AMPA-receptor activation is involved in the antiamnesic effect of DM 232 (unifiram) and DM 235 (sunifiram)
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- Galeotti, N., Ghelardini, C., Pittaluga, A. et al. Naunyn-Schmiedeberg's Arch Pharmacol (2003) 368: 538. doi:10.1007/s00210-003-0812-6
DM 232 and DM 235 are novel antiamnesic compounds structurally related to ampakines. The involvement of AMPA receptors in the mechanism of action of DM 232 and DM 235 was, therefore, investigated in vivo and in vitro. Both compounds (0.1 mg/kg−1 i.p.) were able to reverse the amnesia induced by the AMPA receptor antagonist NBQX (30 mg/kg−1 i.p.) in the mouse passive avoidance test. At the effective doses, the investigated compounds did not impair motor coordination, as revealed by the rota rod test, nor modify spontaneous motility and inspection activity, as revealed by the hole board test. DM 232 and DM 235 reversed the antagonism induced by kynurenic acid of the NMDA-mediated release of [3H]NA in the kynurenate test performed in rat hippocampal slices. This effect was abolished by NBQX. DM 232 increases, in a concentration dependent manner, excitatory synaptic transmission in the rat hippocampus in vitro. These results suggest that DM 232 and DM 235 act as cognition enhancers through the activation of the AMPA-mediated neurotransmission system.
KeywordsDM 232UnifiramDM 235SunifiramKynurenate testAmpakineLearning and memoryPassive avoidance
The α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPA) is a member of the group of ionotropic glutamate receptors, involved in a wide variety of processes in the central nervous system (CNS), including the onset and development of long-term potentiation (LTP), a synaptic event involved in memory formation (Staubli et al. 1994b). Induction of LTP normally requires spatial and temporal addition of fast excitatory synaptic responses with delayed and prolonged ones. AMPA receptors are responsible for both components, since they cause the fast excitatory component by mediating neuron depolarisation, but they also allow the onset of the second, delayed, component, by permitting voltage-dependent NMDA receptor activation (Bliss and Collingridge 1993; Malenka and Nicoll 1993).
Administration of compounds, that either enhance or restore AMPA receptor functions, facilitates learning and memory (Staubli et al. 1994a)
It is therefore conceivable that impairment in functioning of AMPA receptors may be partially responsible for memory deficit.
The discovery that aniracetam modulates AMPA receptor-induced currents opened new perspectives to the approach of promnesic therapy since led to the synthesis of drugs able to facilitate or to prolong the duration of AMPA receptor effects in the CNS.
In the 1990’s, Lynch and colleagues synthesized a class of new compounds, chemically related to the promnesic drug aniracetam, called “ampakines”. These compounds were found to positively modulate glutamatergic AMPA-mediated transmission by slowing the deactivation rate of the receptors and by increasing the size and the duration of excitatory responses in brain slices and in the hippocampus of freely moving rats (Arai and Lynch 1992; Arai et al. 1994).
Moreover, differently from aniracetam, these compounds display promising pharmacokinetic features, since they do not undergo massive metabolism and readily cross the blood-brain barrier.
Recently, a series of 1,4-diazabicyclo[4.3.0]nonan-9-ones, showing cognition-enhancing properties, has been reported (Manetti et al. 2000b). These compounds, which were originally designed as nicotinic agonists (Manetti et al. 1997), may be considered as structural analogues of piracetam, since they carry the 2-oxopirrolidine nucleus (Gualtieri et al. 2002); later it has been reported that this cyclic moiety is not important for activity, since also 1,4-diacylpiperazines, the product of their molecular simplification, maintain the same activity and potency (Manetti et al. 2000a). The most potent compounds of both series, DM 232 (unifiram) and DM 235 (sunifiram) respectively (Chart 1), were able to revert memory impairment induced by scopolamine and other amnesing agents (Ghelardini et al. 2002a, 2002b); however, no significant interaction with receptor or channels known to be involved in the modulation of the cholinergic system had been evidenced, (Ghelardini et al. 2002b) and therefore, the mechanism of action of these compounds remained to be clarified.
Compounds such as 1-BCP or CX516 (Fig. 1), which belong to the group of “ampakines”, have cognition-enhancing properties acting as positive allosteric AMPA-modulators (Yamada 2000; Arai et al. 2000); from the chemical point of view, they are amides derived from the condensation of arylacyl residues with nitrogen-containing saturated heterocycles. Unifiram and sunifiram share structural similarities with the molecules of ampakines (Gualtieri et al. 2002); this observation prompted us to verify whether our compounds have a similar mode of action.
