Effects of Aβ_25-35 and Aβ₄₂ on α7 and α4β2 nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes
Elise Abigail McVarish (Neuroscience, 2004)
Advisor: Nancy Kleckner

Recent studies have demonstrated a potential role for nicotinic acetylcholine receptors (nAChR) in the pathology of Alzheimer's Disease (AD). Alzheimer's disease results from the degradation of the cholinergic system in the brain leading to cognitive dysfunction phenotypically represented by severe memory disruption. The AD brain produces the protein beta amyloid1-42 (Aβ₄₂) at a higher rate than healthy brains ultimately causing the formation of amyloid plaques; a fragment of the protein, Aβ_25-35 has been shown to be neurotoxic in culture. Neurotoxicity can be attenuated with application of an α4β2 nAChR agonist. Furthermore, α7 coimmunoprecipitates with Aβ plaques; it has been demonstrated that Aβ₄₂ binds to α7 nAChR with differing effects depending on concentration and application. The present study endeavored to investigate how these proteins might directly affect the aforementioned receptors. nAChR subtypes α7 and α4β2 were expressed in Xenopus laevis oocytes and tested for activation in response to Aβ₄₂ and Aβ_25-35. Neither protein independently activated either receptor but when perfused in conjunction with ACh there was a marked diminution in response as compared to the ACh control response.

Effects of exposure to nicotinic receptor agonist and secreted form of α-­cleaved amyloid precursor protein following β-amyloid-induced toxicity in hippocampal culture
Timothy D. Talbot (Neuroscience, 2004)
Advisor: Nancy Kleckner

Alzheimer's Disease (AD) is a neurodegenerative disorder that is characterized by memory loss and dementia as a result of the formation of β-amyloid (Aβ)plaques within the hippocampus. It is the direct exposure of this toxic APP-cleaved peptide to neuronal cultures that causes cell death through a believed interaction with specific nicotinic acetylcholine receptors (nAChR). This study looked at similar ways in which Aβ-induced toxicity could be attenuated using nicotine or sAPPα on hippocampal neurons cultured from embryonic day 18 rats. The toxicity of Aβ was consistently observed in a dose-dependent manner with concentrations ranging from 0.5-30 μM, and respective decreases in viable cell populations by 20-65%. Evidence of neuroprotection was not displayed in any experiments utilizing nicotine or the alternatively cleaved APP product, both of which had previously been shown to attenuate some of the Aβ-related cell death by other researchers. Pre-incubations for 24 hours with the two substances showed little to no difference in comparison to same timed combinations with Aβ. It is proposed that the cultures used in this study may have lacked the necessary nAChR expression levels for either the nicotine or the sAPPα  product to take on any significant protective effect.    

              

Isolation and mapping of identified buccal ganglia neurons in Helisoma trivolvis.

Abdul Bachani, '03

Complex motor activity is controlled by activity of underlying neuronal networks called the central pattern generator (CPG). The feeding CPG in the pond snail Helisoma trivolvis is a highly plastic system and is comprised of three subunits - S1, S2, and S3. Glutamate has been identified as the key neurotransmitter in this system. Glutamate from B2, an S2 interneuron thought to modulate the feeding pattern, has been shown to inhibit the activity of two key motor neurons - B5 and B19, of S1 and S3, respectively. This is unlike the actions of glutamate in the vertebrate systems, whereby its effects are almost excitatory. Not much is known about the inhibitory effects of glutamate and the pharmacology and mechanisms underlying it. Various studies using the intact nervous system have been aimed at studying the same, however, studying neurons in isolation would give us a better idea of whether the effects of glutamate are direct, and also study its effects in isolation from the natural environment, without the interference of the normal signals that theses neurons received. Also important was mapping the interneuron B2 so as to enable us characterize and understand the diverse functions and effects that glutamate has in the Helisoma nervous system, and hence enhance our understanding of the same. Isolation of neurons B5 and B19 was done in an attempt to study the neurons in isolation from their normal environment, and intracellular staining techniques - using Lucifer yellow and Cobalt Chloride, were employed to map out the interneuron B2. The cells did not survive in culture past a couple of days and the staining techniques were not as successful. Though preliminary results are promising, these techniques need to be perfected and ironed out in order to further this study.  

