mGluR

MCPG can burn group I (mGluR1 and mGluR5) and group II receptors (mGluR2 and mGluR3) [4].
(S)-MCPG (100 μM) had no observable influence on spine production or removal mechanisms in WT slice cultures. (S)-MCPG inhibits TBS-induced increases in spine turnover and interferes with activity-dependent central stabilizing mechanisms in hippocampal slice cultures [1].

(RS)-MCPG (α-MCPG; 500 μM) boils TBS-induced changes in EGABA in juvenile or newborn neurons [3]. Pretreatment with low-dose (RS)-MCPG (25 nM; ic; daily; 5 days) significantly abolished amphetamine-induced locomotor activity in 10-day-old truck and female Sprague-Dawley lineage scaffolds [4].

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Partial reversal of spine dynamics deficits in Fmr1-KO mice by a metabotropic glutamate receptor antagonist [1]
We then investigated whether these defects could be reversed, and tested the effects of the metabotropic glutamate receptor antagonist MCPG (100 μm) on spine dynamics. In WT slice cultures, MCPG had no detectable effects on basal spine formation or elimination mechanisms (Fig. 3A and B). However, it prevented the increase in spine turnover triggered by TBS (Fig. 3A and B; new spines: 23.8 ± 3.3%, n = 6, 233 spines vs. 42.4 ± 7.9%, n = 5, 240 spines, P < 0.05; lost spines: 20.8 ± 2.4% vs. 43.7 ± 8.8%, P < 0.05). It also interfered with the mechanisms of activity-dependent spine stabilization. In WT mice, MCPG significantly reduced the differential stability of enlarged vs. non-enlarged spines following TBS (Fig. 3C; circles: ns: non-significant difference, two-way anova), consistent with evidence suggesting an involvement of metabotropic receptors in LTP induction mechanisms (Anwyl, 2009). In Fmr1-KO mice, MCPG had three main effects. First, it restored the level of basal turnover by enhancing both newly formed and lost spines per 24 h (Fig. 3A and B: new spines: 17.0 ± 3.2% vs. 9.3 ± 1.3%, n = 6, 8, P < 0.05; lost spines: 15.2 ± 2.5% vs. 9.5 ± 1.5%, n = 6, 8, P < 0.05), consistent with the idea that the decreased turnover observed in Fmr1-KO mice could be due to an over-activation of metabotropic receptors (Pfeiffer & Huber, 2007). Second, it reduced the increased sensitivity of spine turnover to activity: new spine formation still increased following TBS, but to a lesser extent (Fig. 3A; 27.2 ± 3.6%, n = 5 cells, 237 spines vs. 15.6 ± 3.0%, n = 7, 274 spines, P < 0.05). However, the changes in spine loss following TBS stimulation did not reach statistical significance (Fig. 3B; P = 0.20). Thus, MCPG normalized basal spine dynamics and reduced the sensitivity of spine turnover to activity. Third, MCPG improved the general stability of spines [Fig. 3D; compare with (circles) and without MCPG (squares), P < 0.05, two-way anova]. However, MCPG failed to improve the defects in activity-dependent spine stabilization. Enlarged and non-enlarged spines showed the same stability following TBS, and there was no preferential stabilization of enlarged spines (Fig. 3D; circles, ns: non-significant, n = 5 cells, 237 spines analysed). Thus, MCPG improved the general stability of spines but did not restore mechanisms of activity-dependent spine stabilization.[1]

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The effects of the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG) on performance in a water maze and in context-specific associative learning were examined in rats previously implanted with cannulae. MCPG (20.8 micrograms) injected intraventricularly (i.c.v.) before testing impaired the performance of rats in the spatial version of the Morris water maze, but 1/10 of this dose did not. Memory retention, evaluated 24 hr post-training, was also affected by the high dose of MCPG. However, performance in a cued version of the water maze was not impaired by the high dose, excluding effects of the drug on perceptual faculties. The effects of the MCPG were further characterized on performance in another hippocampus-dependent spatial learning task, the context-dependent fear conditioning task. MCPG (20.8 micrograms, i.c.v.) did not interfere with conditioned freezing to context in this task. For comparison, a group of rats was injected with the NMDA receptor blocker MK801. MK801 at a dose that disrupted the performance in the spatial version of the Morris water maze (0.08 mg/kg), significantly reduced freezing compared to controls. These experiments indicate that MCPG-sensitive metabotropic receptors may be required for only a restricted subset of spatial learning tasks, while NMDA receptors may play an integral role in all spatial learning.[2]

