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Mechanism of block and behavioral effects of the N-methyl-D-aspartate receptor antagonists memantine and ketamine

Dissertation
Author: Shawn Edward Kotermanski
Abstract:
Pharmacological inhibition of NMDA receptor activity by ketamine is accompanied by pyschotomimetic side-effects; however, the Alzheimer's disease therapeutic memantine blocks NMDA receptor activity without debilitating side-effects. This dissertation provides electrophysiological and behavioral characterizations of these two NMDA receptor antagonists in an attempt to understand the unique therapeutic utility of memantine. The following work explores memantine and ketamine inhibition at NMDA receptors, their main site of action, with a focus on the mechanism of inhibition and receptor subtype selectivity in physiologically relevant conditions. This research shows NMDA receptors possess a second binding site at which memantine, but not ketamine, can inhibit activity. The research also shows the dramatic effect physiological concentrations of magnesium has on the ability of these drugs to inhibit NMDA receptor activity. Behavioral and cognitive effects of memantine and ketamine are also assessed and compared directly in rat. The effects of memantine and ketamine in rat were found to be similar at the low doses tested and more divergent as dose increased. Furthermore, memantine's effects appeared to be more pronounced and longer-lasting than those of ketamine. These findings demonstrate the importance of considering the physiological environment in which a drug acts, as well as the principles of drug action, when examining the effects of a drug on central nervous system activity.

TABLE OF CONTENTS 1.0

BACKGROUND AND INTRODUCTION ................................................................1

1.1

GENERAL INTRODUCTION...........................................................................1

1.2

NMDA RECEPTOR PROPERTIES .................................................................2

1.2.1

Gene products and expression patterns......................................................2

1.2.2

NMDA receptor structure ............................................................................4

1.2.2.1

Subunit topology ...................................................................................4

1.2.2.2

Stoichiometry ........................................................................................6

1.2.3

NMDA receptor function .............................................................................7

1.2.3.1

Channel activation ................................................................................7

1.2.3.2

Ion permeation ......................................................................................8

1.2.3.3

Block by endogenous Mg 2+ o .................................................................8

1.2.4

NMDA receptor pharmacology ...................................................................9

1.2.4.1

Competitive antagonists .......................................................................9

1.2.4.2

Channel blockers................................................................................10

1.3

NMDA RECEPTOR ROLE IN PHYSIOLOGY AND PATHOLOGY .......12

1.3.1

NMDA receptor role in synaptic transmission .........................................12

1.3.1.1

Learning and memory ........................................................................12

1.3.2

Pathological consequences of NMDA receptor activity ...........................14

1.3.2.1

Excitotoxic cell death ..........................................................................15

1.3.2.2

Neuropsychiatric and neurodegenerative disorders ........................16

1.3.3

Behavioral effects of NMDA receptor inhibition .....................................19

1.3.3.1

Human effects......................................................................................19

1.3.3.2

Ketamine as an animal model of schizophrenia...............................20

1.3.4

NMDA receptors as therapeutic targets ...................................................21

1.3.4.1

Memantine and Alzheimer’s disease .................................................21

2.0

BINDING AT SUPERFICIAL SITE ON NR1/2A NMDA RECEPTORS CONTRIBUTES TO PARTIAL TRAPPING OF MEMANTINE ........................................23

2.1

ABSTRACT ........................................................................................................23

2.2

INTRODUCTION .............................................................................................24

2.3

MATERIALS AND METHODS ......................................................................26

2.3.1

Cell culture and transfection......................................................................26

2.3.2

Solutions .......................................................................................................27

2.3.3

Electrophysiology ........................................................................................27

2.3.4

Analysis ........................................................................................................29

2.4

RESULTS ...........................................................................................................32

iv

2.4.1

Time course of blocker dissociati on from NR1/2A receptors in the absence of agonist .......................................................................................................32

2.4.2

Partial trapping is not the result of open-channel escape .......................36

2.4.3

NR1/2A receptor inhibition in the absence of agonist .............................38

2.4.4

Voltage dependence of trapping ................................................................43

2.4.5

Effects of [ion] o on memantine trapping ...................................................47

2.5

DISCUSSION .....................................................................................................50

2.5.1

Partial-trapping development....................................................................50