Identification of the inhibitory glutamate receptor pathway in motor neurons involved in the feeding mechanism of the pond snail, Helisoma trivolis.

Kate Percarpio, '03

       A primary neurotransmitter in the vertebrates and invertebrate glutamate and its receptors are an essential focus of study in the scientific community. The much examined vertebrate glutamate receptor primarily activates excitation of the cell whereas invertebrate glutamate receptors mostly cause cellular inhibition. The purpose of this study was to identify the mechanism through which the binding of glutamate to invertebrate glutamate receptors causes the opening of ion channels and subsequent inhibition of the cell. In the pond snail, Helisoma trivolvis, glutamatergic neurons, B5 and B19, are integral to the central pattern generator of the feeding behavior. Intracellular recording technique was used to monitor activity of B5 and B19. A bath perfusion system was used to apply glutamate and possible antagonists to the cells. While glutamate inhibited the cells, known blockers of several inhibitory intracellular pathways, picrotoxin, pertussis toxin, NDGA, and BPB, were tested for their effects on cellular activity. A known blocker of ligand-gated chloride channels, picrotoxin toxin, did not block the inhibitory effects of glutamate in B5 and B19. Pertussis toxin and NDGA, both blockers of the lipoxygenase pathway, did not fully permeate the membrane of the cells and did not block the inhibitory pathway. BPB, a blocker of the lipoxygenase pathway, when applied to B5 was shown to have permeated the membrane but did not lock the inhibition by glutamate. This study concludes that inhibition by glutamate in the neurons B5 and B19 does not act through a Cl^-gated ion channel, but instead through a G protein-linked intracellular pathway. The intracellular pathway is not, however, the lipoxygenase pathway of the arachidonic acid metabolism.

The Role of Calcium in Kainate-Induced Toxicity of Cultured Rat Fetal Forebrain Neurons                                                                    Cristina Arrigo Cellurale, '02 

       Glutamate, the major excitatory neurotransmitter in the brain, is thought to cause excitotoxicity through calcium influx at NMDA receptors. Other receptors, such as kainate-sensitive AMPA/KA receptors, are also implicated in excitotoxic cell death. A particular subset of these receptors, calcium-permeable AMPA/KA receptors, are believed to playa large role in kainate-induced toxicity of both cortical and cerebellar granule neurons due to their permeability to calcium. In cerebellar granule neurons exposed to kainate, calcium entry via voltage-gated calcium channels and release from intracellular stores appears to be protective. The present study investigates the role of calcium in kainate-induced cell death of rat cortical neurons in culture. The pharmacological agents nifedipine, a blocker of L-type voltage-gated calcium channels, and ryanodine and thapsigargin, two inhibitors of intracellular calcium release, were used to assess the role of calcium via these pathways. In addition, CNQX, an inhibitor of non­NMDA receptors, as well as Joro Spider Toxin, a selective inhibitor of calcium­permeable AMPA/KA receptors, were used to elucidate the receptors through which kainate induces cell death. Cells were exposed to 100 μM kainate alone and in the presence of drug for one hour, with appropriate controls. MK-801 (10 μM) was added to all conditions to prevent activation of NMDA receptors. Following twenty-four hours, cells were assayed for viability using the trypan blue exclusion method and the colorimetric assay for conversion of MTT to formazan by live cells. Kainate caused death in approximately 10-20% of cortical neurons. Nifedipine (7 μM) and ryanodine (0.5 μM) applied in conjunction with kainate exerted no effect on cell viability, whereas thapsigargin (0.5 μM) alone decreased cell viability. In the presence ofkainate, CNQX (10 μM) effectively blocked kainate-induced death, whereas Joro Spider Toxin (500 nM) exerted no effect on viability. The results of the present study suggest that kainate induces death of a specific subset of cortical neurons via a pathway other than calcium influx through calcium-permeable AMP AIKA receptors. In addition, cortical neurons appear vulnerable to depletion of intracellular calcium stores as a result of thapsigargin treatment. Unlike cerebellar granule neurons, in the specific subpopulation of cortical neurons that is susceptible to kainate toxicity, calcium entry through voltage-gated calcium channels and release from intracellular stores are neither deleterious nor protective in this paradigm.

