Charlotte Jacquemot
email: charlotte.jacquemot@ens.fr

Christophe Lalanne

Agnès Sliwinski

Page Piccinini

Emmanuel Dupoux

Anne-Catherine Bachoud-Lévi

Piccinini

Improving Language Evaluation in Neurological Disorders: The French Core Assessment of Language Processing (CALAP)

Keywords: aphasia, language disorders, scale, diagnosis, functional model of language, cognitive rehabilitation, validation

Aphasia is a devastating brain disorder, detrimental for medical care and social interaction. The early diagnosis of language disorders and accurate identification of patient-specific deficits are crucial for patients' care, as aphasia rehabilitation is more effective when focused on patient-specific language deficits. We developed the Core Assessment of Language Processing (CALAP), a new scale combining screening and detailed evaluation to rapidly diagnose and identify patient-specific language deficits. This scale is based on a model of language processing distinguishing between the comprehension, production, and repetition modalities, and their different components: phonology (set of speech-sounds), morphology (how the sounds combine to form words), lexicon (words), syntax (how words combine to form sentences), and concept (semantic knowledge). This scale was validated by 189 participants who underwent the CALAP, and patients not unequivocally classified as without aphasia by a speechlanguage pathologist underwent the Boston Diagnosis Aphasia Evaluation as the gold standard. CALAPscreening classified patients with and without aphasia with a sensitivity of 1 and a specificity of 0.72, in 3.14 Ϯ 1.23 min. CALAP-detailed evaluation specifically assessed the language components in 8.25 Ϯ 5.1 min. Psychometric properties including concurrent validity, internal validity, internal consistency and interrater reliability showed that the CALAP is a valid and reliable scale. The CALAP provides an aphasia diagnosis along with the identification of patient-specific impairment making it possible to improve clinical follow up and deficit-based rehabilitation. It is a short and easy-to-use scale that can be scored and interpreted by clinicians nonexpert in language, in patients with fatigue and concentration deficits.

Public Significance Statement

This validation study showed that the Core Assessment of Language Processing (CALAP), a new scale for assessing language disorders based on a functional language model and combining screening and detailed evaluation, is a valid and reliable tool for language assessment in patients with neurological disorders. CALAP-screening can be used to diagnose aphasia and CALAP-detailed evaluation efficiently identifies the patient-specific language deficits, making it possible to improve both clinical follow up and deficit-based rehabilitation.

Aphasia is one of the most devastating disorders in patients with brain impairments, regardless of etiology (stroke, traumatic brain injury, or neurodegenerative disease; [START_REF] Denier | Aphasia in stroke patients: Early outcome following thrombolysis[END_REF][START_REF] Kirshner | Frontotemporal dementia and primary progressive aphasia, a review[END_REF][START_REF] Mesulam | Primary progressive aphasia[END_REF]. Aphasia limits social interaction [START_REF] Laska | Recognition of depression in aphasic stroke patients[END_REF], which hinders cognitive rehabilitation efforts [START_REF] Duncan | Management of adult stroke rehabilitation care[END_REF][START_REF] Kauhanen | Aphasia, depression, and non-verbal cognitive impairment in ischaemic stroke[END_REF], and delays the return to work, with a major impact on care pathways and health care costs [START_REF] Ellis | The one-year attributable cost of poststroke aphasia[END_REF].

Following the diagnosis of aphasia, rehabilitation is more effective when intensive and focused on the impaired component than when unspecific [START_REF] Breitenstein | Intensive speech and language therapy in patients with chronic aphasia after stroke: A randomised, open-label, blinded-endpoint, controlled trial in a health-care setting[END_REF]Hillis, 1998;[START_REF] Jacquemot | Specificity in rehabilitation of word production: A meta-analysis and a case study[END_REF]. In order to propose intensive deficit-based rehabilitation, identification of the patient-specific language deficits is critical. However, there is currently no validated scale for identifying the specific components impaired within a functional model of language processing. On the one hand, long scales, such as the Boston Diagnosis Aphasia Evaluation (BDAE; [START_REF] Goodglass | The assesment of aphasia and related disorders[END_REF]-considered as the gold standard for comprehensive language assessment-are used for assessing oral comprehension, production, repetition, and writing and reading abilities. They distinguish between the various types of aphasia (e.g., Broca, Wernicke, etc.). The BDAE requires several sessions and about two or three hours with a trained speech speech-language pathologist for its administration and rating, precluding its use in patients with fatigue. On the other hand, a very short scale such as the Language Screening Test (LAST) briefly assesses oral comprehension, production, and repetition in 2 min [START_REF] Flamand-Roze | Validation of a new language screening tool for patients with acute stroke: The Language Screening Test (LAST)[END_REF]. The LAST is used to detect language disorders in acute stroke and to orient toward thrombolysis or not [START_REF] Denier | Thrombolysis in stroke patients with isolated aphasia[END_REF]. However, none of these scales assesses language components within a functional model of language processing, precluding their use to guide aphasia rehabilitation [START_REF] Caramazza | For a theory of remediation of cognitive deficits[END_REF].