2.5.2

Memantine inhibition of NR1/2A recep tors in the absence of agonist ...51

2.5.3

Voltage dependence of trapping ................................................................53

2.5.4

Superficial memantine binding site may overlap the external cation binding site of NR1/2A receptors ..............................................................................54

3.0

MG 2+ IMPARTS NMDA RECEPTOR SU BTYPE SELECTIVITY TO THE ALZHEIMER’S DRUG MEMANTINE ...................................................................................56

3.1

ABSTRACT ........................................................................................................56

3.2

INTRODUCTION .............................................................................................57

3.3

MATERIALS AND METHODS ......................................................................58

3.3.1

Cell culture and transfection......................................................................58

3.3.2

Electrophysiology ........................................................................................58

3.3.3

Data analysis ................................................................................................59

3.4

RESULTS ...........................................................................................................60

3.5

DISCUSSION .....................................................................................................65

4.0

DIRECT COMPARISON OF THE LOCOMOTOR AND COGNITIVE EFFECTS OF THE NMDA RECEPT OR ANTAGONISTS MEMANTINE AND KETAMINE IN RATS ...............................................................................................................69

4.1

ABSTRACT ........................................................................................................69

4.2

INTRODUCTION .............................................................................................70

4.3

MATERIALS AND METHODS ......................................................................72

4.3.1

Animals ........................................................................................................72

4.3.2

Drugs and drug administration .................................................................72

4.3.3

Experimental procedures ...........................................................................73

4.3.4

Exploratory activity test .............................................................................73

4.3.5

Spontaneous alternation task.....................................................................74

4.3.6

Statistical analysis .......................................................................................75

4.4

RESULTS ...........................................................................................................76

4.4.1

Memantine effects on behavior in the exploratory activity test .............76

4.4.2

Memantine effect on behavior in the spontaneous alternation task ......82

4.4.3

Ketamine effects on behavior in the exploratory activity test ................84

4.4.4

Ketamine effects on behavior in the spontaneous alternation task ........90

4.4.5

Comparison of the effects of memant ine and ketamine on behavior in the exploratory activity test .......................................................................................92

4.4.6

Comparison of the effects of memant ine and ketamine on behavior in the spontaneous alternation task ..............................................................................96

4.4.7

Comparison of the effects of memantine and ketamine on general behavior in the exploratory activity test ..................................................................98

4.5

DISCUSSION ...................................................................................................100

v

4.5.1

Memantine and ketamine effects on be havior in the exploratory activity test.......... ....................................................................................................................100

4.5.2

Memantine and ketamine effects in the spontaneous alternation task 102

4.5.3

Examination of the behavioral e ffects elicited by memantine and ketamine ....................................................................................................................103

5.0

GENERAL DISCUSSION ......................................................................................106

5.1

INTEGRATION OF BEHAVIORAL AND ELECTROPHYSIOLOGICAL FINDINGS .........................................................................................................................106

5.2

POSSIBLE CONSEQUENCES OF NR2C AND/OR NR2D RECEPTOR INHIBITION .....................................................................................................................110

5.3

POSSIBLE BENEFITS OF PART IAL VERSUS FULL TRAPPING .......113

5.4

POSSIBLE EFFECTS ON OT HER NEUROTRANSMITTER SYSTEMS..... ....................................................................................................................114

5.5

CONCLUDING COMMENTS .......................................................................116

APPENDIX ................................................................................................................................117

BIBLIOGRAPHY .....................................................................................................................118

vi

LIST OF TABLES

Table 1. Residual current and unbinding time c onstant of memantine and ketamine at concentrations used to measur e voltage dependence of trapping .................................................45 Table 2. Effect of Mg 2+ o on IC 50 s for memantine and ketamine inhibition of NMDA receptors. .......................................................................................................................................................65 Table 3. Effect of memantine on aver age velocity of ambulation in the exploratory activity test .......................................................................................................................................................79 Table 4. Effect of ketamine on average velocity of ambulation in the expl oratory activity test 88 vii

LIST OF FIGURES

Figure 1.

Topology and transmembrane structure of the NR1 and NR2 subunits of NMDA receptors. .........................................................................................................................................6 Figure 2.

Double-pulse protocol. ................................................................................................30 Figure 3.