Mitochondria Ca²⁺ Accumulation: The roles of the Permeability Transition Pore, the Na⁺/Ca²⁺ Exchanger and Reactive Oxygen Species Generation in Glutamate­-Mediated Excitotoxicity          Thomas H. Armet, '01 

       Glutamate (glut), one of the most important neurotransmitters present in the human brain is important in controlling all bodily functions. However, an over stimulation of glutamate can lead to a type of neuron death called excitotoxicity caused by increased intracellular levels of Ca²⁺ in neurons. Excitotoxicity has been linked to brain damage caused by stroke and neurodegenerative disease such as Alzheimer's Disease and Parkinson's Syndrome. It has been known for some time that both NMDA and non­ NMDA receptors play an extensive role in glut-mediated excitotoxicity. But current research points to the role of the mitochondria, more specifically the Na⁺/Ca²⁺ exchanger (NCE), the production of reactive oxygen species (ROS). The NCE and ROS have been shown to perpetuate the opening of the permeability transition pore (PTP), which has been linked to the secondary rise in [Ca²⁺]_c deemed delayed calcium deregulation (DCD) which is believed to be a key player in excitotoxicity. Excitotoxicity was induced in the lab with O.5mM glut application to neuronal forebrain cultures of E 19 Sprague-Dawley rats. The resultant excitotoxicity was combated using NMDA receptor antagonists (100uM APV) and non-NMDA receptor antagonists (10uM CNQX) that both produced neuroprotective trends, although not significant (n=5). Due to time constraints, only one mitochondrial agent was tested, CGP-37157 a NCE inhibitor. CGP-37157 demonstrated a neuroprotective trend at 5uM and 25uM but not at 100uM. CGP-37157, APV and CNQX all provided similar levels of neuroprotection. This research suggests that blockage at the receptor level produces similar neuroprotective results as blockage at the mitochondrial level. Eventually this and similar research concerning excitotoxicity will lead to the production of more advanced neuroprotective agents.

d-Tetrandrine's Selective Inhibition on NMDA NRl/2A Subtypes Expressed in Xenopus laevis Frog Oocytes

Carolyn Su Chew, '01

 

Ever since the Yung-Chia period (307-313 AD.), Fang ji has served in anti-rheumatic, analgesic, diuretic, and anti-inflammatory roles. The bisbenzylisoqinoline alkaloid which is isolated from the Fang ji, d-tetrandrine (TET), is responsible for its main therapeutic actions. TET's anti-inflammatory was studied against NMDA NR1/2A and NRl/2B receptors, receptors that may induce cell death in strokes, ischemia, hypoxia, and anoxia. In the experimental study done with Xenopus laevis oocytes expressing recombinant NMDA NRl/2A and NRl/2B subtypes, TET inhibited with a greater efficacy the NMDA NRl/2A subtype ( 90.2 +/- 1.6% (n=4)) over NR1/2B subtype receptors (39.6 +/- 9.4% (n=4)). TET also gave an IC₅₀ of 0.03 +/- 18.80 mM against the NR1/2A subtype. The dose-response relationship between 100 uM NMDA/1 uM glycine-induced current at increasing concentrations of TET demonstrated a weak dose­ dependent response dependency (r²=0.39) due to TET's multi-phasic behavior. Results of the voltage sensitivity and trapping experiments suggested that TET may not act within the channel. However, the trapping experiment showed that 0.3 mM of TET, TET could be trapped within the NMDA NRl/2A subtype channel. A significant inhibition was seen in the NMDA/glycine-induced current following the application of NMDA/Glycine/TET and just TET (p=0.0361, n= 4). As a result, TET does have the potential to exhibit neuroprotective effects. Further studies should be conducted to examine the mechanistic properties of TET with the NMDA NRI/NR2A subtype channel and also look at if TET crosses the blood-brain-barrier. If can cross the blood-brain-barrier, TET could be used therapeutically for brain NMDA-induced neurotoxicity.

