Calcium-permeable AMPA/kainate receptors mediate toxicity and . activates both AMPA and kainate receptors (24), in a dose-dependent manner (EC50 = The α-aminohydroxymethylisoxazolepropionic acid receptor is an ionotropic transmembrane receptor for glutamate that mediates fast synaptic transmission in the central nervous system (CNS). It has been traditionally classified as a non-NMDA-type receptor, along with .. The starting signal for AMPAR endocytosis is an NMDAR-dependent calcium. Both a general antagonist of AMPA/kainate receptors (CNQX) and a that the toxicity of these mutants is calcium-dependent and provide direct.
Toxicity Calcium Dependent. Is AMPA/Kainate
These types of experiments have shown that different pathways are being activated or regulated depending on the location of the signal origin. This information is then transported to the nucleus. This allows the transcription factors in the nucleus to respond differently based in the phosphorylation state of Jacob. NMDA receptors are also associated with synaptic plasticity. In addition, both synaptic and extrasynaptic are involved in expressing a full LTD. Another factor that seems to affect NMDAR induced toxicity is the observed variation in subunit makeup.
Although both subunits are found in synaptic and extrasynaptic NMDARs there is some evidence to suggest that the GluN2B subunit occurs more frequently in extrasynaptic receptors. This observation could help explain the dualistic role that NMDA receptors play in excitotoxicity.
Despite the compelling evidence and the relative simplicity of these two theories working in tandem, there is still disagreement about the significance of these claims. Some problems in proving these theories arise with the difficulty of using pharmacological means to determine the subtypes of specific NMDARs.
Excitotoxicity has been thought to play a role in the degenerative properties of neurodegenerative conditions since the late s. Most notably excitotoxic events involving NMDA receptors have been linked to Alzheimer's disease and Huntington's disease as well as with other medical conditions such as strokes and epilepsy. These side effects are, in part, observed because the NMDA receptors do not just signal for cell death but also play an important role in its vitality.
Activation of NMDA receptors requires binding of glutamate or aspartate aspartate does not stimulate the receptors as strongly. D -Serine has also been found to co-agonize the NMDA receptor with even greater potency than glycine. Recently, it has been shown that D -serine can be released both by neurons and astrocytes to regulate NMDA receptors. This property is fundamental to the role of the NMDA receptor in memory and learning , and it has been suggested that this channel is a biochemical substrate of Hebbian learning , where it can act as a coincidence detector for membrane depolarization and synaptic transmission.
Positive allosteric modulators include:. An example of memantine derivative is neramexane which was discovered by studying number of aminoalkyl cyclohexanes , with memantine as the template, as NMDA receptor antagonists.
Neramexane, which can be seen in figure 6, binds to the same site as memantine within the NMDA receptor associated channel and with comparable affinity. It does also show very similar bioavailability and blocking kinetics in vivo as memantine. Neramexane went to clinical trials for four indications, including Alzheimer's disease. Other weak partial agonists of the glycine site of the NMDA receptor such as rapastinel GLYX and apimostinel NRX are now viewed for the development of new drugs with antidepressant and analgesic effects without obvious psychotomimetic activities.
Antagonists of the NMDA receptor are used as anesthetics for animals and sometimes humans, and are often used as recreational drugs due to their hallucinogenic properties, in addition to their unique effects at elevated dosages such as dissociation. When certain NMDA receptor antagonists are given to rodents in large doses, they can cause a form of brain damage called Olney's lesions. NMDA receptor antagonists that have been shown to induce Olney's lesions include ketamine , phencyclidine , and dextrorphan a metabolite of dextromethorphan , as well as some NMDA receptor antagonists used only in research environments.
So far, the published research on Olney's lesions is inconclusive in its occurrence upon human or monkey brain tissues with respect to an increase in the presence of NMDA receptor antagonists.
Some common agents in which weak NMDA receptor antagonism is a secondary or additional action include:. The NMDA receptor is regulated via nitrosylation and aminoadamantane can be used as a target-directed shuttle to bring nitrogen oxide NO close to the site within the NMDA receptor where it can nitrosylate and regulate the ion channel conductivity. Unlike many other NO donors, alkyl nitrates do not have potential NO associated neurotoxic effects.
Alkyl nitrates donate NO in the form of a nitro group as seen in figure 7, -NO 2 -, which is a safe donor that avoids neurotoxicity. The nitro group must be targeted to the NMDA receptor, otherwise other effects of NO such as dilatation of blood vessels and consequent hypotension could result. Provisional studies in animal models show that nitromemantines are more effective than memantine as neuroprotectants, both in vitro and in vivo.
