NMDA receptor (Kd = 1.4 μM)

The D-enantiomer is a potent and specific antagonist of NMDA glutamate receptors (RECEPTORS, N-METHYL-D-ASPARTATE). The L form is inactive at NMDA receptors but may affect the AP4 (2-amino-4-phosphonobutyrate; APB) excitatory amino acid receptors.

D-AP5 is an antagonist of NMDA receptor. Long-term potentiation (LTP) and spatial learning are both adversely affected in vivo in parallel dose-dependent ways by chronic intracerebroventricular D-AP5 infusion. Brain concentrations of D-AP5 do not result in any detectable sensory impairment when spatial learning is prevented [2]. During the trial, a progressive decrease in swimming speed was linked to the infusion of D-AP5. When D-AP5-affected animals are unable to learn, they experience sensorimotor abnormalities in spatial tasks that get worse over time. The delayed-match-place protocol of the water maze revealed delay-dependent spatial memory deficits in rats treated with D-AP5 [3].

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This series of experiments investigated whether the NMDA receptor antagonist D-2-amino-5-phosphonopentanoate (D-AP5) could induce impairments of spatial learning across a dose range comparable to its impairment of hippocampal long-term potentiation (LTP) in vivo. Estimations of the extracellular concentration of D-AP5 in hippocampus using microdialysis were also made to compare whether these impairments occur at concentrations similar to those required to impair LTP in the in vitro hippocampal slice. Rats were chronically infused with D-AP5 into the lateral ventricle at a range of concentrations (0-50 mM) via osmotic minipumps. They were first trained to find and escape onto a hidden platform in an open-field water maze task. After the behavioral learning, they were anesthetized with urethane and an attempt was made to evoke and monitor hippocampal LTP. Extracellular samples of D-AP5 in hippocampus were then taken using microdialysis, and finally, the animals were killed and tissue samples dissected. The microdialysis and tissue samples were analyzed for D-AP5 content using HPLC with fluorescence detection. The results established, first, that D-AP5 impairs spatial learning in a linear dose-dependent manner, highly correlated with its corresponding impairment of hippocampal LTP in vivo. No concentration of D-AP5 was observed to block LTP without affecting learning. Second, the microdialysis estimates indicated that, subject to certain assumptions, D-AP5 causes these impairments at extracellular concentrations comparable to those that impair LTP in vitro. Third, comparison of the whole tissue and microdialysis samples revealed a concentration ratio of approximately 30:1, indicating that 97% of the intracerebral D-AP5 is inaccessible to the dialysis probes. Infusion of 20 mM EGTA was found to cause a sevenfold increase in D-AP5 in the dialysis perfusates, suggesting that at least part of the inaccessible D-AP5 is trapped by a calcium-dependent mechanism. Two further behavioral control studies indicated that the D-AP5-induced impairment of spatial learning is unlikely to be secondary to a drug-induced motor disturbance, and that the performance of the D-AP5 group whose concentration was just sufficient to block hippocampal LTP completely was statistically indistinguishable from that of a group of rats with bilateral hippocampal lesions induced by ibotenic acid. Taken together, these findings offer support for the hypothesis that activation of NMDA receptors is necessary for certain kinds of learning.[2]