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Morris water maze [2]
During the 4 days of training, both the MCPG-treated (at the high dose) and control animals showed significant improvement in performance, indicated by the decrease in both the time of reaching the platform [F(3,108)=107.5; p<0.01] and the distance of the animal from the platform [F(3,108)=77.4; p<0.01] (Fig. 1). Animals given MCPG, however, were significantly slower to learn the task. MCPG-treated rats took longer to reach the platform [F(1,35)=9.6; p<0.01] and their distance from the platform was longer over the training period [F(1,35)=9.9; p<0.01] than vehicle-injected animals. On days 2 and 3, MCPG rats had a significant longer escape latency (p<0.01, Fig. 1(A)) and their measure of search error was significantly longer on each of the first 3 days (p<0.01; Fig. 1(B), Newman–Keuls post-hoc test). On day 4, MCPG-treated animals reached the same level of performance as control animals.

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Contextual fear conditioning [2]
As an alternative measure of learning, we tested the effects of MCPG on groups of rats in a context-specific associative learning. In the training phase of fear conditioning, rats were placed in a shocking chamber and given three footshocks during one 6-min session for 2 days. Rats were then returned to the shocking chamber 24 hr later and monitored for freezing behavior. Infrared photocells monitored the animal's activity and in the test phase a video camera recorded the session to be reviewed by an experimenter off-line. Fig. 5(A) shows the average activity of the animals in the first day of training. Both groups decreased their activity due to the occurrence of the shock [F(5,70)=5.6; p<0.01], but there was no statistical difference between the two groups. Neither motor activity nor freezing were influenced by MCPG during the test on day 3 compared to controls (Fig. 5(B)). Both groups froze more often after the first minute and less towards the end of the 6-min session [F(5,70)=7.6; p<0.01]. These results show that the impairment caused by MCPG 20.8 μg in the spatial learning water maze does not correlate with a similar effect on contextual learning.

Confocal imaging and electrophysiology [1]
Four days after transfection, slice cultures were imaged repetitively using an Olympus Fluoview confocal microscope as described (Dubos et al., 2012). Secondary or tertiary dendritic segments (35–45 μm) on apical dendrites of CA1 pyramidal neurons in the stratum radiatum were visualized at 0, 5, 24, 48 and 72 h. Z-stack images were analysed using Osirix software and cross-checked with another experimenter. All protrusions were included in the analysis, and stability was assessed as the fraction of spines still present at later times. Spines were considered to enlarge if the spine head width increased by more than 0.1 μm between the 0 and 5 h time points. All data are given with n being the number of dendritic segments analysed considering one dendritic segment per neuron and one neuron per slice culture. When appropriate, we also give the total number of spines analysed in the pool of experiments.
For electrophysiological recordings, hippocampal cultures or acute slices (4–6-week-old mice) were maintained in an interface chamber as described (De Roo et al., 2008b), perfused in a medium with the following composition (in mm): NaCl, 124; KCl, 1.6; MgCl, 1.5; CaCl2, 2.5; KH2PO4, 1.2; NaHCO3, 24; glucose, 10; ascorbic acid, 2; bubbled with 95% O2 and 5% CO2. Theta-burst stimulation (TBS) consisted of five trains at 5 Hz, each composed of four pulses at 100 Hz, repeated three times at 10-s intervals. Field excitatory postsynaptic potential slopes and amplitudes were measured using IGOR software.