Materials and methods
Animals and brain tissue
Male Swiss albino mice (23–30 g) and adult male rats (Sprague Dawley, 200–250 g) were used. The mice were housed fifteen per cage whereas the rats were individually housed in stainless-steel cages. The cages were placed in the experimental room 24-h before the test for adaptation. The animals were fed a standard laboratory diet and tap water ad libitum and kept at 23±1°C with a 12-h light/dark cycle, light on at 7 a.m. The animals always had free access to food and water. All experiments were carried out according to the guidelines of the European Community Council for experimental animal care. Rats were killed by decapitation and the hippocampi were rapidly removed and placed in a physiological salt solution (see below) at 2–4°C. All experiments were performed blind.
The test was performed according to the step-through method described by Jarvik and Kopp (1967). The apparatus consists of a two-compartment acrylic box with a lighted compartment connected to a darkened one by a guillotine door. Mice, as soon as they entered the dark compartment, received a punishing electrical shock (0.3 mA, 1 s). The latency times for entering the dark compartment were measured in the training test and after 24 h in the retention test. The maximum entry latency allowed in the training and retention sessions was respectively 60 and 180 s.
Hole board test
The hole board test utilises a 40-cm square plane with 16 flush-mounted cylindrical holes (diameter 3 cm) distributed 4 by 4 in an equidistant, grid-like manner. The plane of the hole board is made of black metal; the separation of the holes from each other is 5.5 cm; the distance of the outermost holes from the edge of the board is 5 cm. The mice were placed in the centre of the board one by one and left to move about freely for a period of 5 min each. Two photoelectric beams, crossing the plane from mid-point to mid-point of opposite sides, thus dividing the plane into 4 equal quadrants, automatically signalled the movement of the animals on the surface of the plane. Miniature photoelectric cells, in each of the 16 holes, recorded the exploration of the holes (head plunging activity) by the mice.
Rota rod test
The apparatus consisted of a base platform and a rotating rod of 3-cm diameter with a non-slippery surface. This rod was placed at height of 15 cm from the base. The rod, 30 cm in length, was divided into 5 equal sections by 6 disks. Thus up to 5 mice were tested simultaneously on the apparatus, with a rod-rotating speed of 16 r.p.m. The integrity of motor coordination was assessed on the basis of endurance time of the animals on the rotating rod according to the method described by Kuribara et al. (1977). One day before the test, the animals were trained twice. On the day of the test only the mice that were able to stay balanced on the rotating rod between 70 and 120 s (cut-off time) were selected for testing. Performance time was measured before and 15, 30 and 45 min after intraperitoneal administration of the investigated compounds.
Release experiments from slices
Slices (0.400 mm thick) were prepared from the hippocampal ventro-medial portion by a McIlwain tissue chopper and then labelled with 0.08 μM [3H]NA, 20 min at 37°C, in a physiological salt solution having the following composition (mM): NaCl, 125; KCl, 3; MgSO4, 1.2; CaCl2, 1.2; NaH2PO4, 1; NaHCO3, 22; glucose, 10 (aeration with 95% O2 and 5% CO2); pH 7.2–7.4. The incubation medium also contained 0.1 μM of the serotonin uptake inhibitor 6-nitroquipazine, to prevent false labelling of serotoninergic terminals. After washing with tracer-free medium, slices were transferred to parallel superfusion chambers (1 slice/chamber) and superfusion was started at 1 ml/min, at 37°C (t=0 min); when indicated, the physiological solution was replaced with a medium from which Mg2+ ions were omitted. After 60 min of superfusion, seven 5-min samples were collected. Samples and superfused slices (solubilised with Soluene) were then counted for radioactivity. Antagonists were added to the medium after 30 min, and were present till the end of superfusion (from t=30 min to t=95 min); nootropics were added concomitantly with the antagonists. NMDA or 12 mM KCl-enriched medium (NaCl substituting for an equimolar concentration of KCl) was added for 3 min, starting at minute 73 of superfusion; under this condition, the evoked release was within the fourth 5-min fraction.