To fill this gap, we developed and validated the French Core Assessment of Language Processing (CALAP), a short easy-to-use scale for the assessment of oral language. The CALAP is based on a functional model of language composed of several components reported in Figure 1A [START_REF] Caramazza | How many levels of processing are there in lexical access[END_REF][START_REF] Jacquemot | Breaking the mirror: Asymmetrical disconnection between the phonological input and output codes[END_REF][START_REF] Jacquemot | Specificity in rehabilitation of word production: A meta-analysis and a case study[END_REF][START_REF] Levelt | Models of word production[END_REF][START_REF] Miceli | The interaction of lexical and sublexical processes in reading, writing and repetition[END_REF] and focused on the evaluation of oral language. The model, purposely simplified, makes the distinction between the comprehension, production, and repetition modalities. Each modality is composed of several components: speech sounds are processed through the phonological component, the combination of sounds to form the words is performed through the morphological component, the recognition or selection of words involves the lexical component, and the words are combined to form sentences through the syntactic component. Finally, semantic knowledge (which includes common knowledge of facts, events, concepts of the world, and personal experience) involves the concept component. The CALAP has two steps: (a) a CALAP-screening phase to detect aphasia and to provide indications, for (b) a CALAP-detailed evaluation phase identifying the impaired language components (Figure 1B). The impairment of a particular component gives rise to a specific language deficit that the CALAP-detailed evaluation can identify through the different subtasks of the scale.

The aim of this study was to determine whether the screening part of the CALAP could be used to diagnose aphasia and whether the detailed evaluation could identify impaired language components, thereby making it possible to improve the effectiveness of rehabilitation.

Method

CALAP Material and Administration

The CALAP is a French-language scale. The material supplied for the CALAP includes the following: a booklet for CALAP administration, a booklet of pictures, and detailed instructions for the rater (see the CALAP administration booklet and CALAP pictures booklet in the online supplemental materials). Figure 1C displays an example of each CALAP task and the number of trials in each task and subtask.

The scale is composed of two different and independent phases: CALAP-screening and CALAP-detailed evaluation. The CALAPdetailed evaluation is administrated only if the total CALAPscreening score is lower than the cutoff score of 22 (Figure 1B; see the Results section on external validity). The scale is self-explanatory. The response of the patient is either perfectly correct (otherwise incorrect), making it possible for clinicians inexperienced in language assessment to score and interpret the responses given. Perfectly correct refers only to the expected responses written in the administration booklet. In both screening and detailed evaluation for comprehension and repetition tasks, a score of 2 (perfectly correct), 1 (perfectly correct after one repetition of the trial by the experimenter), or 0 (incorrect) is assigned for each trial. The experimenter repeats the trial if, and only if, requested by the patient. For production and concept, because there is no trial to be repeated by the experimenter, the score of each trial is either 2 (perfectly correct) or 0. Since healthy participants always needed less than five seconds to respond in each trial, beyond this time delay of 5 s, the answer is considered incorrect (0 point). For the CALAP-screening, a total score is computed (0 -24 pts) whereas for the CALAP-detailed evaluation, because the aim is to evaluate each component separately for identifying the impaired ones, the score of each subtask is not summed. The maximum scores for the screening and for each subtask of the detailed evaluation are reported Figure 1B.

CALAP-Screening

The screening phase assesses language comprehension, production, and repetition. The trials are purposely set so as to be difficult, to increase the screening sensitivity.

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Comprehension is assessed in a sentence-picture matching task including six complex sentences (mean word number: 9.16 Ϯ 1.47; mean syllable number: 10.6 Ϯ 1.14) with a noncanonical grammatical construction: relative clause, negative form and passive construction. The participant is asked to decide whether the sentence heard matches the picture. A "yes" response is expected in half of the trials.

Production capacity is assessed in a picture description task including three pictures that the participant is asked to describe in one sentence. The expected sentences are highly constrained and have noncanonical constructions: a reflexive verbal construction, a reciprocal verbal construction, and a passive form.

Repetition capacity is assessed with a sentence repetition task including three long complex sentences with relative clauses Figure 1. CALAP material and underlying rationale. (A) Simplified model of oral language processing and distinguishing between the comprehension, production, and repetition modality, and, within each modality, the different components: phonology (set of speech sounds), morphology (how the sounds combine to form words), lexicon (words), syntax (how words combine to form sentences) and concept (semantic knowledge). From "Specificity in Rehabilitation of Word Production: A Meta-Analysis and a Case Study," by C. Jacquemot, E. Dupoux, L. Robotham, and A. C. Bachoud-Lévi, 2012, Behavioural Neurology, 25, p

. •••. Copyright 2012 by •••.
Adapted with permission. (B) CALAP structure, including the screening and detailed evaluation of each component of the functional model of language processing. (C) CALAP overview: a single trial, together with its scoring grid, is reported for each task, for both screening and detailed evaluation. The instructions given to the participant are shown in italics. The number of trials in each task and subtask is reported in parentheses. This document is copyrighted by the American Psychological Association or one of its allied publishers.

