the glutamate hypothesis of schizophreniapositive schizophrenic symptoms, the glutamate hypothesis...

PHARMACOLOGY AND PATHOPHYSIOLOGY eNS Drugs 1997 Jon; 7 (1)' 4UJ7 1172-7047/97/IXXll-ffi47/SlO.50/O

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The Glutamate Hypothesis of Schizophrenia Therapeutic Implications

Masahiko J. Ishimaru 1 and Michio Toru2

1 Department of Molecular and Cellular Neuroscience, Medical Research Institute of Tokyo Medical and Dental University, Tokyo, Japan

2 Department of Neuropsychiatry, Medical School of Tokyo Medical and Dental University, Tokyo, Japan

Contents

Summary ........................... . 1. Basis for the Glutamate Hypothesis of Schizophrenia . . .

1.1 The Dopamine Hypothesis - Validity and Limitations . 1.2 Glutamate Levels in Schizophrenic Brains and CSF 1.3 Phencyclidine-Induced Psychosis .......... . 1.4 Glutamate Receptors in Schizophrenic Brains . . . . 1.5 Interaction of Glutamatergic and Dopaminergic Systems

2. Therapeutic Implications of the Glutamate Hypothesis . 2.1 Antipsychotics and the Glutamatergic System .. 2.2 Therapeutic Target Sites on the NMDA Receptor . 2.3 Glycinergic Therapies for Schizophrenia . . . . . .

3. Some Interesting Questions . . . . . . . . . . . . . . . . 3.1 What is the Basis of Glutamatergic Hypoactivity? 3.2 Is the Glycine Site Saturated In Vivo? .. . . . . . 3.3 Is D-Serine an Endogenous Agonist for the Glycine Site?

4. Conclusion ............................ .

. 47

. 48

.48 48 49 52 54 55 55 55 56 60 60 61 61 62

Summary The glutamate hypothesis of schizophrenia has been developed based on the observation that psychotic symptoms induced by phencyclidine and related agents, which are antagonists at the N-methyl-D-aspartate (NMDA) glutamate receptor, closely resemble both the positive and negative symptoms of schizophrenia. In contrast to the dopamine hypothesis, which explains primarily with positive schizophrenic symptoms, the glutamate hypothesis may provide a more comprehensive view of the illness.

Postmortem brain assays also support the glutamate hypothesis by demonstrating alterations in glutamate receptors in several brain areas in patients with schizophrenia, especially remarkable increases in the receptors in frontal and parieto-temporal association fields. This increase in glutamate receptors may reflect postsynaptic up-regulation in response to a deficiency in glutamatergic neuronal activity. Thus, the glutamate hypothesis implies that schizophrenic

48 Ishimaru & Toru

symptoms might be ameliorated by augmenting glutamatergic neural transmission.

Recent clinical studies have been conducted using glycine, milacemide or D-cycloserine as adjuncts to or as a replacement for antipsychotics, in an attempt to augment NMDA receptor-mediated glutamatergic functions by activating the glycine modulatory site. Glycine adjuvant treatment was moderately effective in some patients in all studies and significantly improved negative symptoms in a placebo-controlled study. Replacement of antipsychotics by milacemide, a supposed glycine prodrug that crosses the blood-brain barrier, did not ameliorate schizophrenic symptoms. This lack of effect may be due to the fact that milacemide is predominantly an inhibitor of monoamine oxidases rather than a glycine agonist. Adjuvant administration ofD-cycloserine, a partial agonist ofthe glycine site, was inconclusive, with improvement in negative symptoms occurring in one study and overall exacerbation in most patients in another study.

Well designed placebo-controlled studies using orally active full agonists of the glycine regulatory site should be pursued in the future. Also, intervention at other sites within the NMDA receptor complex should be considered.

1. Basis for the Glutamate HypotheSiS of Schizophrenia

1.1 The Dopamine Hypothesis - Validity and Limitations

The dopamine hypothesis was the first durable biological framework for understanding the aetiology and treatment of schizophrenia. This hypothesis was an unexpected fruit of antipsychotics, drugs that had dramatically changed clinical and research practice in the field of psychiatry. Chlorpromazine was introduced in 1952 as the first effective antipsychotic observed by modem scientists,[I] and its development triggered a burst of discoveries of various other antipsychotics. It was subsequently shown that all these antipsychotics were dopamine receptor antagonists,[2.3] with preferential affinity for the D2 receptor.[4] In addition, amphetamines were found to induce psychotic symptoms that closely mimicked the positive symptoms of schizophrenia[5] by facilitating dopaminergic neural transmission. [6]

Based on these findings, the dopamine hypothesis of schizophrenia has been largely accepted as the basis for the positive symptoms of the illness. However, clinicians have noticed that classical antipsychotics are generally ineffective in amelio-

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rating the negative symptoms[7] that are prominent in patients with a chronic course.l8]

Antipsychotics render schizophrenia manageable, but do not completely cure the disorder. Explanations other than dopaminergic hyperactivity are required for further understanding of the illness, as well as for therapeutic advancement. As reviewed below, accumulating evidence indicates that the glutamate hypothesis is one such candidate that may provide a more comprehensive view of schizophrenia, into which dopaminergic hyperactivity may be incorporated.

1 .2 Glutamate Levels in Schizophrenic Brains and CSF

Glutamate is the major excitatory neurotransmitter in the mammalian brain. [9. 10] Its effects are mediated through binding to a number of receptors: N-methyl-D-aspartate (NMDA), kainate, aamino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and metabotropic. Glutamate is responsible for not only the classical fast synaptic transmission through non-NMDA receptors, but a long term potentiation through the NMDA receptor. This latter effect underlies neuronal plasticity.lll]

Dysfunction in glutamatergic neural transmission is assumed in various neuropsychiatric disor-

eNS Drugs 1997 Jan; 7 (1)

Glutamate Hypothesis of Schizophrenia

ders.D2,13] In 1980, Kim et al.[14] observed a highly significant decrease in the level of glutamate in the CSF of patients with schizophrenia compared with controls (i.e. patients with non-specific neurological disorders or healthy volunteers). From these findings they postulated that impaired glutamatergic function underlies schizophrenia. Although these authors were the first to propose the glutamate hypothesis of schizophrenia, their original finding was not supported by the studies that followed. In subsequent studies, CSF glutamate levels in patients with schizophrenia were reported to be no different from,[15,16] or even higher than, those of individuals who had nonspecific neurological diseases.!17]