  

Ability of Glutamate Antagonists to Reduce Akinesia and L-dopa- Induced Dyskinesia in 6-0HDA Lesioned Rats

Reyna Pijanowski, '01                                                  

Steve Mague, '01                                                                           

 

Recent research suggests that Parkinson's disease (PD) is associated with enhanced glutamate transmission as well as a decrease in dopamine function. This glutamate overactivity is assumed to worsen akinesia and may also be implicated in L-dopa-induced dyskinesia; therefore glutamate antagonists should help alleviate these problems. In Experiment 1, chronic administration of 25 mg/kg L-dopa for 23 days failed to produce dyskinesias, as measured by changes in L-dopa-induced contralateral rotational behavior in unilaterally 6-0HDA-lesioned rats. In Experiment 2, akinesia was measured in unilaterally 6-0HDA rats by a decrease in stepping with the contralateral paw. As expected, acute injections of L-dopa (8 and 16 mg/kg L-dopa with 6.25 mg/kg benserazide) significantly increased, but did not normalize contralateral stepping. Indicating the involvement of glutamate, acute injections of (+)-5-methyl-10,11-dihydro­-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) (0.15, 0.2 mg/kg), and L­-dopa/MK-801 combination MK-801, a glutamate N-methyl-D-aspartate (NMDA) receptor blocker, alone or in combination with 8-16 mg/kg L-dopa also increased but did not normalize contralateral stepping. However, a 2.5 mg/kg dose of 1-[4-aminophenyl]­-4-methyl-7,8-methylene-dioxy-5H-2,3-benzodiazepine, GYKI 52466, a glutamate Alpha­-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor blocker, was ineffective at increasing stepping on the contralateral side. Our results suggest there are beneficial consequences to using NMDA antagonists in the acute treatment of akinesia in PD, supporting the hypothesis that glutamate overactivity plays a significant role in PD.

  

An Investigation of the Subtype-Selective Antagonism of N-Methyl-D-Aspartate Receptors by new Antiepileptic Agent, R-2-Acetamido-N-Benzyl-3-Methoxypropionamide

Gregory M. Troughton, '01

  

Harkoseride is a new anticonvulsant that shows much promise as an anti epileptic agent. Preliminary tests with Harkoseride report that it produced a time and dose dependent reduction

of behavioral seizure score of hippocampal kindled rats. Previous experiments with Harkoseride have provided evidence that argues both for and against interactions at the NMDA receptor and the strychnine-insensitive glycine site. Recombinant NMDA receptors expressed in Xenopus laevis oocytes were used in effort to determine if Harkoseride's clinical profile was linked to the strychnine-insensitive glycine site and the NR1-2A or NR1-2B receptors. Harkoseride reduced NMDA and glycine induced currents in NR1-2B receptors (IC₅₀=1.89 mM) and showed no block of NMDA and glycine currents at NR1-2A receptors. Inhibition of NRl-2B receptors was independent of glycine concentration. H-209, a possible metabolite of Harkoseride, exhibited similar properties to Harkoseride, also inhibtiting NMDA and glycine induced currents at NRl­-2B receptors. Harkoseride's moderate potency for NRl-2B receptors and its independence of the strychnine-insensitive glycine site suggest that its mechanism of action may be similar to other noncompetitive NMDA antagonists. Further research with Harkoseride should focus on NRl-­2A, NRl-2C and NRl-2D NMDA receptors. Research should also be conducted on the inhibitory profile of H-209.