Memantine and newer derivatives could become very important weapons in the fight against neuronal damage. Negative allosteric modulators include:. The NMDA receptor is modulated by a number of endogenous and exogenous compounds: The main problem with the development of NMDA antagonist for neuroprotection is that the physiological NMDA receptor activity is essential for normal neuronal function. Complete blocking of all NMDA receptor activity therefore results in adverse side effects such as hallucination , agitation and anesthesia.
To be clinically accepted the NMDA receptor antagonist must block excessive activation without blocking the normal function. Competitive NMDA receptor antagonists, which were developed first, are not a good option because they compete and bind to the same site NR2 subunit on the receptor as the agonist, glutamate, and therefore block normal function also. These antagonists can be displaced from the receptor by high concentration of glutamate which can exist under excitotoxic circumstances.
Uncompetitive block refers to a type of block that increased concentration of glutamate cannot overcome and is dependent upon prior activation of the receptor by the agonist, i. They block open ion channels but the problem is when the ion channels close they get trapped inside resulting in undesirable side effects because of blocking normal as well as excessive activity.
It's therefore considered to be clinically unacceptable. Phencyclidine , which has slightly shorter dwell time but still too excessive, causes hallucination and is therefore not a good agent either for neurodegenerative diseases. Ketamine is another example of drug with slightly shorter dwell time but still excessive and it is used as anesthetic.
Because of these adverse side effects of high affinity blockers the search for clinically successful NMDA receptor antagonists for neurodegenerative diseases continued and focused on developing low affinity blockers. That way the drug obtained would only block excessively open NMDA receptor associated channels but not normal neurotransmission.
It is a derivative of amantadine which was first an anti-influenza agent but was later discovered by coincidence to have efficacy in Parkinson's disease.
Chemical structures of memantine and amantadine can be seen in figure 5. The compound was first thought to be dopaminergic or anticholinergic but was later found to be an NMDA receptor antagonist. Memantine is the first drug approved for treatment of severe and more advanced Alzheimer's disease , which for example anticholinergic drugs do not do much good for.
Many second-generation memantine derivatives have been in development that may show even better neuroprotective effects, where the main thought is to use other safe but effective modulatory sites on the NMDA receptor in addition to its associated ion channel. Memantine 1-amino-3,5-dimethyladamantane is an aminoalkyl cyclohexane derivative and an atypical drug compound with non-planar, three dimensional tricyclic structure.
Figure 8 shows SAR for aminoalkyl cyclohexane derivative. Memantine has several important features in its structure for its effectiveness:. In patch-clamp measurements memantine has an IC 50 of 2. The methyl side groups play an important role in increasing the affinity to the open NMDA receptor channels and making it a much better neuroprotective drug than amantadine. Several derivatives of Nitromemantine, a second-generation derivative of memantine, have been synthesized in order to perform a detailed structure activity relationship SAR of these novel drugs.
One class, containing a nitro NO 2 group opposite to the bridgehead amine NH 2 , showed a promising outcome. Lengthening the side chains of memantine compensates for the worse drug affinity in the channel associated with the addition of the —ONO 2 group . Excitotoxicity is implied to be involved in some neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis.
This can possibly be achieved by uncompetitive antagonists, blocking the receptors ion channel when excessively open . Memantine is an example of uncompetitive NMDA receptor antagonist that has approved indication for the neurodegenerative disease Alzheimer's disease. In memantine is still in clinical trials for additional neurological diseases.
Therefore, the NMDA receptor functions as a "molecular coincidence detector ". Its ion channel opens only when the following two conditions are met: NMDA receptors are modulated by a number of endogenous and exogenous compounds and play a key role in a wide range of physiological e. NMDAR antagonists like ketamine , esketamine , tiletamine , phencyclidine , nitrous oxide , and xenon are used as general anesthetics.
These and similar drugs like dextromethorphan and methoxetamine also produce dissociative , hallucinogenic , and euphoriant effects and are used as recreational drugs. Memantine , a low-trapping NMDAR antagonist, is approved in the United States and Europe for the treatment of moderate-to-severe Alzheimer's disease,  and has now received a limited recommendation by the UK's National Institute for Health and Care Excellence for patients who fail other treatment options.