Despite this inconsistency (which is of unknown origin), the idea of glutamatergic hypofunction in schizophrenia encouraged further studies of the brains of patients with the disorder, in which various methods were employed for detecting glutamatergic abnormalities. In several studies, measurement of brain glutamate levels in postmortem samples from patients with schizophrenia failed to show a significant change.!15,18] On the other hand, a recent preliminary study using proton magnetic resonance spectroscopy reported that the prefrontal glutamate level measured in vivo was significantly lower in never-treated schizophrenic patients than in healthy volunteers.!19]

Nevertheless, a measurement of the total glutamate level in a given brain area, either postmortem or in vivo, is difficult to interpret, since only a small fraction of the total glutamate represents that which functions as a neurotransmitter. Sherman et al. assayed the synaptosomal fraction (i.e. that containing the pre-synaptic neurons) derived from the neocortex of patients with schizophrenia and observed a significant reduction in glutamate release induced by veratridine,[20] NMDA and kainate.[21] Recently, Tsai et alP2] performed a postmortem study of brains from patients who had had schizophrenia that focused on N-acetylaspartyl-glutamate (NAAG), a neuropeptide that is highly concentrated in glutamatergic neurons.!22] NAAG is a partial antagonist of NMDA receptors and, more-

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over, is cleaved to glutamate and N-acetylaspartate by a specific peptidase, N-acetyl-a linked acidic dipeptidase (NAALADase). In the prefrontal cortex and hippocampus of patients who had had chronic schizophrenia, levels of NAAG were increased whereas NAALADase activity and levels of glutamate and aspartate were decreased compared with samples from unaffected individuals.[22] From these findings it can be postulated that a deficiency in NAALADase leads to an increase in NAAG as well as to a decrease in glutamate, both of which might impair glutamatergic neural transmission. These findings suggest that a deficiency in glutamatergic neural transmission occurs in the schizophrenic brain.

1,3 Phencyclidine-Induced Psychosis

Observations that phencyclidine induces psychosis provided strong evidence for the glutamate hypothesis of schizophrenia. Phencyclidine, like the related compound ketamine, was originally developed as a dissociative anaesthetic.[23] It had to be abandoned, however, because the adverse effects associated with its use, especially postoperative agitation and delirium that could last for hours, were too serious to be tolerated.[24]

Early reports had already pointed out that phencyclidine-induced psychotic symptoms could provide a useful model of schizophreniaps.26] The connection between phencyclidine psychosis and schizophrenia was noted when the admission rate for what appeared to be unusually long, severe and treatment-resistant initial schizophrenic psychoses suddenly tripled at a mental health centre in Washington DC, US, in 1973. All the patients admitted with these symptoms turned out to have smoked 'Angel Dust' , a streetterm for phencyclidine. [27] In addition to the symptoms previously observed in healthy volunteers who were given phencyclidine such as deficits in ego boundaries and feelings of depersonalisation, derealisation and estrangement,[25.28.29] the patients showed the following characteristics: (i) not only positive symptoms (hallucinations and delusions), but also negative symptoms (blunt affect, poverty of speech, apathy

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Cell membrane

Glutamate (NMDA)

I

Competitive antagonists CPP CPPene Selfotel

Ion channel

AP-5 Noncompetitive antagonists Open channel blockers

PCP Ketamine Dizocilplne rcp CyclaZOCine

Polyamine site agonists Spermine Spermidine

Glycine site ligands Full agonist

D-Serine (endogenous agonist?)

Partial agonlsts D-Cyclosenne (agonistic) (+)-HA-966 (antagonistic)

Antagonists Kynurenate 7-CIKA

lshimaru & Toru

Fig. 1. A schematic diagram of the N-methyl-D-aspartate (NMDA) receptor ion channel complex and multiple recognition sites associated with it. Endogenous ligands for each recognition site are shown above the representation of the receptor. while exogenous ligands that are mentioned in the text are listed below. Abbreviations: AP-5 = 2-amino-phosphonovaleric acid; 7-CIKA = 7-chlorokynurenic acid; CPP = 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid; CPPene = 3-(2-carboxypiperazin-4-yl)-1-propenyl-1-phosphonic acid; dizocilpine (MK-801) = (+)-5-methyl-1 0, 11-dihydro-SH-dibenzo-[a,d)cycloheptan-S, 1 O-imine malate; (+ )-HA-966 = (+ )-3-amino-1-hydroxypyrrolid-2-one; PCP = phencyclidine; sellotel (CGS-197SS) = cis-4-phosphonomethyl-2-piperidine carboxylic acid; rcp = N-[1-(2thienyl)cyclohexyl)piperidine.

and attention deficit); (ii) the symptoms generally did not respond to treatment with antipsychotics; and (iii) in some individuals, the symptoms lasted for days to weeks despite abstinence from the drug. [27 ,301

Administration of phencyclidine to schizophrenic patients evoked or worsened their symptoms for a period that lasted for days[31] to weeksJ251 Phencyclidine-intoxicated patients could be differentiated from those with schizophrenia only by a lack of premorbid psychopathologyJ321 Therefore, phencyclidine psychosis was considered to provide a more comprehensive model of schizophrenia than amphetamine psychosis, which mimics only the positive symptoms of schizophrenia,l331


However, the mechanism of phencyclidine psychosis remained unclear for many years. The main reason for this was the diverse mechanisms of action of the drug; it interacts with various transmitter systems such as dopamine, noradrenaline (norepinephrine), acetylcholine, serotonin (5-hydroxytryptamine; 5-HT) and sigma opioid as well as with cation channels. l341 Phencyclidine-induced 'amphetamine-like' behaviours in animals, such as increased locomotor activity, stereotypy and rotation, were ascribed to its indirect dopaminomimetic effectsJ351

Subsequently, Lodge and colleaguesl36,371 demonstrated that phencyclidine (and ketamine) blocks NMDA-induced depolarisation of spinal neurons



while having no effect on non-NMDA mediated depolarisation.l36,37] This observation was supported by a number of studies that followed, and eventually phencyclidine was shown to be an ion channel blocker, It enters the ion channel associated with the NMDA receptor in its open state, is trapped at the binding site located deep within the channel, and prevents calcium ions from entering the cytoplasm (fig. 1))34] Other noncompetitive NMDA receptor antagonists, such as dizocilpine (MK-80 1), N-[ 1-(2thienyl)cyclohexyl]piperidine

51

(TCP), ketamine and cyclazocine, share these properties and the resultant anticonvulsant and neuroprotective effects.[34] All these compounds produce phencyclidine-like abnormal behaviours in animals,[38] as well as phencyclidine-like psychotic symptoms in humans. [39-42] Similarly, Javitt and Zukin[43] reviewed previous studies and suggested that, in the dose range relevant to the psychotomimetic effect, phencyclidine would act exclusively on the NMDA ion channel.[43] Phencyclidine-like psychotic symptoms are also ob-

Table I. Glutamate receptors in the brains of patients with schizophrenia, assessed at postmortem using radiolabelled receptor assays

