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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Curr Protoc Neurosci. Author manuscript; available in PMC 2017 April 8.
Published in final edited form as:
PMCID: PMC4866814

Electrophysiological Measurement of Cannabinoid-Mediated Synaptic Modulation in Acute Mouse Brain Slices

Rita Báldi, Ph.D., Dipanwita Ghose, Ph.D., Brad A. Grueter, Ph.D., and Sachin Patel, M.D., Ph.D.corresponding author


Endocannabinoids (eCBs) are a class of bioactive lipids that mediate retrograde synaptic modulation at central and peripheral synapses. The highly lipophilic nature of eCBs and the pharmacological tools available to interrogate this system require unique methodological consideration, especially when applied to ex vivo systems such as electrophysiological analysis in acute brain slices. Here we discuss protocols for measuring cannabinoid and eCB-mediated synaptic signaling in mouse brain slices including analysis of short-term, long-term, and tonic eCB signaling modes, and the unique considerations for working with eCBs and TRPV1/cannabinoid ligands in acute brain slices.

Keywords: Endocannabinoid, Electrophysiology, Mouse, Cannabinoid, CB1 receptor


The type 1 cannabinoid receptor (CB1) is one of the most widely distributed G-protein coupled receptor in the brain (Kano et al., 2009). It is highly enriched within the perisynaptic presynapse and signals primarily via Gi/o-proteins (Yoshida et al., 2006). CB1 is most heavily expressed in the axon terminals of a subset of GABAergic interneurons, which express the neuropeptide cholecystokinin (CCK), as well as in axon terminals of glutamatergic pyramidal cells of cortical-like structures, and within striatal neurons of the basal ganglia (Katona and Freund, 2012). Activation of CB1 via natural and synthetic cannabinoids results in either reversible or irreversible synaptic depression of glutamate and GABA release (Castillo et al., 2012; Kano et al., 2009; Ohno-Shosaku et al., 2012).

In addition to being the target of plant-derived and synthetic cannabinoids, the CB1 receptor is the target of endogenous cannabinoids (Kano et al., 2009). Endocannabinoids (eCBs) are bioactive signaling lipids, which play a key role in retrograde inhibition at central and peripheral synapses. Two primary eCBs, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), have been well-studied and their role in synaptic modulation relatively well-defined. Although both 2-AG and AEA can activate centrally expressed CB1 (and CB2) cannabinoid receptors, they appear to subserve distinct forms of neuromodulation. For example, the most well-studied form of eCB-mediated retrograde suppression, depolarization-induced suppression of excitation/inhibition (DSE/DSI) is mediated via postsynaptic release of 2-AG (Tanimura et al., 2010), as are some forms of LTD (Chevaleyre and Castillo, 2003; Lerner and Kreitzer, 2012; Tanimura et al., 2010). AEA has been implicated in tonic eCB signaling and in several forms of long-term synaptic depression at both excitatory (LTD) and inhibitory (LTDi) synapses (Azad et al., 2004; Chavez et al., 2010; Grueter et al., 2010; Puente et al., 2011). Lastly, although 2-AG is relatively selective for CB1 and CB2 receptors, AEA is also a known agonist at transient receptor potential vanilloid 1 (TRPV1) receptors and possibly other ion channels such as small-conductance calcium activated potassium channels (SK channels) and some subtypes of sodium channels (Chavez et al., 2010; Grueter et al., 2010; Theile and Cummins, 2011; Wang et al., 2011).

When attempting to measure either CB1 receptor function or eCB-mediated synaptic signaling, several methodological issues can arise. These stem primarily from the chemical nature of most cannabinoids and eCBs, which are highly lipophilic molecules. Here we discuss basic protocols used for analyzing synaptic CB1 receptor function, short- and long-term eCB synaptic signaling, and tonic eCB signaling. Critical methodological and experimental determinants of successful detection of drug effects and eCB release are also discussed. The protocols described assume researchers have skills and equipment required for performing electrophysiological recordings from brain slices.

BASIC PROTOCOL 1: Cannabinoid Receptor Modulation of Synaptic Transmission

Determination of CB1 receptor synaptic signaling may be of interest when aiming to elucidate presynaptic mechanisms regulating neurotransmitter release at a particular synapse being investigated or as a first step in elucidating whether eCB signaling may modulate synaptic strength at a given synapse. Dynamic changes in CB1-mediated synaptic modulation of particular synapses can also be quantitatively determined between different drug treatments, environmental exposures, or even different strains of mice. Here we describe a basic protocol for assessing CB1 receptor function in acute mouse brain slices using electrophysiological approaches.


  1. ACSF (see Reagents and Solutions)
  2. Acute brain slices (see Support Protocols)
  3. Electrophysiological recording equipment (whole-cell or field-potential recording)
  4. CB1 agonists (Commercial sources include Cayman Chemical, Tocris, and others)