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Western blot [1]
The effect of BpV on AKT phosphorylation on the S473 site was tested in hippocampal slice cultures maintained 12–14 days in vitro and treated with 15 nm BpV, as well as on 4–6-week-old mice injected i.p. with BpV [30 μg/100 g in saline (200 μL)]. The tissue was lysed on ice 30 min or 1–4 h after treatments for slices and animals, respectively, using a solution containing (in mm): NaCl, 150; Tris-HCl, 50, pH 7.4; EDTA, 2; dithiothreitol, 1; 1% Triton X-100, 10% glycerol, complete protease inhibitor tablet, completed with phosphatase inhibitors (in mm): sodium orthovanadate, 1; para-nitrophenylphosphate, 20; β-glycerophosphate, 20; okadaic acid, 50 ng/mL. Lysates were sonicated, and 30 μg of total protein put on Nupage 4–12% Bis-Tris gel. Gels were run in 3-(N-morpholino)propanesulfonic acid sodium dodecyl sulfate buffer and transferred to nitrocellulose membranes. Phospho-AKT immunoblotting was performed using 1 : 3000 dilution of the primary rabbit monoclonal antibody against phosphoSer473. Densitometric analysis of the pAKT band of 60 kDa was normalized by β-actin immunoblot and expressed as a function of WT values (monoclonal, 1 : 20 000).

Animal/Disease Models: Morris water maze learning in male Lister-Hooded rats [2]
Doses: 20 μg
Route of Administration: intracerebroventricular injection; 20 μg; 4 days
Experimental Results: Impaired rat performance in the spatial version of the Morris water maze , but 1/10 of that dose did not.\n

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\n\nAnimals and Morris water maze [1]
\nTwenty male adult Fmr1-KO mice (FVB.129P2-Pde6b+ Tyrc-ch Fmr1tm1Cgr/J) and 20 male adult WT controls (FVB.129P2-Pde6b+ Tyrc-ch/AntJ) were used. Mice were individually identified and housed according to the Swiss legislation on animal protection. One Fmr1-KO and one WT mice were used for pilot tests; the final sample (19 Fmr1-KO and 19 WT) was divided into two groups: nine mice were treated with BpV and 10 received the vehicle. The mice were trained in a Morris water maze with four daily trials during four consecutive days. At day 5, a probe trial was run in the absence of the escape platform. Immediately after, mice received either an i.p. injection of BpV [30 μg/100 g in saline (200 μL)] or an i.p. injection of 200 μL saline. One hour later, all mice started a novel, four-trial training session with the platform in the opposite quadrant (reversal learning). Reversal training continued for two more days; treatment with BpV or saline was repeated accordingly. At day 8, a second probe test was run. Swim paths were videorecorded and analysed by a videotracking system. Variables assessed were escape latencies, times spent in target quadrants and platform place proximity indices (PIs), calculated by measuring the average distances to the platform [(PI probe1 − PI probe2)/(PI probe1 + PI probe2)×100].\n

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\n\n\nAnimals were allowed at least 7 days to recover from surgery and were handled daily during this period. They were housed individually for the remainder of the experiment. At the time of behavioral testing, the xstylets were replaced by 33-gauge injection needles connected to Hamilton microsyringes. MCPG or vehicle was administered intraventricularly (i.c.v.) in a volume of 5 μl delivered at a rate of approximately 1 μl/min. The needle was left in place for an additional 1–2 min after completion of the injection to allow diffusion of the drug. The high dose (20.8 μg) or the low dose (2.1 μg) of MCPG were prepared daily and stored at room temperature. At the end of each experiment cannula placements were examined by Nissl staining. [2]