NMDA receptor inhibition by memantine and ketamine. ...........................................33 Figure 4. Time course of partial trapping development. .............................................................35 Figure 5.

Non-trapped memantine does not escape from open channels. ...................................38 Figure 6. Competitive binding hypothesis of pa rtial trapping of memantine. ............................39 Figure 7. NMDA receptor inhibition by memantine but not ketamine at the superficial site. ...42 Figure 8.

Effect of membrane voltage on fractiona l recovery of memantine and ketamine inhibition. ......................................................................................................................................46 Figure 9. Effect of [ion] o on memantine trapping. ......................................................................49 Figure 10. Effects of Mg 2+ o and NMDA receptor subunit com position on memantine inhibition. .......................................................................................................................................................61 Figure 11. Effects of Mg 2+ o and NMDA receptor subunit composition on ketamine inhibition. 64 Figure 12. Effect of memantine on ambulatory dist ance in the exploratory activity test. ..........77 Figure 13.

Effect of memantine on number of rear ings in the explor atory activity test. ............81 Figure 14.

Effect of memantine on behavior in the spontaneous alternation task. .....................83 Figure 15. Effect of ketamine on ambulatory dist ance in the exploratory activity test. .............85 Figure 16. Effect of ketamine on number of r earings in the exploratory activity test. ...............89 Figure 17.

Effect of ketamine on behavior in the spontaneous alternation task. ........................91 Figure 18. Comparison of memantine’s and ketamine’s effect on ambulatory distance in the exploratory activity test. ................................................................................................................93 Figure 19. Comparison of memantine’s and ketamine’s effects on number of rearings in the exploratory activity test. ................................................................................................................95 Figure 20.

Comparison of memantine’s and ketamine’s effect on behavior in the spontaneous alternation task. .............................................................................................................................97 Figure 21.

Comparison of memantine’s and ketamine ’s effect on genera l behavior in the exploratory activity test. ................................................................................................................99

viii

1.0

BACKGROUND AND INTRODUCTION 1.1

GENERAL INTRODUCTION Most excitatory synaptic communication occurring within the vertebrate central nervous system is mediated by the glutamate receptor fa mily of ligand-gated ion channels. N -methyl- D -aspartate (NMDA) receptors are a subtype of glutamate re ceptors that possess uniqu e properties that allow them to contribute to a variety of physiol ogical (development, learning and memory) and pathological (ischemic cell death, many neur odegenerative disorders) processes (Bliss and Collingridge, 1993; Dingledine et al., 1999; Olne y, 2003). Alteration of the ability of NMDA receptors to transmit information reliably can re sult in behavioral and cognitive disturbances (Lodge et al., 2002). Pharmacological modulation of NMDA r eceptor-mediated neurotransmission has generally been accompanied by debilitating psyc hotomimetic side-effects (Palmer, 2001; Lipton, 2004b). While not widely used clinically, some NMDA receptor antagonists (such as ketamine and phencyclidine (PCP) have been crucial in furthering our understa nding of the underlying pathologies of schizophrenia. Ketamine, as well as PCP, reliably reproduces positive (hallucinations and delusions), negative (decreased affect), and disorganizational (thought disorder) symptoms of sc hizophrenia in healthy human adults as well as worsening symptoms in already afflicted individuals (Krystal et al., 1999b). Memantine, another NMDA receptor

1

antagonist, with a mechanism of inhibition comparable to that of ketamine, lacks psychotomimetic side-effects and also has show n therapeutic utility by decreasing the cognitive decline associated with late stages of moderate-t o-severe Alzheimer’s dis ease. The goal of this work is to evaluate the mechanism of action a nd behavioral effects of NMDA receptor inhibition by memantine and ketamine to improve our unde rstanding of the ther apeutic potential NMDA receptor antagonism may hold. 1.2