Protocol steps

  1. Prepare acute mouse brain slices containing brain region of interest (described in Support Protocols).
  2. Prepare drug solutions:
    1. Standard ACSF should be prepared and contain pharmacological agents used to isolate either GABAergic or glutamatergic synaptic transmission.
    2. Add 0.2–0.5g/L fatty-acid-free bovine serum albumin (BSA) to ACSF to enhance the solubility of hydrophobic drugs.
    3. Determine which CB1 agonist will be used. Table 1 contains a list of most commonly used CB1 agonists with receptor affinities and concentrations used in published literature.
      Table 1
      Drug affinities and final drug concentration ranges used in acute brain slice preparations to detect CB1-dependent synaptic depression (Pertwee et al., 2010).
      Step 2c: In some cases eCBs such as 2-AG and AEA are exogenously applied to brain slices. This approach works best when directly applied via picrospritzing or through a micropipette in the vicinity of the cell being recorded. Bath application of CB1 agonists can lead to false-negative results due to hydrolysis of 2-AG and AEA by hydrolytic enzymes active in brain slices. An alternate approach is to use more metabolically stable eCB analogs such as meth-anandamide and 2-AG-ether (Noladin Ether).
    4. Freshly dissolve hydrophobic drug in DMSO and vortex solution. In most cases 1000X stock solutions should be made to limit DMSO to 0.1% by volume. Using higher concentration stock solutions may cause precipitation of drug when added to ACSF. DMSO containing drug solutions should be added in drop wise fashion while ACSF is stirring. In some cases drug-containing DMSO may briefly precipitate when added to ACSF but then turn clear within a few seconds.
    5. Vehicle containing ACSF should contain the same concentration of DMSO as drug-containing ACSF.
  3. Oxygenate ACSF using open-ended tubing (1.5–1.7 mm inner diameter). The use of bubble-stones will cause a large number of very small bubbles to emerge from the ACSF, and is not recommended.
  4. Begin electrophysiological recordings in the presence of vehicle (DMSO)-containing ACSF. Flow rate should be maintained at 2–3ml per minute.
    Step 4: CB1 modulation of excitatory transmission can be measured using whole-cell patch clamp, intracellular recordings, and field-potential recordings, while CB1 modulation of GABAergic transmission generally requires whole-cell or intracellular recordings. CB1 modulation of synaptic transmission can be observed while monitoring evoked, spontaneous, or in some (but not all) cases, miniature synaptic currents. Choice of measurement depends on specific experimental design.
  5. After a stable baseline is recorded switch to CB1 agonist containing solution.
  6. Continue monitoring evoked or spontaneous currents for up to 1 hour or until new steady-state response level is reached. Due to the lipophilic nature of CB1 agonists, synaptic depression can take up to 30–45 minutes to reach its maximal effect. Termination of experiments prior to reaching stable post-drug responses could result in underestimation of drug effect.
    Step 6: As an alternative to measuring CB1 agonist-induced changes in evoked or spontaneous synaptic currents over time, brain slices can be incubated in CB1 agonist or control ACSF for 45–120 minutes prior to recording spontaneous or miniature synaptic currents in the continues presence of CB1 agonist or ACSF, respectively. This is a between cell experimental design, but has the advantage that multiple cells can be recorded from a single slice. However, this approach requires the use of spontaneous or miniature current recordings rather than evoked responses, and thus cannot be done using field-potential recordings.
  7. After completing the experiment replace ACSF from tubing and recording chamber with hot water, and then rinse all tubing and recording chamber with 100% ethanol, followed again by ACSF for at least 10 minutes prior to initiating another recording. Some recording chambers are made of adhesives that are deteriorated by organic solvents, in such cases briefly rinse the recording chamber with 70% ethanol (no more than 30 seconds) followed immediately by hot water and then ACSF.

BASIC PROTOCOL 2: Evaluation of Tonic Endocannabinoid Signaling

The homeostatic role of tonic eCB signaling was describes recently and has been demonstrated at both GABAergic and glutamatergic synapses in various brain regions (Foldy et al., 2013; Kim and Alger, 2010; Lee et al., 2015; Ramikie et al., 2014). Evidence suggests that both AEA and 2-AG can act as tonic eCB signals at different synapses. Tonic eCB signaling can be revealed by the blockade of CB1 receptors, or via inhibition of eCB degrading enzymes. The method used to measure tonic eCB signaling depends on the scientific hypothesis being tested (see Step 1 annotation).


  1. ACSF (see Reagents and Solutions)
  2. Acute brain slices (see Support Protocols)
  3. Electrophysiological recording equipment (whole-cell or field-potential recording)
  4. Drugs
    1. CB1 antagonist (AM251, SR141716; 5–10 μM)
    2. Monoacylglycerol lipase (MAGL) inhibitor JZL-184 (500nM–1μM)
    3. Fatty acid amide hydrolase (FAAH) inhibitor PF-3845 (1μM) or URB597 (1μM)
    4. Gq-coupled GPCR agonists (e.g. M1/3 Muscarinic agonist Oxotremorine-M; 1μM)