This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.

(mean word number: 10.33 Ϯ 1.52; mean syllable number: 14 Ϯ 1.73).

CALAP-Detailed Evaluation

Digit span. Forward and backward digit spans are used to assess verbal short-term memory (STM) and working memory, respectively. Verbal STM can store a limited amount of verbal information for a brief period of time, whereas working memory is used to manipulate the information stored in STM [START_REF] Cowan | What are the differences between long-term, short-term, and working memory[END_REF]; but see also [START_REF] Aben | About the distinction between working memory and short-term memory[END_REF][START_REF] Richardson | Measures of short-term memory: A historical review[END_REF]. In the digit span task, participants are presented with a series of two digits and must immediately repeat them [START_REF] Wechsler | Wechsler Adult Intelligence Scale-Revised manual[END_REF]. The length of the series is increased each time the participant succeeds. In case of failure to repeat the series, another series of the same length is provided. If the participant successfully repeats this series, the test continues, if not, it stops. The digit span is defined as the longest series of digits that the participant can successfully repeat. In the forward version, the participant repeats the digits in the order in which they are presented. In the backward version, the participant repeats the digits in reverse order. Distinguishing between verbal STM and working memory is not relevant in the context of the CALAP scoring, so a digit span score is determined by adding the forward and backward scores independently from the CALAP scores in language subtasks.

Concept. Conceptual knowledge or semantic memory refers to a person's general knowledge of the world and to his personal experience. It is not dependent on language capacity, but may affect language performance if impaired. Conceptual knowledge is assessed through a nonverbal picture-matching task including four trials [START_REF] Caramazza | The organization of conceptual knowledge: The evidence from category-specific semantic deficits[END_REF]. In each trial, a target picture is presented with three other pictures. The target picture must be associated with the one among the three pictures which is functionally matched [START_REF] Jacquemot | Specificity in rehabilitation of word production: A meta-analysis and a case study[END_REF]; concept-depicted mean familiarity 1 value: 2.45 Ϯ 0.9; [START_REF] Alario | A set of 400 pictures standardized for French: Norms for name agreement, image agreement, familiarity, visual complexity, image variability, and age of acquisition[END_REF]; mean imaginability 2 value: 4.39 Ϯ 0.23; [START_REF] Bonin | Normes de concrétude de valeur d'imagerie, de fréquence subjective et de valence émotionnelle pour 866 mots[END_REF]; mean emotional 3 value: 3.12 Ϯ 0.72; [START_REF] Bonin | Normes de concrétude de valeur d'imagerie, de fréquence subjective et de valence émotionnelle pour 866 mots[END_REF].

Comprehension. For the comprehension task, four subtests assess the phonological, lexical, morphological, and syntactic components of the language-processing model (see Figure 1; Badecker, Nathan, [START_REF] Badecker | Varieties of sentence comprehension deficits: A case study[END_REF]Jacquemot, Dupoux, Decouche, & Bachoud-Lévi, 2006;[START_REF] Jacquemot | Comprehending spoken words without hearing phonemes: A case study[END_REF][START_REF] Martin | The neuropsychology of sentence processing: Where do we stand?[END_REF][START_REF] Miceli | Morphological errors and the representation of morphology in the lexical-semantic system[END_REF]. In these subtests, a yes response is expected in half of the trials. Each subtest consists of six trials and begins with a training trial. The phonological component is assessed with a task in which the participant is asked to discriminate between two-syllable pseudowords (nonsense words phonologically legal in French). When different, the two pseudowords differ by a single phonological feature. The lexical component is assessed with a lexical-decision task in which the participant must determine whether or not the item is a French word (mean item length: 4 Ϯ 0.89). The items presented are words with a very low frequency (mean frequency: 0.47 Ϯ 0.73 data from www.lexique.org), and pseudowords differing from a word by a single phoneme. The morphological component is assessed through an anomaly detection task including six sentences (mean words per sentence: 5.33 Ϯ 1.032; mean syllables: 8.33 Ϯ 1.50) that may or may not contain a morphological error. The participant must decide whether or not the sentence is correct. Morphological errors are incorrect conjugation forms that, although incorrect, are not aberrant in French. The syntactic component is assessed with an anomaly detection task including six sentences (mean words per sentence: 5.16 Ϯ 1.47; mean syllables: 8.33 Ϯ 1.50) that may or may not contain a syntactic error. Syntactic errors are incorrect gender and number agreements and an incorrect reflexive verbal construction not aberrant in French.