Reference Year Ligand/displacer No. of patients Results (patients/controls)

Kainate (KA) Nishikawa et al.[50] 1983 [3H1KAlGlu 12110 Frontal cortex (SA 8,9+ 10+46) i; frontal cortex (SA

11+12,45+47), putamen H

Kerwin et al.[51] 1988 [3H1KAlGlu (ARG) 11/9 Left hippocampus !; right hippocampus H

Toru et al.[52] 1988 [3H1KAlGlu 14/10 Parietal cortex (SA 39) i; parietal cortex (except for SA 39), occipital cortex H

Deakin et al. [53] 1989 [3H1KAlGlu 14/14 Frontal cortex (SA 11) i; frontal cortex (SA 10), temporal cortex (SA 21 ,22,38) hippocampus, amygdala H

Kerwin et al,l54] 1990 [3H1KAlGlu (ARG) 7/8 CA3, CA4, dentate gyrus, parahippocampal gyrus, left CA 1, left CA2 !; right CA 1, right CA2 H

AMPA Kerwin et al.[54] 1990 [3H1CNQx/Glu (ARG) 7/8 CA4, left CA3 !; CA 1, CA2, right CA3, dentate gyrus,

parahippocampal gyrus H

Kurumaji et a1.155] 1992 [3H1AMPAlGlu 13110 Frontal cortex, temporal cortex, parietal cortex, occipital cortex, limbic cortex H

Freed et al.[56] 1993 [3H1AMPAlGlu 12115 Frontal cortex, caudate nucleus, nucleus accumbens H

N-Methyl-D-aspartate (NMDA)

NMDA recognition site

Kerwin et al. [54] 1990 [3H1Glu/NMDA (ARG) 7/8 Hippocampus (CA1-4, dentate gyrus, parahippocampal gyrus) H

Ion channel

Kornhuber et al. [57] 1989 [3Hldizocilpine 13112 Putamen i; frontal cortex, area entorhinalis, hippocampus H

(MK-801 )/dizocilpine

Suga et al. [58] 1990 [3Hldizocilpine/ 12110 Temporal cortex (SA 22+38+41+42+52), parietal cortex (SA dizocilpine 40,5+7+31) i; frontal cortex, temporal cortex (SA 20+21 ,36),

parietal cortex (SA 39,1+2+3+5+43), occipital cortex H

Simpson et al.[59] 1992 [3H1TCP/ketamine 13114 Frontal cortex (SA 11) i; frontal cortex (SA 10), temporal cortex (SA 38), amygdala H

Glycine binding site

Ishimaru et al.[60] 1994 [3Hlglycine/glycine 13110 Parietal cortex (SA 39,40,1+2+3+5+43), occipital cortex (SA 17,18+ 19), frontal cortex (SA 6) i; frontal cortex (except for SA 6), temporal cortex, parietal cortex (SA 5+7+31) H

Abbreviations and symbols: AMPA = a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; ARG = autoradiography; SA = Srodmann's area; CNQX = 6-cyano-7-nitroquinoxaline-2,3-dione; Glu = glutamate; TCP = N-[1-(2thienyl)cyclohexyllpiperidine; i indicates an increase; ! indicates a decrease; H indicates no change; + indicates that samples from these areas were analysed together.

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52

served with competitive NMDA receptor antagonists, such as 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPp),[44J 3-(2-carboxypi perazin -4-y 1 )-1-prop en y 1-1-phosphonic acid (CPPene),l45J and selfotel (CGS 19755).l46J

Taken together, it is concluded that blockade of NMDA receptors at any level leads to a psychotic state that closely resembles schizophrenia. Based on the finding, NMDA receptor antagonists have been employed in an attempt to reproduce the behavioural or pathological changes of schizophrenia in animal models.[47-49)

1 .4 Glutamate Receptors in Schizophrenic Brains

Radiolabelled receptor assays and related studies performed in the postmortem brains from patients who had had schizophrenia have provided another line of evidence that suggests that a glutamatergic abnormality may be involved in the illness (tables I and II).

1.4. 1 Non-NMDA Receptors An increase in [3H]kainate binding in the pre

frontal cortex of schizophrenic patients compared with unaffected individuals was reported by 2 separate groups, indicating an increase in the number

Ishimaru & Toru

of these non-NMDA receptors.l50,53) [3H]Kainate binding was also increased in the angular cortex, which is a part ofthe parietal association field (see fig. 2).[52J In contrast, the number of AMPA receptors was not changed in the brain areas examined.[55.56]

Kerwin and his colleagues investigated the hippocampus, and observed a significant decrease in the binding density ofkainate[51] and AMPAreceptors,[541 as well as a decrease in GluRI[61,62] and GluR2[62] mRNA that codes for the AMPA/kainate receptor protein. Their findings are interesting in light of the hippocampal abnormality suggested in schizophrenia.165,66] However, studies from other laboratories have not supported the finding of a decrease in hippocampal kainate receptors in terms of [3H]kainate binding[53) or immunoreactivity of the AMPA/kainate receptor subtypes.l63]

1.4.2 NMDA Receptors

NMDA receptors were investigated using various ligands.

The binding of [3H]dizocilpine (a ligand that labels the ion channel of the NMDA receptor) was reported to increase in the putamen[571 and in 3 cortical areas that included the parieto-temporal association fields[581 in brains from schizophrenic pa-

Table II. Glutamate receptor subunits in the brains of patients with schizophrenia, assessed at postmortem using gene expression and immunochemical assays

Reference

AMPAlkainate Harrison et al.[61J

Eastwood et al. [62J

Breese et al[63J

Year Method Gene probe or antibody No. of patients Results (patients/controls)

1991 ISH Oligonucleotide probe for GluR1 subunit

1995 ISH Oligonucleotide probes for GluR1 and GluR2 subunits

1995 WB Specific antibodies for GluR1, GluR2, GluR3, and GluR5-7 subunits

6/8

14/9

1219

CA3 -1.; CA1, CA4, DG, SUB H

CA3, CA4, DG, SUB (GluR1) -1.; CA3, CA4, DG, SUB, PHG (GluR2) -1.; CA1, PHG (GluR1), CA1 (GluR2) H

Cingulate cortex, hippocampus H

N-Methyl-D-aspartate Akbarian et al.164J 1996 ISH cDNA or its segments for

NR1 and NR2A-D 15/15 NR2C in prefrontal cortex (BA 10) -1.; parieto-temporal

cortex (BA 39) H; l' in NR2D relative to total NR2 in subunits prefrontal cortex (BA 10)

Abbreviations and symbols: AMPA = a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; BA = Brodmann's area; cDNA = complementary DNA; DG = dentate gyrus; ISH = in situ hybridization; PHG = parahippocampal gyrus; SUB = subiculum; WB = Western blot; -1. indicates a decrease; l' indicates an increase; H indicates no change.