Protocol steps

  1. Determine which approach will be used; unmasking tonic eCB signaling via CB1 antagonist application or measuring tonic eCB signaling via eCB degradation inhibition.
    Step 1. Determining the specific approach that will be used is critical to establish prior to experimentation. For example, if one wishes to simply determine whether eCBs or CB1 receptors mediate tonic suppression of glutamatergic or GABAergic transmission at a particular synapse, the CB1 antagonist wash-on approach should be considered first. Alternatively, to determine whether Gq-coupled GPCR activation can trigger tonic eCB signaling, pre-incubation with Gq-coupled agonists prior to CB1 antagonist wash-on approach can be employed. If one wished to determine whether a specific eCB, AEA or 2-AG is capable of acting as a tonic eCB signal to suppress afferent neurotransmitter release, using FAAH or MAGL inhibitors, respectively, is a preferred option. In many cases a combination of approaches will be required for a complete evaluation of tonic eCB signaling.
  2. Prepare acute mouse brain slices containing brain region of interest (described in Support Protocols).
  3. Prepare drug solutions as described in detail in Basic Protocol 1, Step 2–3.
  4. Measuring tonic eCB signaling via eCB degradation inhibition.
    1. Brain slices containing brain region of interest should be divided into 2 holding chambers containing ACSF or HEPES holding ACSF (see Reagents and Solutions).
    2. One chamber should contain drug of choice (JZL-184, PF-3845, URB597), while the other should contain equal concentration of DMSO only.
    3. Place slice in recording chamber continuously perfused with oxygenated (95% O2, 5% CO2) ACSF at a rate of 2–3ml per minute.
    4. Perform whole-cell patch clamp recordings in voltage clamp configuration and wait 3–5 minutes after breaking in the cell before beginning the experiment. Measure spontaneous or, in the presence of tetrodotoxin (TTX, which inhibits action potential firing by blocking voltage-gated sodium channels), miniature excitatory/inhibitory postsynaptic currents (EPSC/IPSC) for 2–4 minutes. TTX. 3–5 cells can be recorded from the same slice but after 1.5 – 2 hours it is recommended to change slice.
    5. Alternate day by day from which experimental condition (vehicle vs. drug treated) will be made the first recordings of the day.
    6. When switching brain slices from drug to vehicle slices, remember to clean the tubing and chamber as described above (Basic Protocol 1, Step 7).
    7. When recording from drug-incubated slices, the perfusion ACSF should contain the same drug concentration as the holding chamber from where the slice was removed.
    8. Collect similar number of cells from each animal in each condition as differences may occur between animals and during slice preparation.
  5. Unmasking tonic eCB signaling via CB1 antagonist application.
    1. For electrical stimulation, bipolar stimulating electrodes or pipettes filed with recording ACSF can be used.
    2. Measure evoked EPSC/IPSCs in voltage clamp or EPSP/IPSPs in current clamp.
    3. Stimulate every 10–15 seconds. If there is any problem with run-down. As evident by progressive reduction in PSC/PSP amplitude over time, try lower frequency or different internal solution.
    4. After a stable baseline has been achieved (5–10 minutes) start the drug wash-on (CB1R antagonist 5–10μM AM251 or SR141716).
    5. Monitor the recording for up to 1 hour after drug application.
      Step 5. In some cases application of the CB1 antagonist will not cause synaptic potentiation, or the effect may be variable between cells. In this case, one approach may be to drive eCB mobilization by applying a Gq-receptor agonist to the perfusion ACSF prior to initiating recordings (Ramikie et al., 2014). Increasing temperature to 32–34°C may also increase tonic eCB signaling. In addition, using internal solutions without EGTA or BAPTA can increase detection of tonic eCB signaling.

BASIC PROTOCOL 3: Evaluation of Endocannabinoid Short-Term Synaptic Depression (DSE/DSI)

Depolarized neurons rapidly release eCBs in a Ca2+ dependent manner (Kano et al., 2009). The result is a short-term depression of synaptic transmission via retrograde signaling through CB1 receptors. This depression of synaptic transmission is termed “depolarization-induced suppression of excitation/inhibition” (DSE/I) depending on whether expressed at excitatory or inhibitory synapses, respectively. DSE/DSI are the most well studied forms of eCB mediated synaptic modulation and has been described at a large number of central synapses (Castillo et al., 2012; Kano et al., 2009; Ohno-Shosaku et al., 2012).


  1. ACSF (see Reagents and Solutions)
  2. Acute brain slices (see Support Protocols)
  3. Electrophysiological recording equipment (whole-cell recording)
  4. CB1 antagonists (Commercial sources include Cayman Chemical, Tocris, and others)

Protocol steps

  1. Prepare acute mouse brain slices containing brain region of interest (described in Support Protocols)
  2. Standard ACSF should be prepared and contain pharmacological agents used to isolate either GABAergic or glutamatergic synaptic transmission.
  3. Place slices in recording chamber continuously perfused with oxygenated (95% O2, 5% CO2) ACSF at a rate of 2–3ml per minute at 30 ± 2°C.
    Step 3: In some cases higher temperatures can facilitate eCB synthesis and may be required for detection of DSE/DSI, which is not present at room temperature. In other cases, where eCB synthesis is robust, use of higher temperatures may reduce the DSE/DSI duration by enhancing eCB degradation/uptake (Kreitzer and Regehr, 2001).
  4. A single neuron is isolated and DSE/I can be evaluated using whole-cell patch clamp techniques.
  5. Voltage clamp neuron at −70mV while stimulating axonal fibers with either electrical stimulation at 0.2Hz. After a stable baseline is recorded, typically 30–40 sweeps, stop the stimulation and depolarize the neuron to 0mV for 2–10 seconds and reverse back to −70mV resuming stimulation for the next 100 traces.
    Step 5: After depolarization, a decrease in amplitude of evoked EPSC/IPSC (according to synapse type) should be noticed. In some cases depolarization to +20 or +30mV may be required to elicit DSE/DSI. In general, DSI required shorter and less robust depolarization than DSE (Yoshida et al., 2011).
  6. To ensure that this DSE/DSI is CB1-mediated, in a separate set of experiments, the same depolarization protocol is used while recording from single neurons from slices pre-incubated for at least 1 hour in the CB1 antagonist (5–10μM AM251 or SR141716). Postsynaptic depolarization should not induce DSE/DSI since CB1 receptors are blocked and the eCBs produced by depolarization cannot bind to their target receptors.
  7. DSE/DSI can also be measured by recording spontaneous EPSCs/IPSCs (depending on which synapse type recordings are obtained from). For this, once a neuron has been patched and whole cell mode reached, baseline spontaneous activity is recorded for 1–2 minutes, followed by postsynaptic depolarization for 2–10 seconds to 0mV, then resuming the measurement of spontaneous currents for an additional 2–3 minutes.
  8. After completing the experiment clean the tubing and recording chamber carefully with concentrated ethanol and hot water (see Basic Protocol 1, Step 7).