  

Characterization oflnhibitory Glutamate Responses in Motor Neurons Involved in Feeding Behavior in the Pond Snail, Helisoma trivolvis

Carly A. Dell'Ova, '00

  

Neurons are organized in circuits, often referred to as central pattern generators (CPGs), that work together to produce behaviors. Studying the CPGs of an organism can help to elucidate how neuronal circuitry works to perform a specific behavior. The pond snail Helisoma trivo/vis uses a CPG composed of three parts, S1, S2 and S3, to control feeding. Two individual neurons, named B5 and B19, have been identified as motor neurons in phases S1 and S3 respectively. Glutamate, released from B2 interneurons, has been shown to inhibit spontaneous activity in both neurons. This inhibitory action of glutamate in Helisoma and other invertebrates contrasts its actions in vertebrate animals, which are almost always excitatory. As this inhibitory action of glutamate in pond snails is a fairly recent finding, not much is known about the pharmacology or mechanisms of the receptors mediating the effect. One way of characterizing newly discovered receptors is to determine which chemical analogs of the natural agonist interact with the receptor. The present study utilized intracellular recording techniques to measure inhibition of spontaneous action potentials in order to examine the selectivity of glutamate analogs for activation or inhibition of the glutamate receptors in cells B5 and B19. Quisqualate and (1S, 3R)-aminocyclopentane-l ,3-dicarboxylic acid (ACPD), vertebrate glutamate metabotropic receptor agonists, mimicked the actions of glutamate in B5 neurons, and ibotenate, a vertebrate ionotropic receptor agonist did not. Quisqualate also inhibited spontaneous action potential firing in B19 neurons, but ACPD and ibotenate did not significantly inhibit action potential firing. D-2-amino-5-phosphonovalerate (D-APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), vertebrate glutamate ionotropic receptor antagonists, did not block the inhibition by glutamate in any of the B5 or B19 neurons tested. These data suggest that neurons B5 and B19 might contain different glutamate receptor subtypes, and that at least one of these receptors (B5) might resemble the invertebrate glutamate receptor in Aplysia which activates an outward potassium current. Further research will be done with non-selective metabotropic receptor agonists and antagonists as well as pertussis toxin and chloride channel blockers to help discern the mechanism by which glutamate, acting at its receptors, causes inhibition of neuronal firing.

 

Signaling pathways involved in "run-up" of NMDA receptor-mediated currents in Xenopus oocytes expressing recombinant NMDA receptors

Kate-Elizabeth Reed, '00

 

Glutamate is an excitatory amino acid involved in synaptic transmission in neuronal pathways in the mammalian nervous system. The N-methyl-D-aspartate (NMDA) receptor, a subtype of glutamate receptor, plays a major role in a variety of physiological processes including neuronal development and long term potentiation of synaptic transmission in the hippocampus. The NMDA receptor contains multiple binding sites at which antagonists, agonists and modulators can interact to affect the activity of the receptor. Due to inconsistent sensitivities in pharmacological studies, it was determined that the receptor can be assembled from different subunits: NR1 (a-h) and NR2 (A-D). NR1-NR2A type of NMDA receptors express well in Xenopus oocytes but exhibit a phenomenon called run-up in electrophysiological studies which confounds research using oocytes. To determine the cause of run-up, various intracellular processes were considered including the activation of protein kinase C, the activation of calcium dependent chloride channels and the role of post synaptic density (PSD) proteins on the NMDA receptor complex. Subtypes of the NMDA receptor (NR1-NR2A) were expressed in Xenopus oocytes and were tested for their sensitivities to NMDA and glycine using pharmacological and electrophysiological techniques. Run-up was seen in the application of NMDA/glycine in barium (Fig.1) and in the application of NMDA/glycine in calcium (Fig.2). However, run-up was never seen when the oocytes had been exposed to either one of the divalents previously. Run-up also occurred in the oocytes after injection with mRNA encoding PSD-93 and PSD-95 when exposed to NMDA/glycine in barium (Fig.3). The exact mechanism behind the run-up phenomenon remains unknown. Further research should involve the elimination of barium and calcium, the inhibition of PKC and the inhibition of the calcium dependent chloride channels.