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Prefrontal association field

('H)Kalnate Medial Iron tal cortex (9+ 1 0+46) ;01

Eye-movement cortex (S) -'()I

Orbital cortex (11 )'511

( lH)TCP Orbital cortex (11) ""I

PH]Glyclne Premotor cortex (6) 1,5

(3) .(1).(2)

Parietal-temporal-occlpital association field

p H)Kalnate Angular cortex (39) ,

('H)Dlzocllplne Supenor panetal cortex (5+ 7+31) 163

Supramarginal cortex (40) 1b.

Supenor temporal cortex (22+3S+41 +42+52) 1631

( IH]GIYClne Angular cortex (39) i6S

Supramarginal cortex (40) 165

Somesthetlc cortex (1 +2+3+5+43) lbS

OCCipital cortex (17+1S+19) 651

(17)

53

Fig. 2. Changes in glutamate receptors in the frontal and parietal-temporal-occipital association fields and related areas that have been found in patients with schizophrenia. The ligand for each receptor is shown (see fig . 1 for relationship between ligand and receptor) . Numbers in parentheses are the Srodmann's area (SA) numbers. The upregulation of glutamate receptor subtypes was observed in the areas of cerebral cortex shaded blue. In the areas shaded grey, a particularly marked effect was seen, either more than 1 receptor subtype was reported to increase (orbital cortex SA 11, angular cortex SA 39) or an extremely large increase was observed (90 to 110% in angular cortex SA 39 and supramarginal cortex SA 40). The prefrontal association field is thought to be involved in executive tasks such as motivation, planning and socialisation. Frontal lobe dysfunction is currently suggested as a cause of the negative symptoms of schizophrenia. In contrast, the parietal-temporal-occipital association field is an area of extensive polymodal convergence, where higher sensory functions are performed. Disarrangement in such an integrative function may underlie the positive symptoms of schizophrenia, such as auditory hallucinations, reality distortion, thought disorder and cognitive deficits. Abbreviation and symbol: TCP = N-[1-(2thienyl)cyclohexyllpiperidine; + indicates that samples from these areas were analysed together.

tients (fig. 2). [3H]TCP binding, which also labels the NMDA ion channel, was increased in the prefrontal cortex (fig. 2),[59] where an increase in [3H]kainate binding had been observed (see section l.4.1))50,53] Strychnine-insensitive [3H]glycine binding associated with the NMDA receptor was

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significantly increased in 6 of the 16 cortical areas examined (fig. 2).160] This increase was more pronounced and widespread than that of [3H]dizocilpine binding observed in the same brain series.l58]

A recent study focusing on gene expression for the NMDA receptor in prefrontal and parieto-


54

temporal cortices reported a significant increase in the NR2D subunits relative to the whole NR2 subunit family in the prefrontal cortex.[64] More information from gene expression studies will be forthcoming in the near future.

An increase in glutamate receptors in the cerebral cortex has been repeatedly observed in the schizophrenic brain (table I, fig. 2) How can this be explained given the glutamatergic hypoactivity that underlies schizophrenia? Reviewing the studies performed in the same brain series, Toru et aU52] demonstrated a significant negative correlation between glutamate levels in several subcortical structures and glutamate receptor binding in cortical areas receiving projections from these subcortical structures. Similar correlations were also revealed between glutamate levels and glycine binding in the parieto-temporal association field.[60] This suggests that the glutamate receptors might be up-regulated in schizophrenic brains in response to a deficiency in glutamatergic neurons.[52] Such a possibility is supported by the observation that glutamate receptors increase in rat hippocampus following electrolytic lesions of the entorhinal cortex[67,68] and removal offimbria-fornix,l69] both of which may result in denervation of glutamatergic fibres that project to the hippocampus.

Results from glutamate receptor studies also indicate that glutamatergic dysfunction may be present in 2 important association fields, prefrontal and parietal-temporal-occipital (see fig. 2). Dysfunction of the frontal lobe is currently associated with negative symptoms of schizophreniaPO,71] In contrast, dysfunction in the parietal-temporaloccipital association field may underlie the pathogenesis of positive symptoms or cognitive deficits, since this brain area is involved in integrative processing of extensive sensory inputs (fig.2).l72,73] Therapeutic intervention that restores glutamatergic neural transmission may help in correcting the dysfunction assumed in these association fields.

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Ishimaru & Toru

1.5 Interaction of Glutamatergic and Dopaminergic Systems

The glutamate hypothesis of schizophrenia as summarised in sections 1.2 to 1.4 is not inconsistent with the dopamine hypothesis of the illness (see section 1.1). Abundant interactions between dopaminergic and glutamatergic systems are known to occur in the regions of the brain that contain dopaminergic cell bodies[74] and nerve terminalsP5,76] Therefore, the glutamatergic hypoactivity that is hypothesised to be the basis of phencyclidine-induced psychosis and schizophrenia may cause psychotic symptoms via dopaminergic modulation. Alternatively, dopaminergic hyperactivity may induce secondary glutamatergic hypoactivity in broad cortical areas, which may be psychotogenic.

Interestingly, systemic administration of phencyclidine to rats increases dopamine turnover in mesocortical and meso limbic terminal areas, while it decreases that in the striatum.[77] This contrast favours phencyclidine-induced psychosis as a model for schizophrenia, since mesocorticall limbic dopaminergic hyperactivity has been assumed to underlie schizophrenic symptomatologyP8,79] Furthermore, in the prefrontal cortex, a putative target of antipsychotics,[78,80] NMDA receptors mediate a tonic inhibitory regulation of dopaminergic transmission)81.82] Thus, phencyclidine may cause schizophrenia-like symptoms by disinhibiting dopaminergic neurons in the prefrontal cortex. Up-regulation in glutamate receptors[50,53] and a decrease in glutamate level[22] observed in the prefrontal cortex of schizophrenic brains may reflect such an impairment in glutamatergic regulation of dopaminergic activity in the area. Since prefrontal glutamatergic neurons project to the striatum and limbic forebrain areas,[83,84] dysfunction in these neurons may be causative of the subcortical dopaminergic hyperactivity suggested in schizophrenic brains)85-87]

However, the implications of the glutamate hypothesis extend beyond a mere modulation of the dopaminergic system. The antipsychoticresistant symptoms observed in phencyclidine-



induced psychosis and schizophrenia are not explained by dopaminergic hyperactivity, which may occur secondary (or be primary) to glutamatergic hypoactivity.!33] Similarly, alterations in glutamate receptors have been demonstrated in various areas of the cerebral cortex, beyond those that receive dopaminergic innervation (table I, fig. 2). Thus, an interaction with the dopaminergic system may be a part of the psychotogenic mechanism of glutamatergic hypoactivity, but not all of it. This suggestion that glutamatergic dysfunction may be independent, at least in part, of dopaminergic hyperactivity may promote a better understanding of schizophrenia. The glutamate hypothesis can incorporate a dysfunction of more global neural circuits, including the dopaminergic system.l13,49,88,89] In this way, it may serve as a reasonable starting point for developing refined hypotheses of schizophrenia, as well as for planning advanced therapeutic strategies for the illness.