BASIC PROTOCOL 4: Evaluation of Endocannabinoid Long-Term Synaptic Depression

eCB-dependent LTD of synaptic transmission results from the production and release of eCBs from the postsynaptic neuron, which then travel in a retrograde manner to bind with presynaptic CB1 receptors, resulting in a depression of synaptic transmission (Castillo et al., 2012; Katona and Freund, 2012). In many cases threshold duration of CB1 receptor activation is required to initiate LTD in place of DSE/DSI. The two phenomenon can be distinguished by the duration of synaptic depression, 10–60 seconds for DSE/I and >30 minutes for LTD. In other cases, different signaling pathways downstream of CB1 receptors are initiated to elicit short-term depression vs. LTD (Chevaleyre et al., 2007). Additionally, some evidence suggests presynaptic activity and calcium flux is required for expression of LTD at GABAergic synapses (Heifets et al., 2008). It is important to note that both chemical and afferent stimulation can induce eCB LTD at various synapses in the CNS, however the types of afferent activity required to induce LTD can vary dramatically between synapses/brain regions. As such, a degree of trial and error may be required to establish effective eCB LTD protocols.


  1. ACSF (see Reagents and Solutions)
  2. Acute brain slices (see Support Protocols)
  3. Electrophysiological recording equipment (whole-cell or field-potential recording)
  4. CB1 antagonists (Commercial sources include Cayman Chemical, Tocris, and others)

Protocol steps

  1. Prepare acute mouse brain slices containing brain region of interest (described in Support Protocols)
  2. Standard ACSF should be prepared and contain pharmacological agents used to isolate either GABAergic or glutamatergic synaptic transmission.
  3. Place slices in recording chamber continuously perfused with oxygenated (95% O2, 5% CO2) ACSF at a rate of 2–3ml per minute at 30 ± 2°C.
  4. A single neuron is isolated and recordings are obtained using whole-cell patch clamp techniques.
  5. Voltage clamp neuron at −70mV. Excitatory afferents are stimulated with an electrode at a frequency of 0.1Hz. Excitatory postsynaptic current amplitude is reported. After a stable baseline is recorded, typically 5–10 minutes, the LTD stimulation protocol is applied (see Table 2). Once the LTD protocol is complete, stimulation at 0.1Hz is resumed and EPSC amplitude recorded for additional 40–60 minutes.
    Table 2
    Selected examples of afferent stimulation protocols used to elicit eCB-LTD in acute brain slices. HFS (high frequency stimulation); 100Hz for 1 second. TBS (Theta-burst stimulation); ten bursts of five stimuli (100Hz within each burst, 200ms interburst ...
    Step 5: Input resistance, access resistance and holding current are monitored throughout the experiment and changes >20 % are deemed unacceptable. Various LTD protocols have been shown to induce eCB-dependent LTD in various brain regions (see Table 2).
  6. To ensure that this LTD is CB1 receptor mediated, in a separate set of experiments, the same LTD protocol is used while recording from single neurons from brain slices pre-incubated with a CB1 antagonist.
    Step 6: This should not induce LTD since the CB1 receptors are blocked and so eCBs that are produced by the postsynaptic neuron cannot bind to their target receptors to decrease synaptic transmission. Pre-incubation of brain slices for >1h in CB1 antagonist solution is important for complete blockade of CB1 receptors.
  7. Expression of CB1 dependent LTD can also be studied by bath application of CB1 agonist followed by CB1 antagonist perfusion. Stimulate excitatory afferents with an electrode at a frequency of 0.1Hz. After a stable baseline is recorded, typically over 5–10 minutes, apply the CB1 agonist and continue the recording for an additional 30–45 minutes. After a stable depression is observed, switch the perfusion ACSF to the one that contains only the CB1 antagonist. Failure of EPSCs to return to baseline suggests CB1 agonist-induced LTD has occurred.
    Step 7: Application of CB1 agonist can result in an LTD of evoked EPSC/IPSC amplitude, which does not reverse after application of the CB1 antagonist. In some cases, CB1 agonist-induced synaptic depression will reverse upon application of a CB1 antagonist, indicating CB1 receptor activation alone is not sufficient to induce LTD at that particular synapse of interest.
  8. After completing experiments that use CB1 agonists or antagonists, replace ACSF from tubing and recording chamber with hot water, and then rinse all tubing and recording chamber with concentrated ethanol as seen above in Basic Protocol 1 (Step 7).

SUPPORT PROTOCOL 1: Tissue Preparation

Preparing good quality brain slices is necessary to be able to collect reliable electrophysiological data and maintain physiological microcircuit properties. Protective cutting methods are based on the replacement of NaCl, which is thought to be the main cause of the poor survival of the neurons causing swelling during the recovery period. Replacement of NaCl by sucrose is a widely used method and it works well in most brain regions in young animals.


  1. 1X Sucrose ACSF (cutting solution)
  2. 1X ACSF (slice holding and recording solution)
  3. Stock solutions of 5X Sucrose ACSF and 10X ACSF are prepared which is then diluted to 1X Sucrose ACSF and 1X ACSF, respectively. 1X solutions are made fresh every day before slicing. For the recipes of each solutions and their storage information see Reagents and Solutions.


  1. Brain dissection: large scissors, 1–2 small scissors, forceps, rongeur brain slicer, razor blades, small beaker, spoon, super glue
  2. Vibratome, razor blade, fine brush, plastic transfer pipette (cut off end to get a wide opening)
  3. Slice holding chambers.
  4. Carbogen supply, tubing, bubble stones
  5. Solution preparation: osmometer.