  

Allosteric Modulation of the Glycine Binding Site on N-Methyl D-Aspartate Receptors by the NR2 Subunits 

Daniel Graham, '98

 

           

       The inotropic N-methyl D-aspartate (NMDA) receptor is ubiquitous within the vertebrate nervous system and plays a vital role in synaptic transmission along excitatory pathways. Furthermore, NMDA receptors have been shown to increase synaptic plasticity which mediates learning and nervous system development (Madison, 1991). Activation of NMDA receptors is initiated by binding of the amino acid neurotransmitter glutamate and the coagonist glycine to their respective recognition sites (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988). Upon activation, the receptor ion channel opens causing an influx of Na+ and Ca2+. The overactivation of NMDA receptors has been shown to induce neuronal cell death by increasing intracellular calcium levels to toxic concentrations. Such excitotoxicity has been observed in myocardial infarctions, stroke, epilepsy, and several other neurological conditions (Choi, 1988).      Two types of subunits, NR1 and N~, interact to form the NMDA receptor complex. The NR1 class is composed of seven different isoforms labelled a-g. Similarly, the NR2 class consists of four different subunits labelled A-D. It is postulated that NMDA receptors are tetramers of these subunits which contain at least one NR1 subunit (Clements and Westbrook, 1991). The different subunit combinations account for the variability in characteristic properties of NMDA receptors. Specifically, NR1a-N~C has a 10-fold greater affinity for glycine than does NR1a-NR2A (Laurie, 1994). Evidence suggests that the N~ subunits allosterically interact with the glycine binding site on the NR1 subunit to regulate glycine affinity. It is likely that the extracellular N-terminus of the N~ subunits is responsible for this interaction. To test this, an attempt was made to exchange the extracellular N-terminus of the NR2A subunit with that of the NR2C subunit. However, more work is required to create these chimeric subunits. It was expected that the NR1a-N~A receptor with the N~C N- terminus (CTMIA) would behave like the NR1a-NR2C receptor in terms of glycine affinity and the NR1a-NR2C receptor with the NR2A N-terminus (ATMIC) would behave like the NR1a-NR2A receptor. In this scenario, it could be deduced that the extracellular N-terminus of the NR2 subunits was responsible for modulating glycine binding. If the ATMIC chimera exhibited glycine affinity similar to the N~C subunit and the CTMIA chimera exhibited glycine affinity similar to the N~C subunit, then some other region of the N~ subunits was responsible for modulating glycine binding at the NR1 subunit.       The N~methyl-D-aspartate receptors (NMDA-R) are classified within the excitatory glutamate receptor family. These receptors are ionotropic receptors whose current is generated by the influx of Ca+2 and other cations. Research has shown that two different classes of subunits must come together in order for a functional NMDA receptor to form. One family, the NR1 subunit family is comprised of seven functional splice variants, NR1a-g. The second subunit that must be present for functional channels to form is the NR2 subunit. There are four known NR2 subunits (A-d); each is coded for by a separate gene (Moriyoshi et al. 1991).  Creation and pharmacological characterization of the chimeric subunits will provide a great deal of insight into the structure-function relationship of NMDA receptors. Furthermore, elucidating the three dimensional conformation of NMDA receptors may aid in the design of antagonistic drugs which show promise in preventing the toxic effects with excessive NMDA receptor activation.