2. Therapeutic Implications of the Glutamate Hypothesis

2.1 Antipsychotics and the Glutamatergic System

The glutamate hypothesis of schizophrenia implies that schizophrenic symptoms should be ameliorated by administration of an agent that restores glutamatergic activity in the brain. However, typical anti psychotics do not primarily have an action on the glutamatergic system, although some indirect effects suggested in vivo, such as a reversal of impaired glutamate releasel20,22] and upregulation of the NMDA receptor-associated ion channel,l90] may contribute to the therapeutic effect of these drugs.

On the other hand, it is tempting to postulate that atypical anti psychotics such as clozapine may affect the glutamatergic system, since clozapine is effective in ameliorating symptoms that are resistant to typical antipsychotics.l91.93] In fact, accumulating evidence has shown that clozapine antagonises behavioural effectsl94.99] and neurotoxicity[IOO] induced by NMDA antagonists. Repeated

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administration of clozapine to animals increased dizocilpine-induced hyperlocomotion and, in parallel, induced an up-regUlation of phencyclidine receptors in the frontal cortex. llOl] Regional distribution of specific [3H]clozapine binding was reported to match glutamatergic innervationl102] and clozapine increased glutamate levels in the rat medial prefrontal cortex.[I03]

Despite all these findings, the data are inconclusive with regard to the way in which clozapine interacts with NMDA receptors. For example, clozapine inhibited [3H]dizocilpine binding in some studies,l 102· 104] while it did not inhibit eH]TCP binding in another study.llOl] Current studies are largely focusing on the possible involvement of multiple receptor systems in the action of clozapine.lI05-107]

Thus, novel agents that augment glutamatergic neural transmission need to be developed in order to derive advanced treatments from the glutamate hypothesis.

2.2 Therapeutic Target Sites on the NMDA Receptor

In light of the finding that antagonism of the NMDA receptor induces psychotic symptoms, this receptor subtype seems to be a promising target of therapeutic attempts for schizophrenia, although alterations in kainate receptors found in postmortem brains suggest that non-NMDA receptors may also play some role in the illness (table I).

The NMDA receptor is a complicated molecule containing several modulatory sites (see fig. 1):

• NMDA binding site • zinc ion (Zn++) modulatory site • magnesium ion (Mg++) modulatory site and

phencyclidine receptor (both of which are located in the ionophore)

• polyamine modulatory site • strychnine-insensitive glycine binding site.

2.2.1 Glutamate/NMDA Binding Site

The recognition site for glutamate or NMDA is obviously not a useful target since agonists at this site are known to be potent neurotoxins.l12,108]


56

2.2.2 Polyamine Modulatory Site The difficulty is that the activity of the NMDA

receptor needs to be augmented to a level that corrects glutamatergic hypofunction, but that avoids excitotoxicity. Polyamines, which constitute a diverse family of endogenous molecules,uo9) may include compounds that show moderate augmentation of glutamatergic activity. In fact, the polyamine site agonists spermine and spermidine enhance NMDA receptor function under certain conditions. [110) Interestingly, both these molecules were reported to inhibit amphetamine-induced dopaminergic hyperactivity in the mesolimbic pathway, but not in the striatum'! II 1) Further understanding of polyamine molecules and their action on the NMDA receptor is required.

2.2.3 Phencyclidine Receptor A compound that prevents phencyclidine from

blocking the NMDA receptor-associated ion channel may be another interesting candidate for a therapeutic agent, especially if glutamatergic hypoactivity is proven to be caused by endogenous phencyclidine-like ligands (see section 3.1 ).[112,113) Metaphit, a phencyclidine receptor acylator, was reported to antagonise phencyclidine-induced abnormal behaviours in the rat,11l4) but this compound is not suitable for clinical use because it has phencyclidine-like behavioural effects of its own.11l5) Development of safer phencyclidine antagonists as well as investigation of modulation at the Zn++ and Mg++ sites might be pursued in the future.

2.2.4 Glycine Binding Site At present, the most promising target for cor

recting glutamatergic hypoactivity seems to be the glycine binding site that is associated with the NMDA receptor.

Glycine dramatically enhances the response of the NMDA receptor in a strychnine-insensitive manner.11l6) This effect has been observed in all of the available assays for NMDAreceptor-mediated functions)117) Activation of the glycine binding site is an absolute requirement for NMDA receptor function;11l8,1l9) therefore, glycine is considered to be a co-agonist at the receptor/ion channel com-


Ishimaru & Toru

plex.!1l7) Even so, glycine may augment NMDA receptor function only moderately,(120) since it acts in a manner that increases the channel opening frequency in the presence of competitive NMDA agonists.!121) The remarkable increase in the glycine binding sites demonstrated in postmortem brain assays (see section 1.4.2) suggests that this site may play a crucial role in the process of schizophrenia)60)

However, there is a major problem with the administration of glycine as a therapy - it does not readily cross the blood-brain barrier, since no highaffinity transport system across the barrier exists for this hydrophilic molecule. (122) Nevertheless, it was shown that brain glycine levels could be increased in the rat brain following an intra-arterial injection of glycine(123) or feeding with a liquid diet containing high levels of glycine.[l24) In humans, administration of glycine was not associated with any significant adverse effects at dosages up to 3 gikg(125) or at 60g per day.(126)

Moreover, intragastric administration of glycine antagonised abnormal behaviours induced by phencyclidine in animals)127) This finding, which was supported by more recent studies in which glycine agonists were administered through intracerebroventricular injection,u28-130) promoted clinical trials involving glycinergic therapy for schizophrenia (table III).