Protocol steps

  1. After the solutions are ready and oxygenated, they are used to prepare brain slices containing area of interest.
  2. For slicing we use a fully automated Leica VT 1200S vibratome.
  3. Cool down the buffer tray by packing crushed ice on each sides of the tray.
  4. Fill up the buffer tray with a combination of cold and frozen “slushy” mixture of 1X Sucrose ACSF cutting solution and continuously oxygenate the solution.
  5. Place the razor blade in the blade holder then put it into place in the vibratome.
  6. Cool down the slicing instruments and put a small 50ml beaker with cold oxygenated 1X Sucrose ACSF on ice.
  7. After the equipment and solutions are ready, briefly anesthetize the mouse using ~5 % isoflurane. Decapitate the mouse and carefully remove the brain and place it in the beaker containing cold oxygenated 1X Sucrose ACSF.
  8. Transfer the brain into a sagittal or coronal brain slicer according to the preferable anatomical plane of the brain area of interest. Dry it off, block off the tissue using sharp blades, remove the remaining solution using filter paper, if necessary, and glue the region of interest on the specimen disk.
  9. Quickly place the specimen disk into the buffer tray of the vibratome and start to adjust the settings for the slicing.
  10. Move the stage up till the razor blade get close to the surface of the brain. Set the cutting start and end position of the razor blade, and start collecting the slices. The thickness of the slices should be adjusted in between 250–400μm.
  11. Once the slices are cut, transfer them into a submerged holding chamber containing oxygenated 1X ACSF (~25°C) and allow recovery for at least 60 minutes prior to recording.

ALTERNATE SUPPORT PROTOCOL 1: NMDG Neuroprotective Tissue Preparation

While sucrose ACSF is a commonly used protective method, it still leaves some issues while preparing brain slices from aging animals. Ting et al. recently described a new protocol where instead of sucrose, they replaced the NaCl with N-methyl-D-glucamine (NMDG) (Ting et al., 2014), which, especially during the early phase of the slice recovery, drastically decreased the cell swelling. The inclusion of 20mM HEPES into the holding ACSF also decreased edema, which appears during the incubation of adult brain slices. If brain slices suffer from poor cell health, either low numbers of cells, or unhealthy cells, this neuroprotective preparation should be considered (Ting et al., 2014).



  1. NMDG ACSF (see Reagents and Solutions)
  2. HEPES holding ACSF (see Reagents and Solutions)


  1. Perfusion and brain dissection: perfusion pump (alternatively: syringe –filled up with cold oxygenated NMDG ACSF right before the perfusion –with a needle), forceps, hemostat, fine dissecting scissors, heavy duty scissors, glass petri dish, spatula, spoon, brain matrix, kimwipes, razor blades, super glue
  2. Vibratome, razor blade, fine brush, plastic transfer pipette (cut off end to get a wide opening)
  3. Slice holding chambers.
  4. Water bath with thermometer: one for keeping the slice incubation chamber at 32–34°C and another at 24°C.
  5. Carbogen supply, tubing, bubble stones
  6. Solution preparation: pH-meter, osmometer.

Protocol steps

  1. Prepare two incubation chambers. One filled up with NMDG-ACSF and placed in the water bath at 32–34°C, the other filled up with HEPES holding ACSF and placed at 24°C. Oxygenate the solutions.
    Step 1: At 34°C the osmolarity of the NMDG ACSF goes up with time. Use it fresh; some cell-types are more sensitive to higher osmolarity.
  2. Follow the steps described in Support Protocol 1 but instead of sucrose, use NMDG- ACSF.
    Step 2: The temperature of the NMDG-ACSF during the perfusion and slicing is not as critical as for sucrose ACSF because NMDG is particularly effective at slowing down the cellular metabolism. Slices can be prepared at higher temperature as it was proposed to be helpful for cell-health (Huang and Uusisaari, 2013).
  3. Alternatively, in between step 6–7 of Support Protocol 1, anesthetize the animal and preform transcardial perfusion with cold oxygenated NMDG-ACSF to maintain better structure. This can help in recordings from deeper brain areas and adult animals.
  4. Protective recovery step: From the vibratome, collect the slices into the pre-warmed NMDG ACSF containing holding chamber for <12 minutes. Temperature and time must be maintained precisely.
    Step 4: This neuroprotective recovery method was developed for adult and aging animals. Ting et al, 2014 suggest that it is not optimized for mice younger than 5–6 weeks of age (where sucrose cutting method works properly) and incomplete wash-out of the NMDG was observed.
  5. After the initial recovery step, transfer the slices into the HEPES holding ACSF containing chamber at 24°C, and try to minimize the carry-over of the NMDG solution during the procedure. Slices can be stored for several hours until transfer to the recording chamber for use.



Stored at 4°C for up to 2 weeks.

ChemicalStock g/LFinal concentration (mM)
Anhydrous CaCl22.782.5


Made and used fresh daily.

For preparing 1X ACSF from 10X stock use 100ml of the stock and dilute up to 1L. Add fresh sodium bicarbonate and glucose to the 1X ACSF in the amounts described below. Stir solution until ingredients are mixed uniformly. Measure the osmolarity using osmometer. Osmolarity should be 295–300 mosmol.

Chemicalg/LFinal concentration (mM)

5X Sucrose ACSF

Stored at 4°C.

ChemicalStock g/LFinal concentration (mM)
Anhydrous CaCl21.112.0

1X Sucrose ACSF

Made and use fresh daily.