NMDA RECEPTOR PROPERTIES 1.2.1

Gene products and expression patterns NMDA receptor subunit proteins are produced from seven different genes that encode three subunit families: NR1, NR2, and NR3 (Moriyoshi et al., 1991; Monyer et al ., 1992; Ishii et al., 1993; Monyer et al., 1994; Ciabarra et al., 1995 ; Sucher et al., 1995; Dingledine et al., 1999; Chatterton et al., 2002; Matsuda et al., 2003). One gene ( GRIN1 ) encodes the NR1 subunit, with eight splice variants produced by alternative splicing (Zukin and Bennett, 1995). Four genes ( GRIN2A-D ) produce distinct NR2 subunits, NR2A-D. Functional NMDA receptors require the presence of NR1 and at least one of the four NR2 subtypes. NR3 subunits are the products of two genes ( GRIN3A-B ) and have been labeled NR3A and NR 3B. Incorporation of NR3 subunits modifies NMDA receptor functioning (Das et al., 1998; Chatterton et al., 2002; Matsuda et al., 2002; Matsuda et al., 2003; Smothers and W oodward, 2007). Electrophysiological studies (Chatterton et al., 2002; Matsuda et al., 2002; Matsuda et al., 2003; Smothers and Woodward, 2007) suggest that NR3 subunits can form excitato ry glycine receptors when co-expressed with

2

NR1, although little is known about the physiological relevance of NR3 subunits in nervous system functioning. NR1 mRNA expression develops along a caudal to rostral gradient. Regional specificity of NR1 splice variants is present at birth and remains fixed through adulthood, with the only developmental change being shifts in the density of the respective splice variants (Laurie and Seeburg, 1994; Laurie et al., 1995). NR2 mRNA expression is both developmentally and regionally regulated (Monyer et al., 1994). Prenatally, NR2B and NR2D subunits predominate, with NR2D expression levels peaking about one week after birth and NR2B levels peaking about three weeks postnatally. NR2A and NR2C subunits begin to appear near birth and increase to peak levels around the third postnatal week. After reaching peak expression, all NR2 and NR1 subunit expression decreases to adult levels (Laurie and Seeburg, 1994). NR1 subunits are found throughout the adult central nervous system (CNS); however, the expression of NR2 subtypes is more regionally restricted. NMDA receptor subunit protein expression for the most part overlaps expression patterns reported for subunit mRNAs (Petralia et al., 1994a; Petralia et al., 1994b; Portera-Cailliau et al., 1996; Wenzel et al., 1996). The NR2A subunit is the most widely expressed subtype in the adult brain. The NR2B subunit is expressed mostly in the adult cortex and the hippocampus, and NR2C is heavily expressed in the cerebellum. NR2D is moderately expressed in the adult midbrain and brainstem, with lower levels in the cortex. Expression of NR2 subunits is neuron subtype-specific within certain brain regions. In the hippocampus, for example, NR2A and NR2B are highly expressed in pyramidal neurons whereas NR2D is found in interneurons (Monyer et al., 1994).

3

1.2.2

NMDA receptor structure A crystal structure of the NMDA receptor does not exist, most lik ely due to the difficulty in crystallizing membrane proteins, especially one as complex as the NMDA receptor. Therefore, the exact structure and location of critical determinants of receptor function are not fully understood. However, evidence exists that shed s light on the structure of NMDA receptors as well as the roles specific amino aci ds play in receptor functioning. 1.2.2.1

Subunit topology The subunit topology of ionotropic glutamat e receptors is conserved among NMDA, α -amino-3- hydroxy-5-methyl-4-isoxazolepropioni c acid (AMPA), and kainite recep tors (Dingledine et al., 1999). The protein structure of an NMDA receptor subunit begins at the extracellular amino- terminal (N-terminal) end, contains three tr ansmembrane regions (M1, M3, and M4) and a reentrant loop (M2) between transmembrane regi on M1 and M3, and ends with an intracellular carboxyl terminal tail (C-ter minal) (Figure 1). NMDA receptors contain a region between th e N-terminal domain and M1 called S1, which, along with the extracellular linker region located between M3 and M4 (S2), forms the agonist binding domain (ABD). The reentrant loop formed by the M2 region lines the narrowest portion of the pore of NMDA receptors and creates the selectivity filter that allows the passage of specific ions through activated receptors. The N-site (named for the asparagine residue located near the apex of the M2 loop) of the M2 region is cri tical for two unique NMDA receptor properties, calcium (Ca 2+ ) permeation and block by endogenous extracellular magnesium (Mg 2+ o ) (Burnashev et al., 1992; Kuner et al., 1996; Wollmuth et al., 1996; Wollmuth et al., 1998). The N-site of NR1 s ubunits strongly regulates Ca 2+ permeability but is less involved in

