NMDA receptor

A caged form of the irreversible NR open-channel blocker MK801 was synthesized by adaptation of a previously used caging procedure (Frankel et al., 2003). The new compound (dizocilpine N-(4,5-dimethoxy-2-nitrobenzyl) carbamate) absorbs UV light (λ <400 nm), and is cleaved by exposure to UV light, liberating MK801 (Fig. 1A). To test whether the caged compound is stable within cells and whether MK801 could be specifically photoreleased by UV flashes in a brain slice preparation, we loaded individual L2/3 neurons of the mouse barrel cortex with cMK801 (1 mm) and monitored the postsynaptic NR current before and following a UV flash (Fig. 1B). The synaptic NR current was monitored during voltage-clamp at +40 mV and was isolated by the addition of 2 μm gabazine and 10 μm NBQX to the superfusion fluid. The NR current was stable before the UV flash. However, after a 10 min baseline, a 1-s-long UV flash produced a gradual decrease of the amplitude of the NR current and an almost complete block within 8–10 min after delivery of the UV flash (3 ± 3% of baseline amplitude, n = 6) (Fig. 1B). This finding suggests that MK801 was efficiently uncaged by a 1-s-long UV flash. The caged compound itself did not seem to have any effect on the NR currents, as the shape, amplitude, and time course of the responses continued to be typical of NR-mediated currents when the flash was not delivered. To ensure that the compound was stable over the duration of synaptic plasticity experiments, we repeated the experiment with 40 min baseline, which yielded equivalent results (4 ± 3%, n = 5) (Fig. 1C). The NR block following photorelease was use-dependent, because stopping synaptic stimulation for 10 min prevented the block of NR current, whereas a progressive block was again observed on resuming stimulation (Fig. 1D).[1]

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To assess the use of cMK801 in synaptic plasticity experiments, we first tested whether postsynaptic photorelease of MK801 could successfully block the induction of t-LTP. To this end, we monitored EPSPs evoked by extracellular stimulation in L4 during whole-cell recording of L2/3 pyramidal cells. LTP was induced by 100 pairings of an EPSP followed by a single postsynaptic spike (with the postsynaptic spike occurring ∼10 ms after presynaptic stimulation). When L2/3 cells were loaded with 1 mm cMK801, but with no flash applied, this pre-before-post pairing protocol induced robust t-LTP (148 ± 4%, n = 5; p < 0.01, t test) (Fig. 2), similar to unloaded cells (Rodríguez-Moreno and Paulsen, 2008). In the same experimental condition, but with a somatodendritic UV flash delivered, the induction of t-LTP was completely blocked (96 ± 6%, n = 6) (Fig. 2). To ascertain that the effect of the UV flash was specific to the cell loaded with cMK801, we delivered UV flashes 200 μm lateral to the recording pipette. In this situation, t-LTP was unaffected (157 ± 6%, n = 6) (Fig. 2). These results indicate that postsynaptic somatodendritic NRs are necessary for t-LTP induction consistent with previous results (Bender et al., 2006; Rodríguez-Moreno and Paulsen, 2008).[1]

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In contrast to the postsynaptic NRs involved in t-LTP, we have previously reported that t-LTD requires presynaptic NRs (Rodríguez-Moreno and Paulsen, 2008). To investigate whether the NRs that are required for t-LTD are located in the axonal or the somatodendritic compartment of the presynaptic L4 neurons, we used paired whole-cell recordings of synaptically connected L4 and L2/3 cells as previously described (Rodríguez-Moreno and Paulsen, 2008). Of 332 pairs recorded, 31 pairs showed a monosynaptic EPSP (latency, 1.9 ± 0.4 ms; jitter, 0.12 ± 0.04 ms), 28 of which were used in plasticity experiments. First, a t-LTD induction protocol was applied to 12 pairs in which the presynaptic L4 neuron was loaded with 1 mm cMK801 (Fig. 3A). A UV flash confined to the somatodendritic region of the L4 neuron delivered 1 min before the start of the pairing protocol did not affect the induction of t-LTD (80 ± 5%, n = 6; p < 0.01, t test) (Fig. 3B,C). In contrast, when the UV flash was delivered to the axonal region of the L4 cell (i.e., in layer 2/3) 1 min before the t-LTD induction protocol, t-LTD was completely blocked (97 ± 4%, n = 6) (Fig. 3B,C). Axonal flash without pairing had no effect on the EPSP (97 ± 7%, n = 3) (Fig. 3D,F). To confirm that the block of t-LTD induction in the axonal region of L4 cells is mediated specifically by uncaging of MK801 and not by the UV flash itself, we repeated the experiment with no cMK801 included in the presynaptic pipette. In this condition, an axonal UV flash delivered 1 min before the t-LTD induction protocol did not affect t-LTD (78 ± 5%, n = 5) (Fig. 3B,C). Moreover, if a 10 min delay was introduced between presynaptic somatodendritic uncaging and the pairing protocol, t-LTD was blocked (96 ± 7%, n = 4) (Fig. 3E,F), suggesting that once photoreleased, free MK801 can diffuse into the axon and block the induction of t-LTD. Together, these results indicate that presynaptic axonal NMDA receptors are required for the induction of t-LTD.[1]