Production. In the production task, two subtests are used to assess the lexical and morpho-syntactic components [START_REF] Bock | Language production: Grammatical encoding[END_REF]. Each subtest consists of four trials and begins with a training trial. The lexical component is assessed through a picturenaming task including long low-frequency words (mean number of syllables: 3.75 Ϯ 0.5, mean frequency: 7.16 Ϯ 10.17). There are two nouns, a verb, and a number. The morpho-syntactic component is assessed with a picture description task. The sentences to be produced are of the subject-verb-object type with low frequency verbs and objects (mean frequency: 48.9 Ϯ 60.64) and contain transitive and reflexive verbal constructions.

Repetition. The repetition task consists of three subtests for assessing the phonological, lexical, and morpho-syntactic components [START_REF] Jacquemot | Breaking the mirror: Asymmetrical disconnection between the phonological input and output codes[END_REF]. The phonological component is assessed with a pseudoword repetition task including four pseudowords (mean number of syllables: 3.5 Ϯ 1.29) that the participant is asked to repeat. The lexical component is assessed with a repetition task including four long low-frequency words (mean number of syllables: 4 Ϯ 0.81; mean frequency: 0.17 Ϯ 0.28). The morpho-syntactic component is assessed with a repetition task including four long sentences (mean number of words: 10.75 Ϯ 1.26; mean number of syllables: 15.25 Ϯ 2.36) with noncanonical constructions: relative clause, subject inversion, interrogative or passive construction, and a nonplausible sentence.

Participants

Consecutive sampling of patients with neurological disorders (n ϭ 101) and healthy volunteers with no known neurological disorders (n ϭ 88) was conducted, respectively, in the Neurology Department of Créteil Hospital (France) and in Paris, between October 2011 and June 2015. All patients and healthy participants were tested with the CALAP (n ϭ 189, 82 men; M age ϭ 49.1 Ϯ 18.4 years; Table 1). For inclusion, the patients had to be French speakers over the age of 18 years. Severe dementia was an exclusion criterion. The causes of patients' neurological disorders are reported Figure 2. The CALAP was either administrated by a speech-language pathologist (n ϭ 4), a neurologist (n ϭ 1), or a neuropsychologist (n ϭ 2). None of them received training for CALAP administration. All patients attended a consultation with a speech-language pathologist at hospital. They were classified as patients unequivocally without aphasia (n ϭ 29) or as patients with possible aphasia (n ϭ 72). The latter underwent the French version of the BDAE [START_REF] Mazaux | HDAE, échelle d'évaluation de l'aphasie adaptation française du Boston diagnostic aphasia examination de H[END_REF], used as the gold 1 Familiarity value refers to the familiarity of the concept depicted, rated on a 5-point scale (1 ϭ very unfamiliar object, 5 ϭ very familiar object).

2 Imageability value refers to the mental image of the concept depicted, rated on a 5-point scale (1 ϭ very difficult and slow construction of the mental image, 5 ϭ very easy and quick construction of the mental image).

3 Emotional value refers to the emotional valence of the concept depicted, rated on a 5-point scale (1 ϭ very unfamiliar object, 5 ϭ very familiar object). This document is copyrighted by the American Psychological Association or one of its allied publishers.

This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.

Statistical Methodology

In this study, all participants performed both the CALAPscreening and CALAP-detailed evaluation. The scores of participants with aphasia (n ϭ 44) and without aphasia (n ϭ 145) were compared in Kruskal-Wallis rank-sum tests. The effect of age and educational level on CALAP-screening and CALAP-detailed evaluation scores was assessed in healthy participants, with Pearson's correlation analyses (two-tailed, n ϭ 88). The validation process is displayed in Figure 2. All analyses were performed with R.3.3.2 software.

External Validity

Aphasia/no aphasia status from BDAE outcome was used to determine the cutoff score for CALAP-screening by receiver operating characteristic (ROC) analysis (n ϭ 189); sensitivity and specificity were calculated as proportions, with 95% bootstrap (2,000 resamples) confidence intervals (CIs).

Concurrent Validity

We assessed the concordance between CALAP-detailed evaluation and BDAE scores, by calculating Pearson correlation coefficients between the CALAP and BDAE scores, separately for the APA NLM comprehension, production, and repetition tasks on the subgroup of patients who were tested both with the CALAP and the BDAE (two-tailed, n ϭ 72 patients, including 44 patients with aphasia).

Internal Validity

We assessed the correlation between the CALAP-screening and CALAP-detailed evaluation scores, by calculating Pearson correlation coefficients (two-tailed, n ϭ 189) between the scores for corresponding tasks in the screening and detailed evaluation (e.g., between the comprehension score in screening and the comprehension score in the detailed evaluation, etc.).

Internal Consistency

Principal component analysis (PCA) was used to explore the relationship between the CALAP-screening tasks with no a priori hypothesis and data reduction possibility. We assessed the weight of each task (comprehension, production, repetition) on scaled and centered data using singular value decomposition. The internal consistency of CALAP-screening, a measurement of reliability based on the internal correlation of screening items, was assessed by calculating Cronbach's alpha (pairwise, n ϭ 189). Internal consistency was also used to measure the correlation between the different items of the CALAP-detailed evaluation. It was assessed by calculating Cronbach's alpha for all tasks of the detailed evaluation, together and separately: digit span, concept, comprehension, production, and repetition.