  

Characterization of the site of action of ifenprodil on N-methyl-D-aspartate receptors composed of NR1/NR2A and NR1/N2B subunits in Xenopus  oocytes                      

Josh Hill, '98

 

           

       The NMDA receptors are clinically important because they are thought to be involved in processes such as neuro-degenerative disease and acute brain damage caused by stroke (Rothman et al. 1987). An over-stimulation of the NMDA receptor by excess glutamate release has been shown to result in a neuronal cell death. Therefore, drugs that exhibit antagonism at the NMDA receptor are of clinical importance.    Ifenprodil is a drug that displays potent antagonism at the NMDA receptor. Furthermore, previous studies have shown that the 1C50 of ifenprodil at receptors comprised of NR1INR2A is different from the 1C50 at NR1INR2B receptors. Gallagher al. (1996) found the affinity of ifenprodil for NR1INR2B receptors to be 300 times higher than its affinity for NR1/NR2A receptors. Williams (1993) found that ifenprodil exhibits different modes of action at NR1INR2A (voltage dependent) and NR1/NR2B (glycine dependent). It is thought that the binding site for ifenprodil is near one of the three polyamine sites on the NR2 subunit (Williams et al. 1995).  The present study sought to determine whether the binding site of ifenprodil was different receptors comprised of NR2A and NR2B subunits. It

  

The Effect of Voltage on the Inhibitory Action of the Antipsychotics Clozapine and Chiorpromazine at the NMDA Receptor

Kristen Kosswig, '98 

 

            Antipsychotic drugs are known antagonists of the NMDA receptor, but their mechanism of action at this receptor has not yet been characterized. The present study examined the voltage dependence of the inhibition of the typical antipsychotic clozapine and the atypical antipsychotic chiorpromazine at the NMDA receptor. Based on previous studies, it was assumed that both drugs would act within the ion channel of the receptor and would thus demonstrate voltage dependence. NR1 /NR2A and NR1 /NR2B NMDA receptor subtypes were expressed in Xeno pus laevis oocytes by injecting the cells with mRNA encoding the subunits forming these receptors. Electrophysiological recording was used to determine the effect of clozapine and chlorpromazine on the NMDA/glycine response at holding voltages of -30 mV, -60 mV, and -90 mV, measured as percent block by the antagonist. Clozapine demonstrated a trend toward an increase of block with hyperpolarization at both the NR1 /NR2A and NR1 /NR2B receptor subtypes. At the NR1 /N~A subtype, the mean percent block of NMDA and glycinemediated currents by 150 ~M clozapine varied from 68.5 f 8.8% at -90 mV to 49.4 f 25.9% at -30 mV. The mean percent block at the NR1 /NR2B receptor subtype varied from 64.6 1 5.2% at -90 mV to 47.9 1 20.6% at -30 mV. Chlorpromazine exhibited significant effects due to voltage at both subtypes. At the NR1 /NR2A subtype (n=6), the mean percent block of NMDA and

  

Inhibitory Glutamate Receptors in Helisoma trivolvis: Pharmacological Characterization and Mechanistic Implications

Anne K. Perry, '98 

 

            Inhibitory glutamate receptors in invertebrates constitute an unusual and incompletely characterized class of glutamate receptors.  Inhibitory glutamate receptors are present extrajunctionally in insect muscle, and in the central nervous system of insects, molluscs, and crustaceans, where they often play a key role in neural networks.  Pharmacology of invertebrate glutamate receptors is dissimilar to vertebrate pharmacology, and often conflicting among invertebrates.  However, some general trends do occur.  Ibotenate seems to activate ionotropic inhibitory glutamate channels, and quisqualate often indicates a putative metabotropic mechanism.  This study further explores the pharmacology as it relates to mechanism in Helisoma trivolvis, a freshwater mollusc.  1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), a vertebrate metabotropic glutamate agonist, will also be used to explore the possibility of metabotropic mechanisms in this species.  In addition, a putative inhibitory glutamate receptor antagonist, proposed to be selective for both ionotropic and metabotropic receptors, was tested in order to further expand the pharmacology of invertebrate inhibitory glutamate receptors, and possibly confirm them as a separate subclass of glutamate receptors. I hypothesize that Helisoma inhibitory glutamate receptors are quisqualate-sensitive, ibotenate-insensitive, ACPD-sensitive, and alpha-amino-pimelate sensitive. No drug trials were performed due to time limitations and equipment problems, but the inhibitory glutamate response was confirmed compared with known responses.