Cochlear NMDARs are the target of intense research to find pharmacological solutions to treat tinnitus. NMDARs are associated with a rare autoimmune disease, anti-NMDA receptor encephalitis , that usually occurs due to cross-reactivity of antibodies produced by the immune system against ectopic brain tissues, such as those found in teratoma.
These are known as anti-glutamate receptor antibodies. Compared to dopaminergic stimulants like methamphetamine , the NMDAR antagonist phencyclidine can produce a wider range of symptoms that resemble schizophrenia in healthy volunteers, in what has led to the glutamate hypothesis of schizophrenia.
NMDAR antagonists, for instance eliprodil , gavestinel , licostinel , and selfotel have been extensively investigated for the treatment of excitotoxicity -mediated neurotoxicity in situations like ischemic stroke and traumatic brain injury , but were unsuccessful in clinical trials. Previous studies have shown that glutamate toxicity may be prevented by antioxidants. Cannabidiol, THC and several synthetic cannabinoids all were demonstrated to be antioxidants by cyclic voltametry.
Cannabidiol and THC also were shown to prevent hydroperoxide-induced oxidative damage as well as or better than other antioxidants in a chemical Fenton reaction system and neuronal cultures.
These data also suggest that the naturally occurring, nonpsychotropic cannabinoid, cannabidiol, may be a potentially useful therapeutic agent for the treatment of oxidative neurological disorders such as cerebral ischemia.
Cannabinoid components of marijuana are known to exert behavioral and psychotropic effects but also to possess therapeutic properties including analgesia 1 , ocular hypotension 2 , and antiemesis 3.
This report examines another potential therapeutic role for cannabinoids as neuroprotectants and describes their mechanism of action in rat cortical neuronal cultures. During an ischemic episode, large quantities of the excitatory neurotransmitter glutamate are released. This event causes neuronal death by over-stimulating N-methyl- d -aspartate receptors NMDAr and 2-amino 4-butylhydroxyisoxazolyl propionic acid AMPA and kainate-type receptors and results in metabolic stress and accumulation of toxic levels of intracellular calcium 4.
Cannabinoids have been suggested to prevent glutamate neurotoxicity by activating cannabinoid receptors 7 , 8 , which can reduce calcium influx through voltage sensitive calcium channels 8 , 9.
A synthetic cannabinoid HU also has been demonstrated to be neuroprotective even though it does not activate cannabinoid receptors.
This compound is an atypical cannabinoid, however, in that it, unlike other cannabinoids, directly antagonizes NMDAr 10 and possesses some antioxidant properties The present study examines classical cannabinoids as neuroprotectants in vitro but focuses on the nonpsychoactive cannabinoid cannabidiol.
Like THC, cannabidiol is a natural component of the marijuana plant, Cannabis sativa , although unlike THC, cannabidiol does not activate cannabinoid receptors and so is devoid of psychoactive effects This study reports that cannabidiol and other cannabinoids such as THC are potent antioxidants that protect neurons from glutamate-induced death without cannabinoid receptor activation.
Cannabidiol, THC, and reagents other than those specifically listed below were purchased from Sigma. Dihydrorhodamine was supplied by Molecular Probes.
Tert -butyl hydroperoxide, tetraethylammonium chloride, ferric citrate, and sodium dithionite were all purchased from Aldrich. Solutions of cannabinoids, cyclothiazide, and other lipophiles were prepared by evaporating a 10 mM ethanolic solution under a stream of nitrogen in a siliconized microcentrifuge tube.
After evaporation, 1 ml of culture media was added, and the drug was dispersed by using a high power sonic probe. Special attention was used to ensure the solution did not overheat or generate foam. After dispersal, all solutions were made to their final volume in siliconized glass tubes by mixing with an appropriate quantity of culture media.
Primary cortical neuron cultures were prepared according to the method of Ventra et al In brief, fetuses were extracted by C-section from a day pregnant Wistar rat, and the fetal brains were placed into phosphate buffered saline.
Cells were counted, were tested for vitality by using the trypan blue exclusion test, and were seeded onto poly- d -lysine coated 24 multiwell plates. This protocol results in a highly neuron-enriched culture Media were conditioned by 24 hr of treatment over a confluent layer of type I astrocytes prepared from 2-day-old Wistar rat pups In brief, cortices were dissected, were cut into small pieces, were digested enzymatically with 0.
The cell suspension then was plated into untreated cm 2 T-flasks, and, after 24 hr, the media were replaced and unattached cells were removed. Once astrocytes achieved confluency, cells were divided into four flasks. Astrocyte cultures were used to condition DMEM for no longer than 2 months.