Interrater Reliability

Two experimenters rated the responses of a subgroup of participants simultaneously and independently: one experimenter administered the CALAP to the participant, and both rated the responses obtained (n ϭ 22 participants, including seven patients with aphasia). The aphasic status of the participants was masked to the raters. The intraclass correlation coefficient (ICC) was calculated to evaluate interrater reliability.

Results

The mean duration of the CALAP-screening was 3.14 Ϯ 1.23 min and the total duration of the CALAP (screening and detailed evaluation) was 11.39 Ϯ 4.1 min (Figure 1B). All scores for both the CALAP-screening and CALAP-detailed evaluation (digit span, concept, comprehension, production, and repetition) were lower in participants with aphasia than in participants without aphasia (Table 2). Age and education had no significant effect on CALAP-screening scores (Pearson correlation coefficient for age: Ϫ0.04, 95% CI [Ϫ0.25, 0.17]; p ϭ .8, and for education: 0.09, 95% CI [Ϫ0.12, 0.3]; p ϭ .4), nor on CALAP-detailed evaluation scores (Pearson correlation coefficient for age: Ϫ0.14, 95% CI [Ϫ0.2 -0.17]; p ϭ .1, and for education: 0.09, 95% CI [Ϫ0.12, 0.3]; p ϭ .2).

External Validity

The ROC analysis identified a cutoff score of 22 (Figure 3) as the best compromise between sensitivity (the ability to identify patients with aphasia correctly; 1, 95% CI [1, 1]) and specificity (the ability to identify patients without aphasia correctly, 0.72, 95% CI [0.65, 0.79]).

Concurrent Validity

The CALAP-detailed evaluation and BDAE scores were highly correlated. Pearson's correlation coefficient4 was 0.62, 95% CI [0.46, 0.75], for comprehension; 0.83, 95% CI [0.75, 0.89], for production; and 0.74, 95% CI [0.61, 0.83], for production (all p values Ͻ0.001).

Internal Validity

The CALAP-screening and CALAP-detailed evaluation scores were highly correlated. Pearson's correlation coefficient5 was 0.56, 95% CI [0.45, 0.65], for comprehension; 0.77, 95% CI [0.71, 0.82], for production; and 0.74, 95% CI [0.61, 0.83], for repetition (all p values Ͻ0.001).

Internal Consistency

For CALAP-screening, the first two components of the PCA (eigenvalues, 2.26 and 0.52), accounted for 93% of the total variance. Each task contributed equally (comprehension Ϫ0.53, production: Ϫ0.59, repetition: Ϫ0.61) to the first principal component (accounting for 76% of the variance). The second component only accounted for 17% of the variance with a larger contribution of the comprehension component (comprehension Ϫ0.84, production: 0.47, repetition: 0.27).

Internal consistency was good for the CALAP-screening score (Cronbach's alpha: 0.88, 95% CI [0.8, 0.86]), and excellent for the CALAP-detailed evaluation score (Cronbach's alpha: 0.94, 95% CI [0.93, 0.95]; [START_REF] Kline | The handbook of psychological testing[END_REF][START_REF] Taber | The use of Cronbach's alpha when developing and reporting research instruments in science education[END_REF]. Calculated separately for the various tasks, Cronbach's alpha was 0.85, 95% CI [0.80, 0.89], for digit span; 0.45, 95% CI [0.34, 0.56], for concept; 0.85, 95% CI [0.83, 0.88], for comprehension; 0.80, 95% CI [0.76, 0.85], for production; and 0.94, 95% CI [0.92, 0.95], for repetition.

Interrater Reliability

Interrater reliability was excellent for CALAP-screening score (ICC: 0.99, 95% CI [0.98, 0.99], p Ͻ .001) and CALAP-detailed evaluation6 scores (ICC of 0.99, 95% CI [0.99, 0.99] for digit span; 0.95, 95% CI [0.88, 0.98] for comprehension; 0.95, 95% CI [0.88, 0.98] for production; and 0.99, 95% CI [0.99, 0.99] for repetition; all p values Ͻ0.001).

Discussion

Efficient and effective functional diagnosis of aphasia remains an unsolved issue. We designed the CALAP, a new short and easy-to-use scale, usable by clinicians nonexpert in language as-

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sessment to detect language disorders, to identify the impaired component within a functional model of language processing in less than 12 min. We showed that the CALAP-screening detects 100% of patients with aphasia in the sample we studied. The psychometric properties of CALAP-screening and CALAPdetailed evaluation scores (external and concurrent validity with the BDAE, internal validity and consistency for screening and detailed evaluation scores, and reliability across raters) were found to be very good.