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\n\nBehavioral methods [2]
\nMorris water maze/spatial task [2]
\nTwo experiments using the metabotropic glutamate antagonist MCPG were conducted with this procedure. In the first experiment, the high dose of MCPG (20.8 μg) was used. Thirty-six rats were trained in the Morris water maze (Morris, 1984), consisting of a circular pool, 120 cm diameter, filled with water rendered opaque by milk and maintained at 26±1°C. The wall of the pool, 60 cm higher than the water level, was painted white. A platform, 10 cm in diameter and submerged by 2–3 cm, represented the escape from the water. The day before testing, animals were given 2 min each of free swimming in the pool to acclimatize them to the water. The rats were placed in a different starting position (among six possible) in each trial. The platform was always left in a fixed location throughout the 4-day training experiment. Animals were allowed 90 sec to locate the platform. Rats that had not found the platform after 90 sec were placed on it and allowed to remain there for the 60 sec intertrial time. Animals were given 4 trials a day for 4 consecutive days. MCPG (n=18) or vehicle (n=18) was injected i.c.v. 5 min before the start of the session every day. A computer-assisted program measured the path and time taken by each animal to reach the platform. While swimming, the proximity of the animal's position with respect to the target was also analyzed by the computer system. This measure, called the “search error”, represents the corrected cumulative distance from the escape platform (Gallagher et al., 1993). A proximity measure was calculated each second by averaging the distance between the rat and the target platform 10 times per second. A correction procedure was used to account for the six different starting points. Cumulative distance was employed on training trials and mean distance from the target was used on the fifth day during the probe trial (for more detail, see Gallagher et al., 1993).
\nTwenty-four hours after the last training session, the animals were tested in a single probe trial for retention of spatial memory. Rats were divided into four groups before the probe trial: one group (MCPG/MCPG) was injected with a further dose of MCPG as on previous days (n=9); a second group (n=9) (MCPG/vehicle) had been injected with MCPG during the previous training sessions but received vehicle injection before the probe trial; a third group (vehicle/vehicle) received vehicle as it did in the past days (n=9); and the final group (vehicle/MCPG) was injected with MCPG after receiving only vehicle before (n=9), to test possible acute effects of MCPG on memory retention. The platform was removed from the pool in this trial, and the rats were allowed to swim for 20 sec. Time spent in each quadrant of the pool (Morris et al., 1986) and the averaged distance of the animal from where the target had been previously (Gallagher et al., 1993), were used to estimate memory retention.
\nA second experiment was run with a new group of rats previously implanted with cannulae into the right lateral ventricle. A dose of MCPG (2.1 μg) 10 times lower than in the previous experiment was used, but all the procedures were identical. MCPG (n=8) or vehicle (n=10) was injected i.c.v. 5 min before each daily session consisting of 4 trials. On day 5 the platform was removed and the animals were tested for memory retention. The MCPG group had only eight animals, because two rats in this group became ill during the course of the experiment and were excluded.\n

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\n\nMorris water maze/cued taskk [2]
\nThree weeks after the end of the spatial task experiment, some of the rats were re-tested in a cued version of the water maze. A 30 cm ×5 cm black tape was placed along the wall of the tank 15 cm above water level and centered around the submerged platform (see inset of Fig. 4). The tape represented the visual cue directing the animals to locate the platform. The tape and platform were left in the same location during each session, but they were moved to a new position every day. Their spatial relationship, however, was always maintained. The animals received 4 trials daily for 4 consecutive days. On the fifth day a probe trial was run as in the spatial task experiment. MCPG (20.8 μg, n=7) or vehicle (n=6) rats were injected i.c.v. every day 5 min before the session. The animals tested in this experiment had been used previously in the spatial version of the water maze. However, most of the animals that we employed had been treated with vehicle in the previous experiment.\n

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\n\nContextual fear conditioningk [2]
\nGroups of rats were placed individually in a rodent conditioning chamber (25×20×17 cm, San Diego Instr.) with a ventilation fan providing background noise. Inside the chamber eight infrared beams, 3 cm apart and 0.5 cm above the grid floor, monitored the animal activity. Shock delivery was controlled by a computer. A video-camera placed in front of the chamber provided recordings of every session for off-line behavioral analyses. The shock was a brief (1 sec, 0.5 mA) delivery of direct current produced by a grid floor shocker. On days 1 and 2, conditioning sessions consisted of three presentations of the shock during a 6-min session. On the third day, animals were placed again in the chamber but no shock was delivered. Motor activity was monitored by the computer system, which measured the number of beams interrupted by the animals in each of 12 30 sec intervals. Freezing, used as an index of conditioned fear, was assessed by an experimenter analyzing the videotapes after the experiment. Freezing was defined as the absence of all movement except respiratory-related movements (Phillips and LeDoux, 1992). The per cent of time spent freezing was calculated for each 1-min interval. Two different experiments were conducted. In the first experiment, rats received either vehicle (n=7) or MCPG (20.8 μg, n=7) i.c.v. 5 min before the start of the session for the 3-day experiment. In the second experiment, different groups of rats were injected s.c. with MK801 (n=8) or vehicle (n=8) 30 min before testing.\n