Drug concentrations, surgery, amino acid analysis, and histology All drugs and anesthetics were made up in deionized water except in a final replicate when they were made up in pyrogen-free water. An equivalent stock concentration of 100 mM D-AP5 was made up in 100 mM NaOH and kept as frozen aliquots. This was diluted with aCSF for a range of concentrations (5, 13, 20, 30, 40, and 50 mM) “spiked” with NaOH to maintain a pH of 7.4. Stock solution of EGTA (500 mM) was made up as an equivalent in NaOH and diluted to 20 mM with Ca*+-free aCSF. Ibotenic acid was dissolved in phosphate-buffered saline (pH 7.4) at a concentration of 10 mg/ml. As D-AP5 does not cross the blood-brain barrier, it was delivered to the brain by chronic infusion using osmotic minipumps (Alza model 2002; pumping rate, 0.5 pl/hr). Animals were placed in a Kopf stereotaxic apparatus to implant the minipumps under tribromoethanol anesthesia (0.29 gm/kg). An incision was made along the midline, the skull was exposed, and an L-shaped stainless steel cannula (23 gauge) was placed in the left lateral ventricle (AP = -0.9, ML = 1.3, DV = -4.5 from skull surface). The cannula was attached to the minipump with Silastic tubing and secured in place with dental acrylic and three watchmaker’s screws. The minipump was placed in the subcutaneous pocket extending from the caudal end of the incision to the shoulder blades. The scalp incision was closed with discontinuous suture, and animals were allowed 2 d postoperative recovery. During the surgery, positions were marked on the skull for the microdialysis probe and the two electrodes. For the ibotenic acid lesions, animals were placed in a stereotaxic frame and anesthetized with tribromoethanol. Animals were lesioned according to the methodology devised by Jarrard (1989) using ibotenic acid (either 0.05 or 0.10 ~1) at 26 sites bilaterally and allowed 2 weeks recovery before behavioral testing. Analysis of D-AP5 and amino acids in both brain tissue and dialysis samples was carried out using a Varian Vista 5500 pumping system 9090 automatic column injector, and a 5 pm Nucleosil C-18 column (250 x 4.6 mm). Separation ofamino acids was achieved with a gradient eluent using a phosphate buffer [buffer A: 50 mM and tetrahyarofuran (THF) (2.5%); pH 5.121 with an organic modifier, methanol [buffer B and THF (1.25%)]. The gradient profile with a pumping rate of 1 ml/ min was as follows: [time (min), %B] 0, 0; 5, 0; 7, 25; 15, 50; 23, 60; 25.90: 28. 110: 32.100: 42.0. Precolumn derivatization with o-nhthaldehydk (L&d&h et al.,’ 19i5) allowed detection of primary amino acids by fluorescence, using an ABS 980 fluorescence detector (excitation wavelength, 230 pm; emission wavelength, 1398 pm). A standard containing known quantities of amino acids and D-AP5 was injected onto the column at the start and end of each daily session of analysis to calibrate retention time and peak area of each molecular component measured. Data were integrated and quantified using a microcomputer-based integration package. The animals were killed at the end of the experiment, and their brains were removed on ice. Tissue from the right and left hippocampus of animals in the dose-response study was dissected out and kept in frozen storage (-20°C) for analysis of D-AP5 and amino acid content. Tissue from the region immediately adjacent to the infusion cannula was retained in formalin, frozen, cut into 30 pm coronal sections, and stained with fast cresyl violet. This allowed verification of the cannula position and assessment of any damage caused by it and/or by drug infusion. The brains from the hippocampally lesioned animals were embedded in egg yolk, frozen, and cut into 30 pm horizontal sections to assess the extent of cell loss.[2]

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Three experiments were conducted to contrast the hypothesis that hippocampal N-methyl-d-aspartate (NMDA) receptors participate directly in the mechanisms of hippocampus-dependent learning with an alternative view that apparent impairments of learning induced by NMDA receptor antagonists arise because of drug-induced neuropathological and/or sensorimotor disturbances. In experiment 1, rats given a chronic i.c.v. infusion of d-AP5 (30 mm) at 0.5 μL/h were selectively impaired, relative to aCSF-infused animals, in place but not cued navigation learning when they were trained during the 14-day drug infusion period, but were unimpaired on both tasks if trained 11 days after the minipumps were exhausted. d-AP5 caused sensorimotor disturbances in the spatial task, but these gradually worsened as the animals failed to learn. Histological assessment of potential neuropathological changes revealed no abnormalities in d-AP5-treated rats whether killed during or after chronic drug infusion. In experiment 2, a deficit in spatial learning was also apparent in d-AP5-treated rats trained on a spatial reference memory task involving two identical but visible platforms, a task chosen and shown to minimise sensorimotor disturbances. HPLC was used to identify the presence of d-AP5 in selected brain areas. In Experiment 3, rats treated with d-AP5 showed a delay-dependent deficit in spatial memory in the delayed matching-to-place protocol for the water maze. These data are discussed with respect to the learning mechanism and sensorimotor accounts of the impact of NMDA receptor antagonists on brain function. We argue that NMDA receptor mechanisms participate directly in spatial learning.[3]

[1]. The effects of a series of omega-phosphonic alpha-carboxylic amino acids on electrically evoked and excitant amino acid-induced responses in isolated spinal cord preparations. Br J Pharmacol. 1982 Jan;75(1):65-75.