The CALAP is a new tool that can be used by neurologists, neuropsychologists, and speech-language pathologists to guide clini-cal practice in diverse contexts. The CALAP-screening is easy to use for the rapid diagnosis of aphasia in clinical settings. The CALAP-detailed evaluation, with its theoretical basis, is useful for both clinical care and follow up. The CALAP-detailed evaluation scores at each subtask (phonology, morphology, lexicon, syntax in comprehension, production, and repetition modalities) have the potential to determine the relative strengths and weaknesses of patients for these various language components and may guide further potential extensive assessment by a speech-language pathologist. For instance, if a patient has a low score in the CALAP subtask "syntax in comprehension," it will be worth further exploring the syntactic deficit through standardized tests focused on syntax comprehension. The CALAP-detailed evaluation can also be used by speech-language pathologists to guide the design of deficit-based rehabilitation. Indeed, the CALAP, which is based on a functional model of language processing, goes beyond the aphasia syndromes of the Broca-Wernicke classification. This previous classification provided a starting point for understanding aphasia. Although it helped for developing therapeutic interventions, as the practice of rehabilitation has proceeded for many years on the basis of this classification, it was not the most effective way [START_REF] Marshall | Classification of aphasia: Are there benefits for practice[END_REF][START_REF] Nickels | Therapy for naming disorders: Revisiting, revising and reviewing[END_REF][START_REF] Nickels | Aphasic naming: What matters?[END_REF]. For instance, in a patient with a Wernicke profile, several components can be impaired (lexicon in comprehension, syntax in comprehension, lexicon in production . . .) but will not be specifically identified with the classical scales. In contrast, rather than classifying patients into Broca-Wernicke syndromes, the CALAP allows identification of language components which are impaired. As rehabilitation of patients with aphasia is more effective when focused specifically on the impairment [START_REF] Jacquemot | Specificity in rehabilitation of word production: A meta-analysis and a case study[END_REF], the CALAP outcome might help in developing a more effective rehabilitation program. For example, if syntax in comprehension is impaired, or at least more impaired than the other components assessed by the CALAP, rehabilitation will be more effective if it CI [94.2,98.7]). This document is copyrighted by the American Psychological Association or one of its allied publishers.

This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.

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focuses on tasks of sentence comprehension which modulate syntactic complexity (active/passive forms, affirmative/interrogative/ negative forms, etc.). By contrast, if "phonology in comprehension" is impaired, rehabilitation will be more effective if it focuses on tasks of increasing difficulty involving pseudoword perception (number of syllables, number of phonemes, phonological complexity, etc.). The CALAP, which can be administrated in one session by clinicians not previously trained and nonexpert in language assessment, is more user-friendly than other scales, such as the BDAE, the Montreal Toulouse 86 [START_REF] Nespoulous | Protocole Montréal-Toulouse d'examen linguistique de l'aphasie: MT-86 module standard initial, M1b. Isbergues[END_REF], or the Comprehensive Aphasia Test [START_REF] Swinburn | The Comprehensive Aphasia Test[END_REF], all of which must be administered by experts in language assessment over several sessions. In addition, in contrast to other scales, the CALAP focuses on the evaluation of oral language without requiring any written response. It can therefore be used for patients with writing difficulties, as frequently observed in patients with left hemisphere lesions [START_REF] Denier | Aphasia in stroke patients: Early outcome following thrombolysis[END_REF]. The short duration of the evaluation also makes it possible to test patients with fatigue and concentration difficulties unable to complete longer scales such as the BDAE.

In addition to aphasia, other disorders such as working memory or attention disorders may affect language performance [START_REF] Jacquemot | Misperception in sentences but not in words: Speech perception and the phonological buffer[END_REF][START_REF] Murray | The effects of varying attentional demands on the word retrieval skills of adults with aphasia, right hemisphere brain damage, or no brain damage[END_REF] and most forms of dementia eventually affect language [START_REF] Patterson | Where do you know what you know? The representation of semantic knowledge in the human brain[END_REF]. However, as the CALAP assesses conceptual knowledge, and working memory independently of language components, it can distinguish between different diseases in patients with brain impairments. For example, patients with aphasia would not be expected to perform poorly in the concept task, whereas semantic dementia patients will have lower scores for the concept task. Patients with working memory deficits are expected to have a lower score in the digit span task than patients with no working memory deficit, and patients with attention disorders would display a rather homogeneous pattern of impairment in all subtasks instead of an impairment focused on a specific subtask. Thus, these different patterns of impairment may be used for detecting disorders other than language deficit.

The CALAP is a valuable tool not only for research allowing clustering patients on their functional impairment rather than on the neurological usual classification, but also for the diagnosis and rehabilitation of language disorders. The CALAP can be administered during patient hospitalization, home visits, or in medical centers. The rapid diagnosis of aphasia and the identification of patient-specific language deficits should have a major impact on clinical practice and on the orientation of deficit-based rehabilitation. The short duration of the CALAP and its self-explanatory nature should make it possible to implement patient care more rapidly. This aspect is particularly important for patients with neurological disorders, in whom interventions must be initiated as soon as possible [START_REF] Dobkin | Rehabilitation after stroke[END_REF][START_REF] Laska | A randomized controlled trial on very early speech and language therapy in acute stroke patients with aphasia[END_REF]. Thus, it can greatly improve routine patient care and positively impact the burden placed upon caregivers. Although the CALAP is an appropriate tool to use in French-speaking environments, the fact that this scale is based on a functional model of language processing, independent from any language, makes it readily adaptable for universal use.