4

mediating block by Mg 2+ o (Burnashev et al., 1992; Wollmuth et al., 1996; Wollmuth et al., 1998). The N + 1-site (an asparagine next to the N-site towards the C-terminal end), of NR2 subunits has been shown to have a greater involvement in mediating block by Mg 2+ o than regulating Ca 2+ permeability (Burnashev et al., 1992; Wollmuth et al., 1996; Wollmuth et al., 1998). Along with forming the binding site for Mg 2+ , the N-site is also involved in the binding of other NMDA receptor channel blockers (Dingledine et al., 1999; Kashiwagi et al., 2002). The intracellular C-terminal region of NMDA receptor subunits contains sites that modulate NMDA receptor function, such as phosphorylation sites (Tingley et al., 1997) and sites for cytoskeletal, as well as cytoskeletal linker proteins (Wyszynski et al., 1997; Ehlers et al., 1998; Lei et al., 2001).

5

Figure 1.

Topology and transmembrane structure of the NR1 and NR2 subunits of NMDA receptors. Although four subunits are likely required for a functi onal receptor, only two subunits are shown here for clarity. N and C designate the extracellular N-terminal and intracellular C- terminal ends of the protein, respectively. The three transmembrane regions (M1, M3, and M4) and the re-entrant pore loop (M2) are portrayed as black rounded cy linders. Residues critical in regulating ion permeability and open-channel block by Mg 2+ o as well as many channel blockers, such as memantine and ketamine, are located near the apex of the M2 region. The ligand binding pockets are depicted as the thick black lines shown N-terminal to M1 (S1) and on the M3 – M4 linker (S2). The binding of glycine (Gly) to the NR1 subunit and glutamate (Glu) to the NR2 subunit are also portrayed.

1.2.2.2

Stoichiometry The number of NMDA receptor subunits in func tional receptors is not known with certainty. Most results suggest that two NR1 and two NR 2 NMDA receptor subunits form a tetrameric receptor complex (Clements and Westbrook, 1991; Laube et al., 1998; Schorge and Colquhoun, 2003). The arrangement of NR1 and NR2 subun its around the channel por e is also unknown. There is evidence that a single NMDA receptor can contain two different NR2 subunits along with NR1 subunits (Sheng et al., 1994; Chazot and Stephenson, 1997; Duna h et al., 1998). The incorporation of multiple NR2 subtypes will result in channel properties distinct from those of channels formed by NR1 subunits and one t ype of NR2 subunit (Wafford et al., 1993; Brimecombe et al., 1997; Vici ni et al., 1998; Tovar and We stbrook, 1999; Cheffings and Colquhoun, 2000; Brickley et al., 2003).

6

1.2.3

NMDA receptor function NMDA receptors possess distinctive properties th at allow them to play a unique role in excitatory synaptic transmission within th e CNS (Dingledine et al., 1999). Among these properties are the requirement of binding by two agonists, glutamate and glycine, for channel activation and ion flux. On ce activated, NMDA receptors al low for the permeation of Ca 2+ into neurons. Intracellular Ca 2+ acts as an important signaling mol ecule inside neurons where it can activate intracellular signal tran sduction cascades that have wi de-ranging and diverse effects on neuronal connections and viab ility. A third uni que property, use- dependent block by endogenous Mg 2+ o , regulates the flow of Ca 2+ through NMDA receptors. 1.2.3.1

Channel activation Ionotropic glutamate receptor ac tivation requires bindi ng of ligands. The ABD is a clamshell- like structure comprised of the S1 and S2 regions of a subunit. Research on AMPA (Armstrong and Gouaux, 2000; Furukawa and Gouaux, 2003) and kainate receptors (Armstrong et al., 1998) suggests that the binding of ligan d (agonist or antagonist ) to the ABD results in closure of the S1S2 clamshell, which translates into moveme nt of downstream portions of the receptor and channel opening. The binding of ag onists or antagonists results in different degrees of clamshell closure that translates to the movement of the S1 and S2 domains relative to the rest of the receptor complex. Agonist binding results in cl amshell closure to a degree that leads to sufficient tension on the pore-forming region to cause channel opening and allow ion flow. Based on the conservation of topology among gl utamate receptors, it is reasonable to assume that NMDA receptor activation occurs in a similar manner. The S1S2 complex of the NR1 NMDA receptor subunit binds th e co-agonist glycine (Kuryato v et al., 1994; Hirai et al.,