2.3 Glycinergic Therapies for Schizophrenia

2.3. 1 Glycine The first study of glycinergic therapy for schizo

phrenia was performed by Waziri,ll31) The impetus for this study was the finding that the activity of serine hydroxymethyltransferase (SHMT) was significantly decreased, while plasma serine levels were increased, in psychotic patients including those with schizophrenia.!151,152) Since the conversion of serine by SHMT is the major source of glycine in the brain,(153) a deficiency of this enzyme might result in a shortage of glycine. Therefore, glycine (5 to 25 g/day) was administered as an adjuvant to 11 patients with chronic schizophrenia who had been receiving continuous antipsychotic


Glutamate Hypothesis of Schizophrenia 57

Table III. Summary of clinical studies that have assessed glycinergic activation in patients with schizophrenia

Reference Year Type of Dosage Study No. of Results study (g/day) duration patients psychotic symptoms extrapyramidal symptoms

(wk) (drug/placebo) rating scale effect" rating scale effect

Glycine Waziri[131] 1988 A then R 5-25 >8mo 11 NA Improved (4111) NA NS Rosse et al.[132] 1989 A 10.8 4 days-8 5M SPRS, CGI, Improved (2/5), AIMS, Improved

SANS aggravated (2/5) SARS (3/5) Costa et al.[133] 1990 A 15 5 6M SPRS Improved (2/6) NA NS Potkin et al.[134] 1992 A 15 6 9M+2F/6M+1F BPRS, CGI, Improved on CGI SARS Unchanged

SANS Javitt et al. [135] 1994 A 2-30 8 7M17M PANSS Improvement in AIMS, ERS Unchanged

negative symptoms

Milacemide Rosse et al.[136] 1990 R 1.2 4 days-4 5M BPRS, CGI, Aggravated (3/5) SARS Unchanged

SANS, WCST Rosse et al. [137] 1991 R 0.4 7 days-4 4M BPRS, CGI, Aggravated (2/4) SARS NS

SANS, WCST

Tammingaet 1992 R 1.2 6 3 (co) SPRS Unchanged NA NS a1.1138]

D-Cycloserine Cascellaet 1994 A 0.25 6 3M+4F SPRS, CGI, Aggravated (417) NA Unchanged al.[139] SANS Goff et al.[140] 1995 A 0.05-0.25 2 8M+1F BPRS, GAS, Improvement in AIMS, Unchanged

SANS, SIRP negative SARS symptoms (SANS) and reaction time (SIRP)

a Where the number of patients in the effect column does not equal the total number of patients, the remainder were unchanged.

Abbreviations: A = adjuvant; AIMS = Abnormal Involuntary Movement Scale[141]; BPRS = Brief Psychiatry Rating Scale[142]; CGI = Clinical Globallmpression[143]; co = crossover; ERS = Extrapyramidal Rating Scale[144]; F = female; GAS = Global Assessment Scale[145]; M = male; NA= not applicable (no scale used); NS = not specified; PANSS = Positive and Negative Symptom ScaleI146]; R = replacement; SANS = Scale for Assessment of Negative Symptoms[147]; SARS = Simpson-Angus Rating Scale for Extrapyramidal Symptoms[148]; SIRP = Sternberg's Item Recognition ParadigmI149]; WCST = Wisconsin Card Sorting Test11501.

medication and were socially handicapped.D3l) The treatment was successful in 4 patients who showed 'a definite salutary response', allowing the dosages of their antipsychotics to be reduced or even discontinued and the patients to become involved in rehabilitation.[13I) This effect lasted for several months.£13l)

These observations opened the way for further studies, although a deficiency in SHMT was not supported by other studies.£154,155) Waziri et al.D 56) later suggested that a deficiency in SHMT might lead to a more complicating metabolic abnormality, in which levels of glycine as well as serine might be elevated.

© Adls International Umited. All rights reserved.

In another open label study by Rosse et al.,[132) glycine was given to 6 patients at a fixed dosage (10.8 g/day) as an adjunct to antipsychotics, and the effect was evaluated by standardised rating scales (see table III.) During the glycine adjuvant period, 2 patients showed improvement whereas 2 others worsened. Another patient showed no clinical response to adjuvant glycine. The remaining patient reported marked subjective improvement at the beginning of the glycine phase, but left hospital before formal ratings could be performed. In I of the 2 responders, deterioration was observed after glycine withdrawal, but a subsequent rechallenge with glycine failed to result in a persistent effect.


58

The other responder showed no worsening after glycine discontinuation. Interestingly, a remarkable improvement was observed in extrapyramidal symptoms in 3 of the patients.[l32]

Costa et aUl33] also administered glycine (15 g/day) as an adjunctto 6 patients with schizophrenia who had been maintained on relatively stable dosages of antipsychotics. Two patients showed remarkable improvement in Brief Psychiatric Rating Scale (BPRS) scores and none of the patients showed significant aggravation of symptoms or major adverse effects. The authors conducted another study involving a larger number of patients in a double-blind manner and observed a slight improvement in symptoms, as reflected in Clinical Global Impression scores but not in the other rating scales employed.[l34] The changes in BPRS showed improvement especially in the score of hostility-suspiciousness, but this did not reach statistical significance.l l34]

More recently, Javitt et aUl35] administered glycine to patients with schizophrenia in a doubleblind, placebo-controlled study where changes were evaluated according to the Positive and Negative Syndrome Scale. A significant decrease in negative symptom scores was observed after the glycine administration period without exacerbation of positive symptoms. In contrast to the previous report,[l32] the patients showed no significant change in Extrapyramidal Rating Scale scores, suggesting that the improvement in negative symptoms did not result from an amelioration in antipsychoticinduced extrapyramidal symptoms.[l35]

Despite some minor inconsistencies, these studies suggest that glycine administration may be a valuable adjunctive tool in the treatment of schizophrenia.

2.3.2 Milacemide Because the relative impermeability of the

blood-brain barrier to glycine seemed to be the major difficulty for a glycinergic activation strategy,[l32] 2 orally active compounds, milacemide and D-cycloserine, were assessed in clinical studIes.

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Ishimaru & Toru

Milacemide is a glycine prodrug that readily crosses the blood-brain barrier and is converted by monoamine oxidase (MAO)-B into glycinamide and glycine)l57] This drug was administered either at a high (1200 mg/day)[l36] or low (400 mg/day) dosage[l37] to patients with chronic schizophrenia. However, clinical worsening was observed in almost all the patients during the milacemide administration period in both studies, which resulted in withdrawal of half of them from treatment (3 of 5 and 2 of 4 patients, respectively; see table III).[l36.l37] On the other hand, Tamminga et aUl38] also administered milacemide (1200 mg/day) in a small, placebo-control crossover study and observed no improvement or deterioration on BPRS scores in schizophrenic patients.

It should be noted that in these studies antipsychotics were replaced by milacemide, while in the other studies, glycine or D-cycloserine were administered as an adjunct to antipsychotics (see table III). In replacement studies, it is particularly important to design an appropriate scheduled for the cessation and wash-out of antipsychotics that had been administered to the patients involved. Rosse et aUl36.137] employed a 4-day medicationfree period before administrating milacemide, whereas in the study by Tamminga et al.,[138] there was a wash-out period of at least 4 weeks until the psychotic symptoms of the patients reached a stable baseline. The difference in these wash-out periods may explain the inconsistency between their results, since in the former studies cessation of antipsychotics might have resulted in exacerbation of schizophrenic symptoms during the period when milacemide was being administered.