[2]. The NMDA receptor antagonist D-2-amino-5-phosphonopentanoate (D-AP5) impairs spatial learning and LTP in vivo at intracerebral concentrations comparable to those that block LTP in vitro. J Neurosci. 1992 Jan;12(1):21-34.

[3]. N-methyl-d-aspartate receptors, learning and memory: chronic intraventricular infusion of the NMDA receptor antagonist d-AP5 interacts directly with the neural mechanisms of spatial learning. Eur J Neurosci. 2013 Mar;37(5):700-17.

The D-enantiomer is a potent and specific antagonist of NMDA glutamate receptors (RECEPTORS, N-METHYL-D-ASPARTATE). The L form is inactive at NMDA receptors but may affect the AP4 (2-amino-4-phosphonobutyrate; APB) excitatory amino acid receptors.

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1 The depressant actions on evoked electrical activity and the excitant amino acid antagonist properties of a range of omega-phosphonic alpha-carboxylic amino acids have been investigated in the isolated spinal cord preparations of the frog or immature rat. 2 When tested on dorsal root-evoked ventral root potentials, members of the homologous series from 2- amino-5-phosphonovaleric acid to 2-amino-8-phosphonooctanoic acid showed depressant actions which correlated with the ability of the substances to antagonize selectivity motoneuronal depolarizations induced by N-methyl-D-aspartate. 3 2-Amino-5-phosphonovalerate was the most potent substance of the series giving an apparent KD of 1.4 microM for the antagonism of responses to N-methyl-D-aspartate. 4 A comparison of the (+)- and (-)-forms of 2-amino-5-phosphonovalerate indicated that the N-methyl-D-aspartate antagonist activity and the neuronal depressant action of this substance were both due mainly to the (-)-isomer. 5 The (-)- and (+)-forms of 2-amino-4-phosphonobutyrate had different actions. The (-)-forms of this substance had a relatively weak and non-selective antagonist action on depolarizations induced by N-methyl-D-aspartate, quisqualate and kainate and a similarly weak depressant effect when tested on evoked electrical activity. The (+)-form was more potent than he (-)-form in depressing electrically evoked activity but did not antagonize responses to amino acid excitants. At concentrations higher than those required to depress electrically evoked activity, the (+)-form produced depolarization. This action was blocked by 2-amino-5-phosphonovalerate.[1]

C5H10NO5P-2

195.1104

197.045

C, 30.47; H, 6.14; N, 7.11; O, 40.58; P, 15.71

79055-68-8

DL-AP5;76326-31-3;L-AP5;79055-67-7

135342

White to off-white solid powder

1.5±0.1 g/cm3

482.1±55.0 °C at 760 mmHg

245.4±31.5 °C

0.0±2.6 mmHg at 25°C

1.536

-2.32

4

6

5

12

200

1

C(C[C@H](C(=O)O)N)CP(=O)(O)O

VOROEQBFPPIACJ-SCSAIBSYSA-N

InChI=1S/C5H12NO5P/c6-4(5(7)8)2-1-3-12(9,10)11/h4H,1-3,6H2,(H,7,8)(H2,9,10,11)/t4-/m1/s1

(2R)-2-amino-5-phosphonopentanoic acid

D-AP5; (R)-2-Amino-5-phosphonopentanoic acid; 5-Phosphono-D-norvaline; (2R)-2-amino-5-phosphonopentanoic acid; d-APV; D-Norvaline, 5-phosphono-; D-(-)-2-Amino-5-phosphonopentanoic Acid;

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)

H2O : ~27.78 mg/mL (~140.92 mM)
DMSO :< 1 mg/mL
Ethanol :< 1 mg/mL

Solubility in Formulation 1: 100 mg/mL (507.28 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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