7

1996; Furukawa and Gouaux, 2003), and the corre sponding site on NR2 binds the co-agonist glutamate (Laube et al., 1997; Anson et al., 1998; Lummis et al., 2002). It is likely that four molecules of agonist (two glutamate and two glycine) are required for channel activation. Physiologically, aspartate and D-serine may al so contribute to NMDA receptor activation by binding to the glutamate and glycin e sites, respectively (Patneau and Mayer, 1990; Priestley et al., 1995; Schell et al., 1995). 1.2.3.2

Ion permeation Activated NMDA receptors exhibit nearly equal permeability to the monovalent cations sodium (Na + ), potassium (K + ), and the less physiologically relevent cation cesium (Cs + ) (Dingledine et al., 1999). NMDA receptors are also highl y permeable to the divalent cation Ca 2+ (Burnashev et al., 1995). The influx of cations through ac tivated NMDA receptor channels results in depolarization of the corresponding neuron. The va ried and potentially de leterious effects of Ca 2+ influx through NMDA receptors requires tight regulation of NMDA receptor-mediated transmission. 1.2.3.3

Block by endogenous Mg 2+ o

The binding of agonist and channel opening is not sufficient to allow ion flow through NMDA receptors. Endogenous Mg 2+ o enters the pore of activated NMDA receptors, binds near the N- site, and blocks the flow of ions through the cha nnel at membrane voltages near rest (Burnashev et al., 1992; Kuner et al., 1996). Mg 2+ o inhibition of NMDA recepto rs limits the influx of Ca 2+

through activated receptors, preventing in physiological situations, the accumulation of intracellular Ca 2+ to toxic levels.

8

The extent of Mg 2+ o inhibition of NMDA receptors is highly dependent on membrane voltage and subunit composition (Ascher and No wak, 1988; Monyer et al., 1994; Momiyama et al., 1996; Dingledine et al., 1999). At voltages n ear rest, physiological concentrations of Mg 2+ o

inhibit a majority of NMDA receptor-mediated neurotransmission. Membrane depolarization that results from the activation of non-NMDA glutamate receptors is necessary to reduce Mg 2+ o

inhibition of NMDA receptors and allow the flow of ions through activated channels. The type of NR2 subunit incorporated into the receptor strongl y affects the degree of Mg 2+ o inhibition (Monyer et al., 1994; Momiyama et al., 1996). Receptors composed of NR1 with NR2A and/or NR2B subunits are more strongly inhibited by Mg 2+ o than receptors that contain NR1 with NR2C and/or NR2D subunits. 1.2.4

NMDA receptor pharmacology Many drugs exist that modulate NMDA receptor function, a nd most drugs decrease NMDA receptor activity. Effective NMDA receptor i nhibitors must be capable of preventing overactivity while preserving physiological levels of activity. Two possibilities for inhibiting overactive NMDA receptors are competitive bindi ng by antagonists and blocking the pore of activated receptors. These two mechanisms of in hibition are associated w ith various advantages and disadvantages. 1.2.4.1

Competitive antagonists Competitive antagonists of NMDA receptors work by binding at the agonist binding site. The binding of antagonist to the agonist binding site prevents the conformational changes of the receptor necessary for channel opening. The requirement of two different agonists for channel

9

activation offers two distinct sites at which comp etitive antagonists can ac t, the glycine binding site on the NR1 subunit and the glutamate binding site on the NR2 subunit. Kynurenic acid derivatives, such as 5, 7-dichlorokynurenic acid (5, 7-DCK), are examples of competitive antagonists at the glycine site of NMDA receptors . Phosphono derivatives of short-chain amino acids, such as 2-amino-5 phosphonopentanoic acid (AP5) and 2-amino-7 phosphonopentanoic acid (AP7), are examples of competitive antagonists at the glutamate site (Priestley et al., 1995; Dingledine et al., 1999). Competitive antagonists, especially at the glycine site, offer a way to inhibit the functioning of all NMDA receptors, because NR1 subunits are required for functional receptors. Competitive antagonists that bind at the glutam ate site, potentially could allow for subunit- specific inhibition of NMDA receptors; however, many glutamate site antagonists are not NR2 subtype selective (L odge et al., 2002). Inhibition by competitive antagonists is compromised in cases where a high concentration of agonist is present. This eff ect is useful in preserving NMDA receptor-mediated synaptic transmission where high concentrations (~1 mM) of glutamate are released that can outcompete the antagonist for binding (Lodge et al., 2002). 1.2.4.2