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Based on trial-to-trial fluctuation analysis (Rodríguez-Moreno and Paulsen, 2008), as well as changes in paired-pulse ratio (Bender et al., 2006), t-LTD at L4-L2/3 synapses has been proposed to be expressed presynaptically through a decrease in presynaptic release probability. Changes in release probability at excitatory synapses can be assessed by analyzing the progressive block of NR-mediated currents by MK801 (Hessler et al., 1993; Rosenmund et al., 1993). As MK801 is an irreversible NR open-channel blocker (Jahr, 1992), NRs are blocked only at synapses that release transmitter, so the trial-to-trial rate of block provides a measure of the release probability (Rosenmund et al., 1993). We wanted to take advantage of caged MK801 to investigate whether a change in release probability can account for the magnitude of synaptic depression. To this end, we used an improved, water-soluble version of cMK801 (TEG-substituted cMK801, TcMK801), which we loaded postsynaptically and monitored EPSPs evoked by extracellular stimulation in L4 during current-clamp whole-cell recording of L2/3 pyramidal cells. t-LTD was induced in one pathway by 100 pairings of a postsynaptic action potential followed after ∼15 ms by an EPSP. This post-before-pre pairing protocol induced robust t-LTD (75 ± 6%, p < 0.01, n = 7) (Fig. 4A). An unpaired naive pathway was used as control, and no changes in EPSP slopes were observed in that pathway (101 ± 6%, n = 7) (Fig. 4A). Thirty to 35 min after the pairing protocol, we switched to voltage-clamp mode and recorded NR–EPSCs at −30 mV in the same cells. NR currents were isolated by the addition of gabazine (2 μm) and NBQX (10 μm) to the superfusion solution to block GABAA and AMPA/kainate receptors, respectively. After 10 min baseline, a 1-s-long UV flash was applied to photorelease MK801. After delivery of the UV flash, a gradual decrease of the amplitude of the NR currents and eventually an almost complete block was observed in the paired (96 ± 3%, n = 7) as well as the unpaired pathway (97 ± 3%, n = 7) (Fig. 4B). However, a slower rate of decay (measured as the number of stimuli necessary to reduce NR–EPSC amplitude) was observed in the paired compared with the unpaired pathway (Fig. 4B). The decay could be adequately fitted with a single exponential function (R2 > 0.90 in all inputs; n = 14) (Fig. 4B). The half-life of the NR–EPSC was 32 ± 4 stimuli for the unpaired pathway (n = 7) and 46 ± 4 stimuli for the paired pathway (n = 7; p < 0.05, t test) (Fig. 4B). These results are consistent with a reduction in the neurotransmitter release probability in the paired pathway. To estimate the relative change in release probability, we compared the difference in reduction of estimated NR current after one stimulation between the paired and unpaired pathways. There was a 27 ± 10% (n = 7) difference between the two pathways, which compares to 25 ± 6% depression in the paired pathway (n = 7). This result suggests that a decrease in release probability can account for t-LTD, indicating a presynaptic locus of expression for this form of t-LTD [1].

Recording conditions. [1]
Barrels were identified under a stereomicroscope. One or two stimulation electrodes were placed at the base of a barrel (L4). Whole-cell patch-clamp recordings were obtained from L2/3 pyramidal cells in the same barrel column with 5–7 mΩ borosilicate pipettes. In voltage-clamp experiments, the pipette solution contained the following (in mm): 140 CsCl; 0.2 EGTA; 10.0 HEPES; 2.0 ATP–Mg; 0.3 GTP–NaCl; 5.0 QX-314, adjusted to pH 7.2 with CsOH. Voltage-clamp experiments were performed in the presence of gabazine (2 μm) and NBQX (10 μm). For current-clamp experiments, the pipette solution contained the following (in mm): 110 potassium gluconate; 40 HEPES; 4 NaCl; 4 ATP–Mg; 0.3 GTP, adjusted to pH 7.2 with KOH. The pipette tip was filled with standard solution and backfilled with cMK801- or TcMK801-containing solution. Cells with a pyramidal-shaped soma were selected for recording using infrared, differential interference contrast optics. All cells were tested for regular spiking responses to positive current injection, characteristic of pyramidal cells. Whole-cell recordings were made using an Axon Multiclamp 700B amplifier. Recordings were low-pass filtered at 2 kHz and acquired at 5 kHz using an ITC-16 board and custom-made software procedures programmed in Igor Pro. Cells were rejected if series resistance changed by >15% during the recording.