For preparing 1X sucrose from 5X stock use 200ml of the stock and dilute up to 1L. Add fresh sodium bicarbonate and glucose to the 1X sucrose in the amounts described below. Stir solution until ingredients are mixed uniformly. About 200ml of 1X sucrose is placed at −20°C to freeze so that this frozen “slushy” mixture can be used for slicing.

Chemicalg/LFinal concentration (mM)
Glucose1. 810


Made and use fresh daily.

Pour MilliQ water into a beaker (less than the final volume) and add each compounds except the MgSO4·7H2O and CaCl2·4H2O (Note that CaCl2·4H2O precipitates at basic pH). Stir it continuously. At this point the pH of the solution is close to 10. Use concentrated HCl acid to set the pH to 7.3 – 7.4. This step needs some time, make sure the pH has stabilized. Add the CaCl2 and MgSO4 from the 2M stock solutions (see tables below for recipe). Fill it up to the final volume and measure the osmolarity. Osmolarity should be ~300mOsm. It is recommended to make it fresh each day. Although 10X stock solution containing KCl, NaH2PO4, HEPES, glucose works well up to 2 weeks at 4°C after careful preparation.

Chemicalg/LFinal concentration (mM)
Titrate the pH to 7.3 – 7.4 (with concentrated HCl acid)
MgSO4·7H2O5 mL10
CaCl2·4H2O0.25 mL0.5

1X HEPES holding ACSF

Made and use fresh daily.

Similarly to the NMDG solution add each compound (except MgSO4·7H2O and CaCl2·4H2O) into the high purity water. Titrate the pH to 7.3 – 7.4 with NaOH. Fill it up to the final volume and set the osmolarity to ~300mOsm. 10X stock solution can be used with careful preparation adding the following compounds: NaCl, KCl, NaH2PO4, HEPES, Glucose. Storage is at 4°C up to 2 weeks. NaHCO3, Na-ascorbate, Na-pyruvate, MgSO4·7H2O and CaCl2·4H2O should be added on the day of use to 1 X solution.

Chemicalg/LFinal concentration (mM)
Titrate the pH to 7.3 – 7.4 (with NaOH)
MgSO4·7H2O (2 M stock)1 mL2
CaCl2·4H2O (2 M stock)1 mL2

MgSO4 and CaCl2 stock

Stored at 4°C.

2 M MgSO4 stock
Chemicalg/100 mLConcentration (M)
2 M CaCl2 stock
Chemicalg/100 mLConcentration (M)

Example Internal solution recipes

Cesium based Internal solution: (in mM) 120 CsMeSO4, 15 CsCl, 8 NaCl, 10 HEPES, 0.2 EGTA, 10 TEA-Cl, 4 Mg2+ ATP, 0.3 Na2+ GTP and 5 QX-314 (pH 7.25–7.35, adjusted with CsOH).

Potassium based Internal solution: (in mM) 120 K+-gluconate, 4 NaCl, 10 HEPES, 20 KCl, 4 Mg-ATP, 0.3 Na-GTP, and 10 Na-phosphocreatine (pH 7.25–7.35, adjusted with KOH).


Background Information

Since the initial identification and CNS localization of CB1 receptors, extensive research has identified cannabinoid and eCB signaling as a widespread neuromodulatory system regulating synaptic strength (Castillo et al., 2012; Kano et al., 2009; Katona and Freund, 2012; Ohno-Shosaku et al., 2012). In addition to responsivity of these receptors to exogenous agonists, including tetrahydrocannabinoid, the primary psychoactive constituent of cannabis plants, CB1 receptors are activated by endogenously released eCB lipids. 2-AG and AEA are two well-studied eCB ligands implicated in synaptic modulation. Synaptic 2-AG is synthesized from free diacylglycerol primarily via diacylglycerol lipase α, which is localized to dendritic spines and some somatic regions apposing invaginating GABAergic synapses (Yoshida et al., 2006; Yoshida et al., 2011). 2-AG is degraded primarily by MAGL localized to presynaptic axon terminals (Dinh et al., 2002). In contrast, AEA can be synthesized via a variety of enzymatic mechanisms (Liu et al., 2008), however the precise route for synthesis of synaptic AEA remains controversial. In contrast, AEA is degraded primarily by FAAH, which is localized to postsynaptic dendrites and cell bodies (Cravatt and Lichtman, 2003). Both 2-AG and AEA can activate CB1 receptors, but the physiological mechanisms triggering selective or redundant release of different eCBs and the interaction between these two eCB systems are only now beginning to be elucidated (Lee et al., 2015). Since their discovery, eCBs have been demonstrated to play a critical role in synaptic modulation throughout the brain and periphery, and thus comprise a ubiquitous and generalized mechanism subserving activity-based retrograde synaptic suppression.

The first form of eCB signaling, DSI/DSE, was described in the cerebellum and hippocampus by the Marty and Alger labs in the early 1990’s (Llano et al., 1991; Pitler and Alger, 1992). 10 years later DSI/DSE was first shown to be dependent upon CB1 receptor function by three labs; by the Nicol lab at inhibitory synapses in the hippocampus, and in the cerebellum by the Regehr and Kano labs (Kreitzer and Regehr, 2001; Ohno-Shosaku et al., 2001; Wilson and Nicoll, 2001). In addition to being CB1 dependent, DSI/E is Ca2+ dependent and can be enhanced by activation of Gq-coupled GPCRs (Ohno-Shosaku et al., 2002). More recently, seminal studies have confirmed that DSE/DSI is mediated by 2-AG signaling (Gao et al., 2010; Tanimura et al., 2010). Since these initial discoveries, expression of DSI/DSE has been demonstrated at numerous synapses in the CNS, and the molecular mechanisms subserving this form of eCB signaling have been extensively investigated (see (Castillo et al., 2012; Kano et al., 2009; Ohno-Shosaku et al., 2012)). DSE/DSI is now considered a prototypical synaptic bioassay for detection of synaptic 2-AG release.