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\n\nDrugsk [2]
\n(+)-α-Methyl-4-carboxyphenylglycine (MCPG) was employed in all experiments. MCPG (2.08 mg) was dissolved in equimolar NaOH (1 M), diluted to final volume with saline (0.9% NaCl) and the pH was then adjusted to 7.6±0.2. MCPG or vehicle was injected i.c.v. in 5 μl volume to each implanted animal. \n\n

[1]. Reversal of activity-mediated spine dynamics and learning impairment in a mouse model of Fragile X syndrome. Eur J Neurosci. 2014 Apr;39(7):1130-7.

[2]. Effects of the metabotropic glutamate receptor antagonist MCPG on spatial and context-specific learning. Neuropharmacology. 1996;35(11):1557-65.

[3]. Regulation of GABA Equilibrium Potential by mGluRs in Rat Hippocampal CA1 Neurons. PLoS One, 2015 Sep 21, 10(9):e0138215.

[4]. The effects of (RS)-MCPG on amphetamine-induced sensitization in neonatal rats. https://scholarworks.lib.csusb.edu/etd-project/3181/.

(S)-α-methyl-4-carboxyphenylglycine is a non-protein α-amino acid, the S-enantiomer of alanine, with the α-hydrogen replaced by a 4-carboxyphenyl group. It is a non-selective type I/II metabotropic glutamate receptor (mGluR) antagonist. It functions as a metabotropic glutamate receptor antagonist. Fragile X syndrome (FXS), characterized by intellectual disability and autistic features, is caused by silencing of the FMR1 gene, which encodes proteins involved in the regulation of synaptic protein synthesis. The deficiency of functional Fragile X intellectual disability proteins is thought to lead to overactivation of synaptic metabotropic glutamate receptor signaling, resulting in altered synaptic maturation and plasticity. However, it remains unclear how activity-dependent spinous process dynamics are affected in Fmr1 knockout (Fmr1-KO) mice, and whether this effect is reversible. This study used repeat imaging techniques to investigate the characteristics of structural plasticity and its signaling pathway regulation in hippocampal slice cultures. We found that the basal spinous process turnover rate was significantly reduced in Fmr1-KO mice, but activity significantly enhanced this turnover rate. In addition, Fmr1-KO mice exhibited loss of activity-mediated spinous stability. The application of the metabolite glutamate receptor antagonist α-methyl-4-carboxyphenylglycine (MCPG) enhanced basal spinous turnover and improved spinous stability, but failed to restore activity-mediated spinous stability. Conversely, enhancement of the phosphatidylinositol-3 kinase (PI3K) signaling pathway (which is associated with multiple aspects of synaptic plasticity) reversed both basal spinous turnover and activity-mediated spinous stability. It also restored the defective long-term potentiation (LTP) mechanism in brain slices and improved reversible learning ability in Fmr1 knockout (Fmr1-KO) mice. These results suggest that modulating the PI3K signaling pathway may help improve cognitive deficits associated with Fragile X syndrome (FXS). [1] In addition to MCPG, our study showed that the PTEN inhibitor BpV is another potential molecule capable of reversing the Fmr1-KO mouse phenotype. BpV application enhances PI3K activity. The PI3K signaling pathway, activated by multiple surface receptors including BDNF, also inhibits LTD (Jurado et al., 2010) and participates in LTP signaling (Tang et al., 2002). Consistent with this, our experiments showed that BpV can restore activity-dependent spinal stability. Therefore, these results suggest that, in addition to overactivation of LTD signaling, another defect in Fmr1-KO mice may involve a deficiency in the LTP mechanism. Our LTP experiments support this explanation and are consistent with the results of several other studies (Lauterborn et al., 2007; Meredith et al., 2007; Hu et al., 2008; see also Krueger and Bear, 2011). However, the specific role of the PI3K signaling pathway in Fragile X syndrome (FXS) remains unclear. Two recent studies have shown that PI3K activity and AKT phosphorylation levels have been upregulated in Fmr1 knockout (KO) mice (Gross et al., 2010; Sharma et al., 2010), a result that contradicts that of another study (Hu et al., 2008). It is important to note that the PI3K-AKT signaling pathway is downstream of many cell surface receptors and participates in various interactions with other systems, which may be achieved through differential regulation of AKT phosphorylation sites (Hemmings and Restuccia, 2012). Although this study cannot precisely elucidate the complex signaling mechanisms involved in PI3K signaling, our data clearly demonstrate that BpV treatment enhances AKT phosphorylation at the S473 site under both in vitro and in vivo conditions, not only reversing the spinal dynamics deficits observed in Fmr1-KO mice but also increasing LTP and improving behavior in the Morris water maze learning task in Fmr1-KO mice. Consistent with these results, the PI3K signaling pathway has been shown to be involved in long-term potentiation (LTP) mechanisms (Tang et al., 2002), in the brain-derived neurotrophic factor (BDNF) signaling pathway, which can also rescue LTP in Fmr1 knockout mice (Lauterborn et al., 2007), regulate synaptic and spinous process formation in hippocampal neurons (Jaworski et al., 2005; Cuesto et al., 2011), and, more importantly, is also defective in a mouse model of Angleman syndrome (a neurodevelopmental disorder with severe cognitive deficits and autistic features) (Cao et al., 2013). Therefore, the PI3K signaling pathway may be a potential target for reversing cognitive deficits. [1]