Channel blockers NMDA receptor antagonism by channel blocke rs is use-dependent, meaning that channel activation is required for these compounds to reach th eir binding site and exer t their effects. The N-site of NMDA receptors contributes to the binding site of several NMDA receptor channel blockers, including Mg 2+ o (Kashiwagi et al., 2002). NM DA receptor inhibition by channel blockers is voltage -dependent, since their binding site is located within the membrane voltage field. Voltage-dependent inhibition results in a d ecrease in a channel bloc ker’s ability to inhibit

10

NMDA receptor activity as membrane voltage depolarizes (MacDonald et al., 1987; MacDonald et al., 1991; Parsons et al., 1993; Parsons et al., 1995; Parsons et al., 1996; Blanpied et al., 1997; Aracava et al., 2005; Wrighton et al., 2008). A channel blocker can either prevent the closing of the channel while bound, or it can allow for channel closure around the bound blocker. The differential effects of blockers on channel gating have led to the classification of channel blocking drugs as either sequential (foot- in-the-door) blockers or trapping blockers, respectively. Sequential channel blockers are not unique to NMDA receptors and have been described for other ligand-gated ion channels (Neher and Steinbach, 1978). The binding of a sequential channel blocker within the channel of activated receptors inhibits not only ion flow but also channel closure (Benveniste and Mayer, 1995; Antonov and Johnson, 1996). A representative NMDA receptor sequential channel blocker is 9-aminoacridine (9-AA). The mechanism responsible for stabilizing the open state of NMDA receptor channels while sequential blockers are bound is not completely understood. Trapping channel blockers bind within the channel of activated NMDA receptors and allow subsequent channel closure and dissociation of agonists, an effect that traps the drug within the pore of the receptor. The rebinding of agonist and channel activation allows unbinding of the trapped blocker and its escape from the open channel (Huettner and Bean, 1988; Lerma et al., 1991; MacDonald et al., 1991; Parsons et al., 1993; Parsons et al., 1995; Parsons et al., 1996; Blanpied et al., 1997; Chen and Lipton, 1997; Sobolevsky and Koshelev, 1998; Sobolevsky et al., 1998; Mealing et al., 1999; Sobolevsky, 1999; Mealing et al., 2001; Kashiwagi et al., 2002; Bolshakov et al., 2003; Wrighton et al., 2008). There are differences in the degree to which trapping blockers remain bound to closed channels. Full trapping channel blockers

11

(such as the dissociative anesth etics ketamine and PCP) are co mpounds that, once trapped within closed NMDA receptors, cannot be released until subsequent channel opening allows escape back to the extracellular environment (Huettner and Bean, 1988; Lerma et al., 1991; Mealing et al., 1999; Mealing et al., 2001; Bolshakov et al., 2003). Partial trapping channel blockers (such as the amino-adamantane derivative memantine) are capable of dissociating from a fraction of NMDA receptors before subsequent channel activa tion allows open channel escape (Blanpied et al., 1997; Chen and Lipton, 1997; Sobolevsky a nd Koshelev, 1998; Sobolevsky et al., 1998; Mealing et al., 1999; Mealing et al., 2001; Bolshakov et al., 2003). The underlying mechanism through which partial trapping channel bloc kers prevent being trapped is unknown. 1.3

NMDA RECEPTOR ROLE IN PHYSIOLOGY AND PATHOLOGY 1.3.1

NMDA receptor role in synaptic transmission NMDA receptor-mediated neurotransmission is tightly regulated. NMDA receptors are coincidence detectors because of the require ment of presynaptic glutamate release and postsynaptic depolarization fo r channel activation and Mg 2+ o unblock to occur (Collingridge et al., 1988; McBain and Mayer, 1994; Albensi, 2007). Postsynaptic depolarization typically arises from activation of other members of the glutam ate receptor family, mainly AMPA receptors. 1.3.1.1