In addition to DSE/DSI, eCBs are also known to mediate LTD at both glutamatergic and GABAergic synapses. eCB-dependent LTD is induced primarily by repetitive afferent stimulation with and without postsynaptic depolarization, and has been found in several areas of the brain (Castillo et al., 2012; Heifets and Castillo, 2009; Kano et al., 2009). While Gerdeman and Lovinger (2002) first reported eCB-dependent LTD at excitatory synapses in the dorsal striatum (Gerdeman et al., 2002), eCB-dependent LTD of inhibitory synapses was first described by the Lutz and Castillo labs in the amygdala and hippocampus, respectively (Chevaleyre and Castillo, 2003; Marsicano et al., 2002). Examples of brain regions in which eCB-LTD exist at excitatory synapses including the dorsal striatum, nucleus accumbens, cerebral cortex, dorsal cochlear nucleus, cerebellum, and hippocampus (see Kano et al., 2009). Endocannabinoid-LTD of inhibitory synapses has been uncovered in the hippocampus, amygdala, ventral tegmental area and dorsal striatum (Heifets and Castillo, 2009). Novel mechanisms contributing to eCB-LTD continue to be discovered at a rapid pace, as exemplified by recent implication of astrocyte CB1 receptor in eCB-LTD (Min and Nevian, 2012).

The protocols described herein will allow investigators to establish synaptic assays for measuring CB1 receptor-dependent synaptic modulation and eCB-mediated signaling processes in acute brain slices. Establishing reliable eCB signaling bioassays is critical for experimental analysis of dynamic changes in synaptic eCB signaling that could occur in response to pharmacological and environmental challenges and in disease models. These assays will also form the basis from which new insights in to the molecular mechanisms regulating eCB signaling at the synaptic levels, and its interaction with other neuromodulatory systems, can be elucidated.

Critical Parameters

  1. Osmolarity and pH of all the solutions should be checked to ensure good recordings. Typically osmolarity of 1X ACSF is adjusted to be slightly higher than that of the internal solution. However, iso-osmolar solutions can also work well.
  2. Age of the animal is a very important factor for eliciting both short and long term plasticity. In some cases, the ability to measure eCB synaptic signaling may decline in older mice.
  3. Composition of internal solutions is also very important for eliciting both short and long term plasticity. Use of calcium chelators such as EGTA and BAPTA could prevent calcium-dependent eCB production and should be minimized/avoided in internal solutions. In some cases, it has been noted that cesium-based internal solutions prevent detection of eCB-mediated signaling (Huang and Woolley, 2012).
  4. Careful preparation of solutions containing CB1 agonists/antagonists and eCB degradation inhibitors is critical. Lipophilic compounds tend to precipitate in aqueous solutions and carriers such as BSA are often required to keep drugs from precipitating. Over-stirring and over-oxygenating can also result in precipitation. Careful monitoring of drug solutions is required throughout the day. All solution should be made fresh daily.


  1. Problem: Application of CB1 agonists did not cause synaptic depression: Make sure drug solution has not precipitated during the course of the experiment. Next, ensure that the concentration is high enough. It is generally recommended that initial studies utilize a high concentration of agonist (see Table 1). Next, ensure that recordings are of sufficient duration. In many cases drug effects can take 30–60 minutes to stabilize. Lastly, if evaluating a synapse that has not been previously shown to be modulated by CB1 receptor activity, perform a positive control in a brain region known to express functional CB1 receptors. GABAergic synapses in the CA1 hippocampus and glutamatergic cortico-striatal synapses have both been extensively examined in several mouse and rat strains and uniformly show synaptic depression in response to CB1 agonist application.
  2. Problem: There is a lack of DSE/DSI at a particular synapse: Many factors contribute to the expression and magnitude of DSE/DSI in acute brain slices. Regional heterogeneity is a key factor, with several brain regions such as the cerebellum and hippocampus expressing high levels of eCB-mediated short-term plasticity. Many other regions also express DSE/DSI, however, in many cases the magnitude is generally lower than in these areas. To maximize detection of DSE/DSI we suggest the followings: 1) utilizing internal patch solutions that do not contain any EGTA or BAPTA, 2) if Cs-based solutions are not effective, try K-based internal solutions, 3) if room temperature is not effective, increase to 32–34 degrees, 4) if using mice older than 6 weeks of age, try younger ages as a positive control, 5) ensure that synapses under investigation express functional CB1 receptor (see Basic Protocol 1), 6) augment ACSF with Gq-GPCR agonist (mGluR1/5 agonist dihydroxyphenylglycine (DHPG; 5–25μM) or M1/3 muscarinic agonist Oxotremorine-M (0.1–1μM).
  3. Problem: There is a lack of synaptically-induced LTD at a particular synapse: As a first step toward troubleshooting synaptically-induced LTD, verify CB1 agonists induce irreversible synaptic depression (see Basic Protocols 1). In addition to the troubleshooting suggestions listed above in 1 and 2, stimulating in the presence of a FAAH or MAGL inhibitor has been shown to enhance eCB-LTD in several studies (Kreitzer and Malenka, 2007; Puente et al., 2011). Other considerations for troubleshooting synaptically-induced LTD are to increase signaling through Gq receptors by adding low concentrations of agonists (i.e. DHPG for Group I mGluR) or blocking glutamate uptake (TBOA) when recording at higher temperatures 32°C.