The failure of milacemide to ameliorate schizophrenic symptoms may not necessarily argue against the validity of glycinergic intervention. This is because milacemide was shown to be an inhibitor as well as a substrate of MAO_B[l58.l59] and also an inhibitorofMAO-A,[159] both of which are the enzymes that convert dopamine to 3,4 dihydroxyphenylacetic acid (DOPAC). It is likely that inhibition of these enzymes led to an accumulation of dopamine,[l60] to the extent that it may



have negated the beneficial effect of glycine. Milacemide also potentiated the anticonvulsant effect of dizocilpine in such a way that the potentiation was not blocked by a selective antagonist of glycine sites,l l61 1 suggesting that the predominant effect of milacemide in vivo is ascribed to a mechanism other than glycinergic activation.

2.3.3 D-Cycloserine

The long term administration of D-cycloserine in the treatment of tuberculosis is frequently associated with a high incidence of neuropsychiatric abnormalities.ll621 Interestingly, it has also been administered to psychotic patients at high dosages (1.0 to 2.0 g/day), which resulted in exacerbation of psychotic symptoms with motor excitement followed by an improved response to subsequent antipsychotic treatment ('symptom provocative' therapylI621). D-Cycloserine has been identified as a partial agonist at the glycine site,[163, 1641 and so has recently been utilised for the treatment of schizophrenia at moderate dosages.

However, Cascella et aLi l391 administered the drug at a dosage of 250 mg/day for 6 weeks to 7 patients and observed a deterioration in 4 of the patients, with intensified positive symptoms and emotional agitation. The authors suggested that this might be due to a biphasic action of D-cycloserine.[ 1391 As a partial agonist, the drug has only limited activity and could antagonise the action of an endogenous agonist at the glycine site when given at high dosages.l1 65,1661 Therefore, they suggested there might exist a therapeutic 'window' for cycloserine,[1391

Indeed, this window was observed in a later study - Goff et aLl l401 reported that patients given a placebo and 4 dosages of D-cycloserine (5, 15, 50 and 250 mg/day) in a consecutive schedule showed a significant reduction in Schedule for Assessment of Negative Symptoms scores as well as in reaction time according to Sternberg's Item Recognition Paradigm at 50 mg/day, while at 250 mg/day the scores were reversed to the level observed at 0 to 15 mg/day. This indicates that the window may occur around 50 mg/day, although the effect of drug accumulation should be taken into


59

account. Another consideration is that the patients in these 2 studies might have differed in the severity of their illness; the former study used inpatients with moderate to severe symptomatology,l'391 while the latter used outpatients who had been managed on a stable medication regimen much longer than those in the former study.ll401 This may partly explain the ineffectiveness of D-cycloserine observed in the former study.

2.3.4 Other Comments on Findings

Although most of the studies reviewed above were preliminary ones conducted with differing experimental designs, the results include some interesting findings (table III). Administration of glycine as an adjunct to antipsychotics was effective in some patients. The effect was moderate in general, but the drug rarely aggravated symptoms. Glycine did not exacerbate extrapyramidal symptoms and sometimes ameliorated them.

It is interesting that improvement in negative symptoms was observed in recent studies following administration of glycine[1351 or D-cycloserine,l 1401 Earlier studies also included several cases in which negative symptoms were ameliorated by glycine.l l3l ,132] In contrast, no quantified improvement in positive symptoms has been reported so far. However, it should be noted that in these studies glycine and D-cycloserine were given as adjuvants to antipsychotics which had been administered for years. The positive symptoms that had responded to antipsychotic treatment might also have been ameliorated by glycine site activation. In this respect, it is interesting that, in the first study by Waziri,[131] 4 of the patients who responded to glycine could be maintained on reduced doses of anti psychotics or even have the drugs discontinued for several months. However, the reliability of this finding is still not clear, since Costa et aLl133] reported that the administration of glycine without concomitant anti psychotics had no antipsychotic effect in patients with acute schizophrenia. More studies are required to clarify whether glycine can successfully replace antipsychotics in controlling positive symptoms.l 1351

eNS Drugs 1997 Jan; 7 (l)

60

It also remains to be clarified whether the moderate effects of glycine were due to its modulatory action on dopaminergic pathways, or the result of correcting the primary deficit in glutamatergic neural transmission.

Since milacemide is not an effective source of glycine and D-cycloserine is only a partial agonist at the glycine site, a full glycine site agonist that readily crosses the blood-brain barrier is required. Such an agent will provide valuable information pertaining to the validity of glycine site activation therapy as well as the glutamate hypothesis of schizophrenia. In future studies, special attention should be paid to: (i) the administration schedule of the drugs; (ii) employment of a placebo-control design; (iii) methods for evaluating the symptomatology; and, especially, (iv) the clinical condition of patients such as sUbtypes of the illness and severity of the symptoms. Considering the wide variety of symptomatology and aetiology subsumed under a diagnosis of schizophrenia,[167] one might question the validity of evaluating drug effects in such a heterogenous population.

3. Some Interesting Questions

3.1 What is the Basis of Glutamatergic Hypoactivity?

As a model for schizophrenia, phencyclidinepsychosis allows at least 2 possible mechanisms that may cause glutamatergic hypoactivity: (i) a deficiency in glutamatergic neurons caused by either presynaptic or postsynaptic abnormalities; or (ii) the presence of an endogenous ligand that acts at phencyclidine receptors to block the function of NMDA receptors. Theoretically, these 2 assumptions may lead to totally different predictions of the consequence of glycine site activation. If glutamatergic hypoactivity is due to a deficiency in glutamatergic neurons, then exogenous glycine would be effective in activating the receptors. If, by contrast, a phencyclidine-like endogenous ligand is involved in schizophrenia, further activation of NMDA receptors by exogenous glycine

© Adis Internafional limited. All rights reserved.

Ishimaru & Toru

might be ineffective, as the phencyclidine-like ligands would readily block the open channels.

At present, there is no definite answer to the question. A deficiency in glutamatergic neurons is suggested by several findings: a reduction in glutamate release from synaptosomes obtained from schizophrenic brains,[2o.21] a reduction in glutamate levels in the brains of patients with schizophrenia,[22] a negative correlation between glutamate levels and the number of its receptors, [52] and alterations in non-NMDA receptors[50-54] that are not likely to be caused by an endogen...ous NMDA antagonist.