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NMDA receptors play a key role in spatial and contextual learning and memory (see Squire, 1992). Our results suggest that mGlu receptors may have only a regulatory or minor role. A recent study found that intrahippocampal injection of MCPG did not affect working memory, but the combined use of MCPG with NMDA receptor antagonists did (Ohno and Watanabe, 1996). Studies using long-term potentiation (LTP) as a model of learning and memory also indicate that the role of mGlu receptors is regulatory rather than central. Although NMDA receptor antagonists can significantly block LTP induction in vitro (Collingridge et al., 1983) and in vivo (Morris et al., 1986; Abraham and Mason, 1988), the effects of the mGlu receptor antagonist MCPG have been reported to vary, sometimes even depending on the technique used (see BenAri and Aniksztejn, 1995). Bashir et al. (1993) reported that MCPG could block LTP induction in hippocampal slices, but other researchers have not confirmed this result (Chinestra et al., 1993; Manzoni et al., 1994; Selig et al., 1995). Similarly, Riedel and Reymann (1993) found that MCPG could block the induction of LTP in vivo, but other researchers (Bordi and Ugolini, 1995; BenAri and Aniksztejn, 1995) did not find this effect. Generally, there is a good correlation between the effect of antagonist compounds on LTP and their effect on spatial memory tests (but see further discussion in Saucier and Cain, 1995 and Bannerman et al., 1995). Since existing data suggest that MCPG causes some impairment in spatial learning tests but not in other tests, LTP may only be partially affected by MCPG (Richter-Levin et al., 1994) or slightly affected. Future research should focus on more effective and selective mGlu receptor antagonists to elucidate the relationship between different types of learning and LTP mechanisms. In summary, our results indicate that the mGlu receptor antagonist MCPG interferes with the performance of rats in the spatial water maze test, but only at high concentrations of the drug, and not in the cue water maze test, which excludes the drug’s effect on sensory/perceptual abilities. MCPG does not affect the performance of rats in the context-specific associative learning task, but the NMDA receptor blocker MK801 severely interferes with the performance of the task, as observed in the spatial water maze test. Future research may be able to use these two behavioral paradigms to differentiate the roles of ionotropic and metabotropic glutamate receptors in learning and memory. [2]