All animal procedures were carried out according to the European Community Guidelines for animal care, DL 116/92, application of the European Communities Council Directive (86/609/EEC). Experiments were carried out on rat hippocampal slices, prepared as previously described (Pugliese et al. 1996). Male Wistar rats (Harlan, Italy; 150–200 g body weight), under anaesthesia with ether, were killed with a guillotine and their hippocampi were rapidly removed and placed in ice-cold oxygenated (95% O2–5% CO2) artificial cerebral spinal fluid (aCSF) of the following composition (mM): NaCl 124, KCl 3.33, KH2PO4 1.25, MgSO4 2, CaCl2 2, NaHCO3 25 and D-glucose 10. Slices (400 μm thick) were cut by a McIlwain tissue chopper and kept in oxygenated aCSF for at least 1 h at room temperature. A single slice was then placed on a nylon mesh, completely submerged in a small chamber (0.5 ml) and superfused with oxygenated aCSF (30–32°C) at a constant flow rate of 2 ml/min−1. The treated solutions reached the preparation in 90 s and this delay was taken into account in our calculations.
Test pulses (80 μs, 0.06 Hz) were delivered through a bipolar nichrome electrode positioned in the stratum radiatum. Evoked extracellular potentials were recorded with glass microelectrodes (2–10 MΩ) filled with 3 M NaCl, placed in the CA1 region of the stratum radiatum. Responses were amplified (Neurolog NL 104, Digitimer Ltd), digitised (sample rate, 33.33 kHz), and stored for later analysis using pCLAMP 6 software facilities (Axon Instruments Inc.). Stimulus-response curves were obtained by gradual increases in stimulus strength at the beginning of each experiment. The test stimulus pulse was then adjusted to produce a field excitatory postsynaptic potential (fEPSP) whose slope was 40–50% of the maximum and was kept constant throughout the experiment. In some experiments both the amplitude and the initial slope of fepsp were quantified, but since no appreciable differences were observed in the effect of drugs, only the measure of the amplitude was expressed in figure.
The following drugs were used: DM 232 (unifiram) and DM 235 (sunifiram) prepared in the Department of Pharmaceutical Sciences of University of the Florence according to the method described by Manetti et al. (2000a, 2000b); [3H]noradrenaline (specific activity 39 Ci/mmol) was purchased from Amersham Radiochemical Centre (Buckinghamshire, UK). Kynurenic acid (KYNA) was obtained from Sigma Chemical Co (St. Louis, MO, USA); NMDA, (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) were from Tocris Cookson (Bristol, UK)
Drugs were dissolved in isotonic (NaCl 0.9%) saline solution immediately before use. Drug concentrations were prepared so that the necessary dose could be administered in a volume of 10 ml/kg−1 by i.p. injection for mice. For electrophysiological experiments DM 232 was dissolved in dimethylsulfoxide (DMSO) and stock solutions were made to obtain concentrations of DMSO of 0.05% and 0.01% in aCSF, respectively. Control experiments, carried out in parallel for an unrelated project, showed that this concentration of DMSO did not affect the amplitude of synaptic potential.
Calculation and statistical analysis
The total tissue tritium was evaluated by adding the amount of tritium released during the collection period and that remaining in the slices at the end of the superfusion period and amounted to 58.55±0.43 nCi. The amount of tritium released into each superfusate fraction was expressed as a percentage of the total tissue tritium content at the start of the respective collection period (fractional rate). The basal release in the first fraction collected amounted to 0.71±0.08/5 min fraction of the total tritium content. The tritium overflow (total outflow minus basal outflow) induced by exposing slices to 100 μM NMDA was 2.50±0.5 nCi, corresponding to 4.26±0.12% of the total tritium content. Effects observed with different drug treatments were evaluated by calculating the ratio between the percent efflux in the fraction corresponding to the maximal effect reached with agonist stimulation (the fourth fraction) and the efflux in the corresponding first fraction collected. This ratio was compared to the corresponding ratio obtained under control conditions (no drugs added). Comparison between means was performed by applying two-tailed Student’s t-test or Dunnett’s test as appropriate. Analysis of variance (ANOVA), followed by Fisher’s Protected Least Significant Difference (PLSD) procedure for post-hoc comparison, was used to verify significance between two means in mouse behavioural data. Data were analysed with the StatView software for the Macintosh (1992). p values of less than 0.05 were considered significant. All experimental results are given as the means ± SEM.
For electrophysiological experiments data were analysed using Clampfit (Axon) and Prism 3.02 software (Graphpad Software, San Diego, CA, USA). Fitting of data points to functions was done by non-linear least-squares algorithms. Data were tested for statistical significance with analysis of variance (one-way ANOVA). When significant differences were observed, Newman-Keuls multiple comparison test (one-way ANOVA) was inferred. A value of p<0.05 was considered significant.