On the other hand, the existence of a phencyclidine-like ligand has also been reported in animals,£i 12,113] suggesting that glycinergic activation may not be helpful in patients with schizophrenia. In fact, it was reported that the stereotypes induced by dizocilpine in rats were not counteracted by a glycine agonist (D-cycloserine) and were even augmented.[168] This led Kretschmer and Schmidt[l69] to doubt the usefulness of glycine administration to schizophrenic patients, a view that is supported by the 'negative findings' of clinical studies. Carlsson et alJ l70] raised a similar question based on their finding that D-cycloserine potentiated, while (+)HA-966 counteracted, the locomotor stimulation induced by an NMDA antagonist (either dizocilpine and CPPene) coadministered with clonidine in monoamine-depleted mice. They argued, as had been suggested by another group,[I7I] that (+)-HA-966, an antagonistic partial agonist of the glycine site, might be effective in treating schizophrenia, whereas D-cycloserine might not be.£170]

However, these results should be carefully interpreted, since the effect of D-cycloserine, a partial agonist of the glycine site, could be biphasic depending on the dosage.D 65,166] This is particularly important if modulation is attempted at the glycine site in vivo. When competing with an endogenous full agonist, D-cycloserine may readily act as an antagonist,[166] even though it has a relatively high intrinsic activity.£164] In fact, there are several studies reporting that abnormal behaviours induced by phencyclidine or dizocilpine were successfully an-



tagonised by glycine[l27] or by full agonists of the glycine site, such as D_serine[l28,130] and D-alanine)129,130] The facilitating effect of phencyclidine on dopamine utilisation in the medial frontal cortex was also inhibited by intracerebroventricular injection of D-alanine,D72]

The precise mechanism of these effects remains unclear, An in vitro study suggested that glycine site agonists counteracted the inhibitory effect of phencyclidine on the NMDA-mediated signal transduction, but probably by some indirect mechanism at a physiologicalleveUl73] In addition, the effect of a glycine site ligand in vivo seems to be far more complex than is predicted by the data obtained in vitro, since the NMDA receptor mediates either enhancement or inhibition of dopaminergic neural transmission depending on the brain area in which it is located)77,171,174]

Whatever the mechanism is, these findings suggest that glycine agonists may be beneficial to patients with schizophrenia even if glutamatergic hypofunction is due to a phencyclidine-like endogenous ligand.

3,2 Is the Glycine Site Saturated In Vivo?

The CSF glycine level is known to be as high as 1 ~mol/L and above in both healthy individuals and patients with schizophrenia.[16] This suggests that glycine levels in vivo might be high enough for glycine to occupy all the glycine sites)1l6] If this is the case, administration of exogenous glycine agonists would have no effect, a hypothesis that would dampen enthusiasm for the glycinergic activation therapy.

Early studies reported that responses mediated by NMDA receptors in brain slices are not enhanced by glycine or D-serine, but could be blocked by glycine antagonists such as kynurenate, 7-chlorokynurenic acid and (+)-HA-966)17S] This favoured the hypothesis that the glycine site is saturated in vivo.

However, under in vitro conditions that closely mimicked those observed in vivo, NMDA receptor-mediated synaptic potentials were enhanced by glycine in neocortical slices)176] This finding


61

was followed by studies in vivo that showed a facilitation ofNMDAresponses by local application of glycine or D-serine.[l77-179] Also, in cerebellar granule cells, where the high affinity glycine transporter is abundant, the NMDA receptor-mediated synaptic currents were absent unless glycine was supplied in the perfused medium) I 80] These observations argue that the glycine sites in vivo stay unsaturated, and provide a rationale for the administration of exogenous glycine agonists for activating NMDA receptors)181]

Recently, brain-specific glycine transporters were cloned in rodents and the co-localisation of these transporters with NMDA receptors was observed)182-184] This suggests that the glycine level in the synaptic cleft may be precisely regulated by these transporter molecules. Kynurenate, an endogenous tryptophan metabolite that competitively antagonises the glycine site,[18S-187] may also play an important regulatory role)188,189] Modification of the function of the glycine transporter, as well as that of endogenous antagonist metabolism, may provide an interesting basis for a therapeutic intervention, although more information about these regulatory mechanisms is required,

3,3 Is D-Serine an Endogenous Agonist for the Glycine Site?

D-Serine is a selective agonist at the glycine site on the NMDA receptor, and has a similar potency to glycine)1l8,190,191] Although D-serine has been employed in experimental studies, it was believed that D-enantiomers of amino acids did not naturally exist in mammalian tissues,[192] except in small amounts.[193,194] However, recent studies have shown that free D-serine is present in substantial quantities in the brain ofrat[l9S] and human)196]

The anatomical distribution of D-serine, as well as its age-related change, was highly correlated with that of the NMDA receptor,[197] Analysis of the cloned NMDA receptor expressed on Xenopus oocytes revealed a lower EDso value (dose of a drug that produces 50% of the maximal response) for D-serine than for glycine, i.e. D-serine is a more potent ligand for the receptor than glycine)198] Fur-


62

thermore, D-serine was found to concentrate in the

synaptosomes that included the uptake system for

the molecule.l l971 Therefore, there is a good possi

bility that D-serine is an endogenous modulator at

the glycine site associated with the NMDA recep

tor.D 811

Considering that D-serine antagonised NMDA

antagonist-induced abnormal behaviours[128, 1301

and dopaminergic hyperactivity in the prefrontal

cortex,[ 1721 this amino acid may be considered as

another promising candidate for glycinergic acti

vation therapy, particularly if an orally administra

ble prodrug for D-serine is developed.

4. Conclusion

In contrast to the dopamine hypothesis, which

was conceived on the basis of the action of anti

psychotics, the glutamate hypothesis of schizo

phrenia has been proposed on the basis of patho

logical findings and a therapeutic approach is

developing from it. Although glutamatergic hypo

activity in the schizophrenic brain seems compel

ling, it remains to be demonstrated that schizophre

nic symptoms are successfully ameliorated by

augmenting glutamatergic neural transmission.

Initial therapeutic attempts have focused on the

glycine binding site of the NMDA receptor. The

results obtained so far are promising, but not con

vincing. Carefully designed controlled studies us

ing either orally active glycine agonists or agents

aimed at other targets are required. The results of

these studies may determine whether the glutamate

hypothesis survives as an authentic theory or fades

away after a temporary boom.

Acknowledgements

The authors thank D.E Wozniak (PhD), G. BrosnanWatters (PhD) and S.c. Yancey (MEd) for reading the manuscript and for making helpful suggestions.

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Correspondence and reprints: Dr Masahiko J. Ishimaru, Department of Psychiatry, Washington University School of Medicine, 4940 Children's Place, Saint Louis, MO 63110, USA. E-mail: [email protected]

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