI | |
Osmometer | Advanced | FISKE 210 | |
PH meter | Mettler Toledo | FE20 | |
Confocol microscope | Zeiss | LSM 700 |