  -assess/z1t-assess/z1t99918/z1t3441d18z xppws Sϭ1 11/22/18 15:56 Art: 2018-2302APA NLMstandard for aphasia assessment in this study. These patients were then classified as patients with aphasia (n ϭ 44) or not (n ϭ 28) on the basis of their BDAE results in the evaluation of oral comprehension, production, and repetition (Flamand-Roze et al., 2011; see Figure2). The local ethics committee approved the study and participants gave written informed consent.

Figure 3 .

 3 Figure3. Receiver operating characteristic (ROC) curve showing the sensitivity (true-positive rate) and 1-specificity (false-positive rate) for CALAP screening. The optimal cutoff point was 21.5. The area under the curve was estimated at 96.4 (95%CI [94.2, 98.7]). This document is copyrighted by the American Psychological Association or one of its allied publishers.This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.

Table 1

 1 Demographic Data for the Participants Design of the validation study and cause of the neurological disorder in patients with and without aphasia (n ϭ 101). This document is copyrighted by the American Psychological Association or one of its allied publishers.This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.

	Participants	Status	N	Age	Education level	Laterality (right-handed)
	Neurology patients	Aphasia	44	64.8 (15.3)	11 (3.6)	38
		No aphasia	57	53.1 (14.8)	12.6 (3.2)	51
	Healthy participants	No aphasia	88	38.7 (15.2)	14.3 (2.3)	81

Table 2

 2 Mean (SD) Scores for CALAP Screening and Detailed Evaluation by Aphasia Status (No Aphasia and Aphasia)Note. The maximum score is reported in parentheses for each task: Screening, Digit span, Concept, Comprehension, Production, and Repetition and for each subtask (in italics). For participants without aphasia, the 95% confidence interval is reported in brackets. All the components assessed in the detailed evaluation are reported. The p values presented are those for Kruskal-Wallis tests for an effect of group (aphasia/no aphasia status) for the various components of the Core Assessment of Language Processing (CALAP).

	CALAP	No aphasia (n ϭ 145) Aphasia (n ϭ 44) Kruskal-Wallis 2	p value
	Screening total (24 pts)	23.0 (1.6) [22.7, 23.3]	15 (5)	96.2	p Ͻ .001
	Digit span total (17 pts)	10.0 (2.4) [9.6, 10.4]	6.2 (2.3)	63.5	p Ͻ .001
	Concept total (8 pts)	8 (.0) [8, 8]	7.5 (1.2)	22.9	p Ͻ .001
	Comprehension total (48 pts) 47.5 (1.1) [47.3, 47.7] Lexicon (12 pts) 11.8 (.7) [11.7, 11.9] Morphology (12 pts) 11.9 (.4) [11.8, 12.0] Phonology (12 pts) 11.8 (.5) [11.7, 11.9] Syntax (12 pts) 12.0 (.2) [12, 12]	41.2 (6.6) 10.4 (1.9) 10.4 (1.9) 10.1 (2.5) 10.3 (2.1)	80.3 44.1 54.9 41.6 85.0	p Ͻ .001 p Ͻ .001 a p Ͻ .001 a p Ͻ .001 a p Ͻ .001 a
	Production total (16 pts) Lexicon (8 pts) Morpho-syntax (8 pts)	15.2 (1.3) [15.0, 15.4] 7.5 (1.0) [7.3, 7.7] 7.8 (.8) [7.7, 7.9]	9.7 (4.7) 4.5 (2.5) 5.2 (2.9)	74.3 67.1 64.7	p Ͻ .001 p Ͻ .001 a p Ͻ .001 a
	Repetition total (24 pts) Lexicon (8 pts) Phonology (8 pts) Morpho-syntax (8 pts)	23.1 (1.6) [22.8, 23.4] 7.9 (.5) [7.8, 8.0] 7.7 (.6) [7.6, 7.8] 7.5 (1.0) [7.3, 7.7]	12.5 (8.1) 5.2 (3.0) 4.5 (3.0) 2.8 (2.7)	86.9 61.6 65.5 93.1	p Ͻ .001 p Ͻ .001 a p Ͻ .001 a p Ͻ .001 a

a Corrected for multiple comparison.

Spearman correlation coefficients are reported in Table 1 in the online supplemental materials.

Spearman correlation coefficients are reported in Table 1 in the online supplemental materials.

Since concept was equally rated by the two experimenters for all the participants and the standard deviation equal to zero, the ICC could not be calculated. This document is copyrighted by the American Psychological Association or one of its allied publishers.This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.