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MK801 uncaging. [1]
Photolysis of the caged MK801/cMK801 was achieved with flashes of focused ultra-violet (UV) light (1 s, ∼120 μm diameter, <460 nm) produced by a 100 W mercury arc lamp (HBO 100, Zeiss in combination with a Uniblitz T123 Driver and LS5 shutter system) connected to the epifluorescence port of the microscope via a fiberoptic light-guide. Light intensity was ∼20 μW/cm2 after passing through a dichroic filter and the 40× objective.

[1]. Antonio Rodríguez-Moreno, et al. Presynaptic induction and expression of timing-dependent long-term depression demonstrated by compartment-specific photorelease of a use-dependent NMDA receptor antagonist. J Neurosci. 2011 Jun 8;31(23):8564-8569.

NMDA receptors are important for synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). To help investigate the precise location of the NMDA receptors that are required for different types of synaptic plasticity, we synthesized a caged form of the use-dependent NMDA receptor antagonist MK801, which we loaded into individual neurons in vitro, followed by compartment-specific uncaging. We used this method to investigate timing-dependent plasticity at layer 4-layer 2/3 synapses of mouse barrel cortex. Somatodendritic photorelease of MK801 in the postsynaptic neuron produced a use-dependent block of synaptic NMDA receptor-mediated currents and prevented the induction of LTP. Compartment-specific photorelease of MK801 in the presynaptic neuron showed that axonal, but not somatodendritic, presynaptic NMDA receptors are required for induction of LTD. The rate of use-dependent block of postsynaptic NMDA receptor current was slower following induction of LTD, consistent with a presynaptic locus of expression. Thus, this new caged compound has demonstrated the axonal location of NMDA receptors required for induction and the presynaptic locus of expression of LTD at layer 4-layer 2/3 synapses. [1]

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We have developed a caged form of MK801 to study synaptic plasticity. Our data confirm that postsynaptic NRs are necessary for t-LTP at L4-L2/3 synapses, induced by pairing single presynaptic and postsynaptic action potentials, which is an effective protocol for induction of t-LTP in developing cortex including hippocampus (Bi and Poo, 1998; Debanne et al., 1998; Meredith et al., 2003; Campanac and Debanne, 2008). In contrast, our results indicate that presynaptic axonal NMDA receptors are required for t-LTD at this synapse, as a spatially localized flash in the axonal but not the somatodendritic region of cMK801-loaded presynaptic cells blocked the induction of t-LTD. These results provide further evidence for the presence of functional NRs in the presynaptic axonal membrane (Berretta and Jones, 1996; Brasier and Feldman, 2008), supporting previous anatomical evidence for presynaptic NRs in the neocortex (Aoki et al., 1994; DeBiasi et al., 1996; Charton et al., 1999; Corlew et al., 2007). The requirement of axonal NRs for induction of t-LTD raises the question of the source of ligand that activates these presynaptic NRs. The ligand(s) might be released by the postsynaptic neurons, as retrograde release of glutamate has been reported (Harkany et al., 2004) or from glial cells (Perea et al., 2009). Another possible source of ligand(s) could be the spill-over from neighboring synapses, although this appears less likely in paired recordings. Further experiments are needed to uncover the source of ligand(s) activating the axonal NRs required for t-LTD. The use-dependent nature of NR block by MK801 (Jahr, 1992; Hessler et al., 1993; Rosenmund et al., 1993) enabled us to use uncaging of MK801 in the postsynaptic neuron to study the locus of expression of t-LTD. Our data provide evidence that MK801 produces a use-dependent block of NRs also when applied intracellularly. A slower rate of block in the depressed pathway compared with a naïve pathway suggests that t-LTD is expressed by reduced transmitter release probability, which is consistent with increased paired-pulse ratio (Bender et al., 2006) and coefficient of variation (Rodríguez-Moreno and Paulsen, 2008) following induction of t-LTD at this synapse. In conclusion, we have successfully used intracellular compartment-specific photorelease of a channel blocker to study the locus of induction and expression of t-LTD, demonstrating that presynaptic axonal NRs are necessary for this presynaptically expressed LTD. Our results also confirm that t-LTP at the L4-to-L2/3 synapse in the barrel cortex is mediated by postsynaptic (L2/3) somatodendritic NRs. Thus, spatially and temporally precise uncaging of ion channel blockers is a useful tool to uncover compartment-specific biological functions. [1]

C26H24N2O6

460.48

217176-91-5

Typically exists as solids at room temperature

O(CC1C=C(C(=CC=1[N+](=O)[O-])OC)OC)C(N1[C@@H]2CC3C=CC=CC=3[C@@]1(C)C1C=CC=CC2=1)=O

cMK801; 217176-91-5; Caged MK801, >=98% (HPLC); (4,5-dimethoxy-2-nitrophenyl)methyl (1S,9R)-1-methyl-16-azatetracyclo[7.6.1.02,7.010,15]hexadeca-2,4,6,10,12,14-hexaene-16-carboxylate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)