Anticipated Results

  1. Basic Protocol 1: It is anticipated that application of CB1 agonist will decrease the amplitude of evoked IPSC/EPSCs after CB1 agonist application provided sufficient time has passed to allow drug to penetrate the brain slice (Figure 1A). An increase in the paired pulse ratio (PPR) should also be observed, and possible increase in the coefficient of variation (CV), relative to the baseline (pre-drug) period. It is anticipated that the effects of the CB1 agonist will be absent or strongly attenuated in the presence of a CB1 receptor antagonist or in CB1 receptor KO mice. If measuring spontaneous IPSC/EPSCs, it is anticipated that cells exposed to CB1 agonist for a sufficient duration to allow full occupancy of CB1 receptors will show a decrease in frequency, but not amplitude of spontaneous currents (Figure 1B). In most (but not all) cases, CB1 receptor activation will cause a more rapid and potent effect on GABAergic synapses than glutamatergic synapses.
    Figure 1
    CB1 agonists reduce evoked and spontaneous glutamate release in the amygdala
  2. Basic Protocol 2: If tonic eCB signaling is present, it is anticipated that bath application of a CB1 receptor antagonist will result in an increase in the amplitude of evoked synaptic currents over a 20–40 minute period (Figure 2). It is anticipated, that if under basal conditions, no tonic eCB signaling is revealed, that addition of Gq-coupled GPCR agonists to the ACSF could trigger tonic eCB release (Figure 2A). If 2-AG mediated the eCB signal, the diacylglycerol lipase inhibitor THL and post-synaptic calcium chelation should block CB1 antagonist-induced synaptic potentiation (Figure 2B). If 2-AG is acting in a tonic manner to suppress afferent neurotransmission, it is anticipated that incubation with the MAGL inhibitor will cause a decrease in frequency (but not amplitude) of spontaneous synaptic currents relative to vehicle-incubated cells. If AEA is acting in a tonic manner to suppress afferent neurotransmission, it is anticipated that incubation with the FAAH inhibitor will cause a decrease in frequency (but not amplitude) of spontaneous synaptic currents relative to vehicle-incubated cells. In some cases a change in amplitude may occur, which could be due to postsynaptic effects of AEA on TRPV1 channels (Chavez et al., 2010; Grueter et al., 2010).
    Figure 2
    Tonic eCB signaling at central amygdala glutamatergic synapses
  3. Basic Protocol 3: It is anticipated that postsynaptic depolarization will cause a transient decrease in the amplitude of evoked synaptic currents lasting tens of seconds, but no longer than 1–2 minutes (Figure 3). DSE/DSI will be eliminated or strongly reduced in slices pre-incubated with a CB1 receptor antagonist. In general, DSI will require shorter periods of depolarization to elicit than DSE.
    Figure 3
    Single cell example of DSE in nucleus accumbens
  4. Basic Protocol 4: It is anticipated that repetitive afferent activity, sometimes requiring concomitant postsynaptic depolarization, will result in eCB-mediated LTD (Figure 4). However, the frequency, pattern, and duration of afferent stimulation required to induce eCB-LTD can vary considerably between brain regions. It is anticipated that LTD mediated by eCB signaling will be partially or completely blocked by pre-incubation with a CB1 receptor antagonist (Figure 4). In many cases, both homosynaptic and heterosynaptic eCB-LTD require activation of mGluR1/5 receptors and increases in postsynaptic calcium, and can therefore be blocked by antagonists of mGluR1/5 and postsynaptic calcium chelation. eCB-mediated LTD should result in an increase in paired-pulse ratio and coefficient of variation.
    Figure 4
    Single cell examples of eCB-LTD in the nucleus accumbens

Time Considerations

In general, the time considerations for the described protocols are defined by the electrophysiological approaches, not the particular type of signaling system being considered. However, there are some key points worth noting. First, examination of short-term eCB signaling required less total time than examination of LTD. Second, examination of eCB agonist effects on spontaneous synaptic currents using a between cell design may reduce overall time since multiple cells can be recorded from the same slice (which is not the case for within cell designs comparing pre-drug and post-drug spontaneous IPSC/EPSC frequencies or evoked synaptic current amplitudes). Lastly, examination of CB1 agonist induced synaptic depression may require longer duration recordings than for water-soluble drugs. Early termination of recordings can underestimate the magnitude of drug effects.


The authors were supported by NIH Grants R01MH100096 and R21MH103515 (S.P.), and R00DA031699 (B.A.G).

Contributor Information

Rita Báldi, Department of Psychiatry, 2213 Garland Avenue, 8415 MRBIV, Vanderbilt University Medical Center, Nashville, TN 37232-0413, Tel. 615-936-7768, Fax. 615-936-4075.

Dipanwita Ghose, Department of Anesthesiology, 2213 Garland Avenue, P445 MRBIV, Vanderbilt University Medical Center, Nashville, TN 37232-0413, Tel. 615-936-1684, Fax. 615-936-0456.

Brad A. Grueter, Department of Anesthesiology, 2213 Garland Avenue, P435H MRBIV, Vanderbilt University Medical Center, Nashville, TN 37232-0413, Tel. 615-936-2586, Fax. 615-936-0456.

Sachin Patel, Departments of Psychiatry and Molecular Physiology & Biophysics, 2213 Garland Avenue, 8425B MRBIV, Vanderbilt University Medical Center, Nashville, TN 37232-0413, Tel. 615-936-7768, Fax. 615-936-4075.


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