Learning and memory Alteration in the strength of synaptic connections is beli eved to be a biophysical mechanism underlying learning and memory. Changes in the strength of a synapse can be expressed as

12

either an increase or a decrease in the ability of a synapse to transmit information. A long-lasting increase in the efficacy of a synapse is termed long-term potentiation (LTP) and a long-lasting decrease long-term depression (LTD) (Bliss and Collingridge, 1993; Rison and Stanton, 1995; Bear and Abraham, 1996; Riedel et al., 2003). NMDA receptor activation has been shown to be critical in the expression of many forms of LTP and LTD at excitatory synapses (Malenka and Bear, 2004). For LTP induction, the intensity of postsynaptic activity must be strong enough to initiate the cascade of events that will strengthen the connection between the pre- and post-synaptic neuron. Cooperativity, associativity, and input-specificity are three properties commonly found to be associated with LTP. Cooperativity refers to the convergence and spatial/temporal pairing of signals that arise from many fibers. Each signal alone is not intense enough to yield LTP; however, the combined activity of all the fibers converging on a postsynaptic site can surpass the threshold of signal strength required to result in LTP. The ability of a weak signal to be potentiated, if its activity is paired with a strong stimulus at a separate but connected site, is called associativity. Input-specificity, which is not a universal property of LTP, refers to the expression of LTP being localized to the activated synapse and not induced at other synapses of the same neuron (Bliss and Collingridge, 1993; Rison and Stanton, 1995). The requirements for the induction of LTP can be linked to the unique properties of NMDA receptors and the organization of the synapse. The presynaptic release of glutamate will activate glutamate receptors within the postsynaptic membrane. Low-intensity activity results in the activation and passage of ions through AMPA receptors, while NMDA receptor activity is inhibited by Mg 2+ o . Higher-intensity activity results in greater AMPA receptor-mediated current and depolarization of the postsynaptic site, relieving Mg 2+ o block of NMDA receptors. The

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requirement for presynaptic glutamate release and postsynaptic depolarization intense enough to reduce Mg 2+ o block of NMDA receptors accounts for th e associative and cooperative properties of LTP. The activation of postsynaptic NM DA receptors results in the influx of Ca 2+ , which activates various second messenger systems localized to the region of the activated postsynaptic site, accounting for the input-specificity of LTP. The activation of second messengers by Ca 2+

influx results in synaptic changes, such as in creasing the flow of ions through postsynaptic receptors, that contribute to the expression of LTP (Bliss and Collingridge, 1993; Rison and Stanton, 1995). While high levels of synaptic activity are required for LTP induction, lower levels of activity can produce LTD (Malenka and Bear, 2004) . NMDA receptors have been implicated in the induction of some forms of LTD, since in hibition of NMDA receptors has been shown to prevent LTD induction (Dudek and Bear, 1992; Thie ls et al., 1996). Like LTP, it appears that increases in intracellular Ca 2+ concentrations, likely through activated NMDA receptors, is critical for inducing LTD (Mulkey and Malenka , 1992). The increase in postsynaptic Ca 2+

Full document contains 143 pages
Abstract: Pharmacological inhibition of NMDA receptor activity by ketamine is accompanied by pyschotomimetic side-effects; however, the Alzheimer's disease therapeutic memantine blocks NMDA receptor activity without debilitating side-effects. This dissertation provides electrophysiological and behavioral characterizations of these two NMDA receptor antagonists in an attempt to understand the unique therapeutic utility of memantine. The following work explores memantine and ketamine inhibition at NMDA receptors, their main site of action, with a focus on the mechanism of inhibition and receptor subtype selectivity in physiologically relevant conditions. This research shows NMDA receptors possess a second binding site at which memantine, but not ketamine, can inhibit activity. The research also shows the dramatic effect physiological concentrations of magnesium has on the ability of these drugs to inhibit NMDA receptor activity. Behavioral and cognitive effects of memantine and ketamine are also assessed and compared directly in rat. The effects of memantine and ketamine in rat were found to be similar at the low doses tested and more divergent as dose increased. Furthermore, memantine's effects appeared to be more pronounced and longer-lasting than those of ketamine. These findings demonstrate the importance of considering the physiological environment in which a drug acts, as well as the principles of drug action, when examining the effects of a drug on central nervous system activity.