Effect of DM 232 and DM 235 in the mouse rota-rod test
Endurance time (s)
DM 232 (0.1 mg/kg−1)
DM 232 (10 mg/kg−1)
DM 235 (0.1 mg/kg−1)
DM 235 (10 mg/kg−1)
The possible involvement of AMPA receptors in the effects of DM 232 and DM 235 in the kynurenate test was then assessed by determining the sensitivity of these reversals to the selective AMPA receptor antagonist NBQX. When applied at 5 μM, NBQX prevented the [3H]NA release induced by AMPA (100 μM), but not the 100 μM NMDA-mediated tritium release from rat hippocampal slices (Pittaluga et al. 1999).
NBQX, inactive on its own, antagonizes the DM 232 reversal of the KYNA antagonism of the NMDA-evoked tritium release (Fig. 4) as well as the DM 235-mediated effect (Fig. 5), suggesting involvement of AMPA receptors in the effects of both compounds.
AMPA and NMDA receptors had previously been localised on noradrenergic axon terminals of the rat hippocampus, where they mediate enhancement of NA release. About 80% of the radioactivity released by AMPA or NMDA consisted of authentic [3H]NA (Pittaluga et al. 1999).
In 4 slices the cumulative concentration-response curve for the increase in fEPSP amplitude by DM 232 was constructed. Estimation of the EC50 values (27±6 nM) was calculated from the best fit of the experimental data to a logistic function (Fig. 6B).
The present results evidence for the involvement of the AMPA receptors in the antiamnesic activity of DM 232 (unifiram) and DM 235 (sunifiram). Both compounds reversed the impairment of memory processes induced by the AMPA antagonist NBQX in “in vivo” studies. “In vitro” experiments, performed on hippocampal slices, also supported the hypothesis of a role of the AMPA receptors for DM 232 and DM 235.
The amelioration of mouse memory processes induced by DM 232 and DM 235 is obtained without any induction of side effects. Both compounds, at the highest effective doses, did not impair motor coordination, as revealed by the rota rod test, nor modify spontaneous motility and inspection activity, as indicated by the hole board test. The lack of induction of hyper or hypo-excitability is also confirmed by the observation that, in the first session, the latency to enter the dark compartment of the light-dark box in the passive avoidance test was not modified by the administration of DM 232 and DM 235. It should be noted that deleterious behavioural effects were not present at doses 100-fold higher than the actives ones indicating that these compounds are endowed with an extremely low toxicity.
DM 232 and DM 235 have been predicted to act as ampakine-like compounds and, as a direct consequence, they should be expected to ameliorate amnesic conditions through AMPA/kainate receptor-mediated mechanisms (Staubli et al. 1994a; Larson et al. 1995) as well as to reverse memory impairments chemically-induced by administering AMPA/kainate receptor antagonists (Kim et al. 1993; Quilfeldt et al. 1994; Filliat et al. 1998). As almost all the quinoxaline derivatives, the competitive AMPA antagonist NBQX fails to discriminate among AMPA/kainate receptor subtypes (Bleakman and Lodge 1998), but, differently from CNQX or DNQX, it displays low affinity for NMDA receptors (Sheardown et al. 1990). Here we show that DM 232 and DM 235 prevent amnesia caused by NBQX, suggesting a possible role of AMPA-mediated system in the ameliorative effects on memory processes exerted by the two investigated compounds. The administration of NBQX induced amnesia of intensity comparable to that produced by well-known amnesic compounds such as scopolamine, mecamylamine and baclofen. This effect is in agreement with previous results performed in the rat passive avoidance test (Burchuladze and Rose 1992) and more recently in the Morris Water Maze task (Filliat et al 1998).
DM 232 and DM 235 did not show any procognitive activity in the passive avoidance test when given alone. However, an improvement in cognition of young animals, which have no memory impairment is difficult to demonstrate. As a matter of fact, not only the above-mentioned compounds, but also well-known nootropic drugs such as piracetam and aniracetam, do not show any memory facilitation in non-amnesic animals (Gouliaev and Senning 1994).