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This study aimed to investigate the role of metabotropic glutamate receptors (mGluR) in the development of amphetamine-induced behavioral sensitization. Eleven-day-old rat pups were given bilateral infusions of the mGluR antagonist (RS)-methyl-4-carboxyphenylglycine (MCPG) for 5 days, followed by systemic injection of amphetamine, and their motor activity was measured. We hypothesize that young rats receiving amphetamine pretreatment and amphetamine stimulation exhibit a significant increase in activity, indicating short-term behavioral sensitization. As predicted, repeated administration of amphetamine during the pretreatment phase resulted in progressively increased motor activity, indicating the development of behavioral sensitization. The effect of MCPG on motor activity appeared to be independent of amphetamine-induced motor activity, and MCPG pretreatment failed to persistently block behavioral sensitization expression in rats re-administered with amphetamine after amphetamine pretreatment. This study suggests that, contrary to previous studies in adult rats, the mGluR system does not appear to persistently mediate the development of amphetamine-induced sensitization in newborn rats. [3]

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The equilibrium potential of GABA-A receptor-mediated current (EGABA) in newborn central neurons is at a relatively depolarized level, which is thought to be due to low expression of K+/Cl- cotransporter (KCC2) and relatively high expression of Na+-K+-Cl- cotransporter (NKCC1). In juvenile hippocampal CA1 pyramidal neurons, laminar theta rhythmic stimulation (TBS) induces a negative shift in EGABA levels. This study investigated the effects of TBS on EGABA levels in neonatal and juvenile hippocampal CA1 neurons and its potential mechanisms. Metabolic glutamate receptors (mGluRs) are thought to regulate the expression levels of KCC2 and NKCC1 in cortical neurons. Therefore, this study also explored the regulatory effects of mGluRs on KCC2 or NKCC1 activity and EGABA levels after TBS. Whole-cell patch-clamp techniques were used to record EGABA levels in sections of hippocampal CA1 pyramidal neurons from Wistar rats. The results showed that in neonatal rats, TBS induced a positive shift in EGABA levels, which was blocked by NKCC1 antisense mRNA, while NKCC1 sense mRNA had no effect. (RS)-α-methyl-4-carboxyphenylglycine (MCPG) is a class I and II mGluR antagonist that blocked TBS-induced EGABA shift in juvenile and neonatal hippocampal neurons. Although blocking mGluR1 or mGluR5 alone can interfere with TBS-induced EGABA shift in neonatal hippocampal neurons, only combined blocking can produce the same effect in juvenile hippocampal neurons. These results indicate that TBS induces a negative shift of EGABA in juvenile hippocampal neurons and a positive shift of EGABA in neonatal hippocampal neurons by altering the expression of KCC2 and NKCC1, respectively. Activation of mGluR appears to be a necessary condition for both shifts to occur, but the specific receptor subtypes involved seem to differ. [4]

C₁₀H₁₁NO₄

209.20

209.069

C, 57.41; H, 5.30; N, 6.70; O, 30.59

146669-29-6

(S)-MCPG; 150145-89-4; 1303994-09-3

1222

White to off-white solid powder

1.39g/cm3

230 °C17 mm Hg(lit.)

95-98 °C(lit.)

221-223°C/10mm

5.44E-08mmHg at 25°C

1.343

3

5

3

15

271

0

CC(C(O)=O)(N)C1=CC=C(C(O)=O)C=C1

DNCAZYRLRMTVSF-UHFFFAOYSA-N

InChI=1S/C10H11NO4/c1-10(11,9(14)15)7-4-2-6(3-5-7)8(12)13/h2-5H,11H2,1H3,(H,12,13)(H,14,15)

4-(1-amino-1-carboxyethyl)benzoic 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)

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.)