After exposure, cells were washed twice with saline and were incubated for 18 hr in conditioned DMEM. Cells were incubated with glutamate in the presence of nM MK for 18—20 hr before analysis. Specific AMPA and kainate receptor ligands also were used to separately examine the effects of cannabinoids on AMPA and kainate receptor-mediated events. When specifically examining kainate receptor activity, cyclothiazide was replaced with 0. Although the neuron preparation technique described above results in a largely neuronal culture, a minority of astrocytic cells remain.
Astrocytes are highly resistant to glutamate toxicity 16 because of their lack of functional NMDAr 17 , 18 , although glutamate toxicity in astrocytes has been observed to prevent AMPA receptor desensitization if cyclothiazide is present It was concluded, therefore, that astrocyte contamination does not contribute substantially to the effects of glutamate in our neuronal cultures.
Tert -butyl hydroperoxide was used because its miscibility with both water and lipids allows oxidation to occur in both cytosolic and membrane-delimited cellular compartments.
Cell toxicity was assessed 18—20 hrs after insult by measuring lactate dehydrogenase release into the phenol red-free culture media. Experiments were conducted with triple or quadruple values at each point, and all plates contained positive glutamate alone and baseline controls. The assay was validated by comparison with a tetrazolium-based viability assay XTT Results were similar with either system, although lactate dehydrogenase release was used in this study because it provided a greater signal to noise ratio than the XTT assay.
Tetraethylammonium chloride in acetonitrile 0. The antioxidant activities of each of the compounds were evaluated by their ability to prevent oxidation of dihydrorhodamine to the fluorescent compound rhodamine. Oxidant was generated by ferrous catalysis diothionite-reduced ferric citrate of tert -butyl hydroperoxide in a Various concentrations of cannabinoids and butylhydroxytoluene BHT were included to examine their ability to prevent dihydrorhodamine oxidation.
Data are reported as mean values plus and minus standard error. To examine NMDAr-mediated toxicity, rat cortical neurons were exposed to glutamate for 10 min in a magnesium-free medium, and the level of lactate dehydrogenase released was used as an index of cell injury.
Similar data also was observed when glutamate was replaced with either AMPA-specific or kainate receptor-specific ligands data not shown. Each experiment was repeated on at least four occasions with essentially the same results. Cannabinoids were present during and, in the case of NMDAr mediated toxicity, after the glutamate exposure periods.
See Materials and Methods for further experimental details. Such depolarization may activate both voltage-sensitive calcium channels 21 and facilitate NMDAr activation 22 , However, a combination of these calcium channel inhibitors did not completely block EDTA-preventable calcium-dependant cell death.
Cannabinoids were present throughout the glutamate exposure period. Significant difference between EDTA and other treatments is indicated with an asterisk. Unlike cannabidiol, THC is a ligand for the brain cannabinoid receptor 24 , and this action has been proposed to explain the ability of THC to protect neurons from NMDAr toxicity in vitro 7. This was confirmed by inclusion of a cannabinoid receptor antagonist, SRA Fig. Neither THC or cannabidiol neuroprotection was affected by cannabinoid receptor antagonist.
Effect of THC, cannabidiol, and cannabinoid receptor antagonist on glutamate induced neurotoxicity. See Materials and Methods for experimental details. Studies have suggested that ROS damage may be involved in glutamate neurotoxicity 5 , 6.
To investigate whether cannabinoids could protect neurons against glutamate by reacting with ROS, the antioxidant properties of cannabidiol and other cannabinoids were assessed. Cyclic voltametry, a procedure that measures the ability of a compound to accept or donate electrons under a variable voltage potential, was used to measure the oxidation potentials of several natural and synthetic cannabinoids.
Anandamide arachidonyl-ethanolamide , which is not a cannabinoid in structure but is an endogenous ligand for the cannabinoid receptor, did not undergo oxidation in this assay Fig.
excitotoxicity, depending on the receptor type that is activated. 1To whom . alone. Toxicity of AMPA applied together with CTZ and of kainate was abolished by CNQX. .. activation (e.g., voltage-gated calcium channels or the sodium- calcium. calcium, and astrocyte toxicity occurred only after 24 hr expo- sure to high ( alone was not toxic, was concentration-dependent for each of NMDA toxicity, they do have functional non-NMDA iono- tropic receptors. induced toxicity has been shown to be calcium dependent; this study demonstrates that .. AMPA/Kainate Toxicity Is Calcium Dependent. Increased calcium.