The hypothesis that DM 232 and DM 235 exert their antiamnesic effect through the activation of AMPA receptors is also supported by “in vitro” results in which both compounds produced a NBQX-sensitive reversal of the kynurenate-induced antagonism in the “kynurenate test”. In 1995, a biochemical test for evaluation of cognition enhancers acting through glutamate receptors of the N-methyl-D-aspartate (NMDA) type was proposed (Pittaluga et al. 1995). In this test, called “the kynurenate test”, nootropics are evaluated for their ability to attenuate kynurenate antagonism of the NMDA-evoked NA release from rat hippocampal slices. The attention was focused on kynurenic acid since it is a broad spectrum endogenous antagonist at ionotropic glutamate receptors, showing high affinity for the glycinergic binding site of the NMDA receptor-complex (Stone 1993; Moroni 1999). Under physiological conditions, the levels of kynurenate in the human brain reach micromolar concentrations (Moroni et al. 1988; Turski et al. 1998), probably allowing the blockade of a very low percentage of NMDA receptors. Abnormally elevated concentrations of kynurenic acid, however, may occur in the CNS during ageing or psychopathologies such as AIDS associated dementia or Alzheimer’s disease (Gramsbergen et al. 1992; Baran et al. 1999; Bara et al. 2000). These conditions are typified by the development of cognitive deficits and have been proposed to be parallel by decrease of glutamate receptor functions. It was, therefore, postulated that learning and memory improvements obtained with some nootropics might be associated to a relief of the antagonism exerted by the endogenous compounds at glutamate receptors, especially the NMDA receptor complex subtypes. Several compounds were found to be active in this test: some of these drugs relieved the kynurenate antagonism probably by acting directly on the NMDA receptor (i.e. D-cycloserine, oxiracetam, CR2249; Pittaluga et al. 1995, 1997) while other compounds reverted the kynurenate antagonism through indirect mechanisms, involving receptor-receptor interaction (Pittaluga et al. 1999, 2001). Aniracetam and the ampakine 1-BCP were tested in this biochemical assay and were found to be potently active. While the effect of aniracetam was insensitive to the presence of NBQX, the reversal of the kynurenate antagonism mediated by 1-BCP was found to partly depend on AMPA receptor activation, since co-administration of the selective AMPA receptor antagonist NBQX abolished it (Pittaluga et al. 2001).
One possible explanation to the DM-induced AMPA-mediated reversal of the “kynurenate test” is that AMPA receptors might influence NMDA receptors function, by directly modulating their activity. Actually, AMPA and NMDA receptors co-localised on noradrenergic terminals and they reciprocally influence their functions. Another possible explanation considers that, once slices are exposed to 100 μM NMDA, a release of endogenous glutamate occurs in the biophase that might induce AMPA receptor desensitisation (reviewed by Holmann and Heinemann 1994). The desensitisation is prevented, possibly, by the presence of ampakine-like compound, leading to a reinforcement of the AMPA-mediated effect and therefore to an apparent reversal of the kynurenate antagonism.
Finally, it could be proposed that DM 232 and DM 235 might influence the AMPA-induced release of neurotransmitters others than noradrenaline, which might in turn facilitate NMDA receptor functions. Such an indirect mechanism underlies the reversal of the “kynurenate test” by the nootropic compound CGP36742. This drug acts as a very weak GABAB receptor antagonist, but it improves cognitive performances at low doses (0.01–1 μM) “in vivo”: its effect in the “kynurenate test” was found to be mediated by disinhibition of somatostatin release in hippocampal slice.
The results obtained by electrophysiological recording in vitro demonstrate that DM 232 brought about a long lasting increase of neurotransmission in the CA1 region of rat hippocampal slices. This effect is concentration-dependent and not reversible upon interruption of drug application. Our data provide experimental evidences supporting the proposition that the long-lasting synaptic enhancements produced by DM 232 are similar to the hippocampal LTP, that represents a model for a cellular mechanism related to learning and memory (Staubli et al. 1994b). DM 232, through unknown mechanism(s) might enhance either the release of the putative neurotransmitter such as glutamate, as already demonstrated for FK960, a putative cognitive enhancer in the hippocampus (Hodgkiss and Kelly 2001) or the response to glutamate at post synaptic level probably on AMPA receptors, since NMDA receptors contribute little to the generation of fEPSP evoked by low frequency stimulation in the presence of physiological concentrations of Mg++ (Novak et al. 1984). Further experiments carried out in the presence of selective AMPA or NMDA antagonists will be devoted to clarify the exact mechanism.
In conclusion, these results indicate that DM 232 and DM235 act as cognition enhancers through the activation of the AMPA-mediated neurotransmission system.