Becerril Ortega

Dr Helene Hirbec
email: helene.hirbec@montp.inserm.fr

Keywords: SCI, spinal cord injury sEPSC, spontaneous excitatory postsynaptic current SynNMDAR, synaptic NMDA receptor TTX, tetrodotoxin Excitotoxicity, NMDA antagonist, recombinant NMDA receptor, whole cell recording, Gacyclidine (GK11)

come

Comparison of the pharmacological properties of GK11 and MK801, two NMDA receptor antagonists: towards an explanation for the lack of intrinsic neurotoxicity of GK11.

INTRODUCTION

Two decades of pharmacological, physiological, and genetic studies have established that NMDA receptors (NMDARs), a subtype of ionotropic glutamate receptors, play a key role in physiological and pathological processes [START_REF] Dingledine | The glutamate receptor ion channels[END_REF]. Activation of NMDARs is thus required for numerous fundamental physiological processes related to synaptic plasticity including learning, memory, long-term potentiation and long-term depression [START_REF] Collingridge | Memories of NMDA receptors and LTP[END_REF]. However, excessive activation of NMDARs can generate an uncontrolled influx of calcium and start off the excitotoxic cascade that is thought to contribute to neuronal cell injury and death in many different acute and chronic neurologic diseases including stroke, traumas, Parkinson's and Alzheimer's diseases [START_REF] Doble | Excitatory amino acid receptors and neurodegeneration[END_REF][START_REF] Arundine | Molecular mechanisms of calcium-dependent neurodegeneration in excitotoxicity[END_REF]. Several studies based on in vitro and in vivo models of ischemic and traumatic brain injury have demonstrated that neuronal cell death is primarily mediated through NMDARs and that blocking NMDARs during or shortly after an excitotoxic event can prevent much of the neuronal death that would have otherwise occurred [START_REF] Rothman | Glutamate and the pathophysiology of hypoxic-ischemic brain damage[END_REF][START_REF] Clark | Blockade of excitatory amino acid receptors protects anoxic hippocampal slices[END_REF][START_REF] Choi | Glutamate neurotoxicity and diseases of the nervous system[END_REF][START_REF] Olney | NMDA antagonist neurotoxicity: mechanism and prevention[END_REF]. Accordingly, both competitive and non-competitive NMDAR antagonists have been investigated as potential neuroprotective agents in clinical trials for different CNS pathologies [START_REF] Davies | Actions of D and L forms of 2-amino-5phosphonovalerate and 2-amino-4-phosphonobutyrate in the cat spinal cord[END_REF][START_REF] Davies | CPP, a new potent and selective NMDA antagonist. Depression of central neuron responses, affinity for [3H]D-AP5 binding sites on brain membranes and anticonvulsant activity[END_REF][START_REF] Priestley | Electrophysiological characterisation of the antagonist properties of two novel NMDA receptor glycine site antagonists, L-695,902 and L-701[END_REF]. However, these trials did not live up the preclinical expectations as they were inefficient and/or had deleterious side effects [START_REF] Muir | Clinical experience with excitatory amino acid antagonist drugs[END_REF][START_REF] Lees | Neuroprotection[END_REF][START_REF] Sacco | Glycine antagonist in neuroprotection for patients with acute stroke: GAIN Americas: a randomized controlled trial[END_REF]. Indeed administration of these compounds in man classically induced cardiovascular as well as psychotropic adverse effects [START_REF] Leppik | Consensus statement on compliance in epilepsy[END_REF]. Additionally, further pre-clinical studies demonstrated that single dose administration of NMDAR antagonists in rats leads to neuronal degeneration in the cingulate cortex [START_REF] Olney | Pathological changes induced in cerebrocortical neurons by phencyclidine and related drugs[END_REF][START_REF] Olney | Excitotoxic amino acids and neuropsychiatric disorders[END_REF][START_REF] Olney | NMDA antagonist neurotoxicity: mechanism and prevention[END_REF].

NMDARs are proposed to be tetrameric protein complexes comprised a NR1 subunit with at least one type of NR2 subunit. Different NR2 subunits confer distinct electrophysiological and pharmacological properties on the receptors and couple them with different signalling machineries involved in specific physiological and pathophysiological pathways [START_REF] Monyer | Developmental and regional expression in the rat brain and functional properties of four NMDA receptors[END_REF][START_REF] Cull-Candy | NMDA receptor subunits: diversity, development and disease[END_REF][START_REF] Vanhoutte | Opposing roles of synaptic and extrasynaptic NMDA receptors in neuronal calcium signalling and BDNF gene regulation[END_REF][START_REF] Liu | Switching of NMDA receptor 2A and 2B subunits at thalamic and cortical synapses during early postnatal development[END_REF]). Moreover recent experiments suggest that synaptic and extrasynaptic NMDA receptors may be enriched in respectively the NR2A and NR2B subunits [START_REF] Liu | NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo[END_REF], and present distinct roles in synaptic plasticity, coupling to intracellular pathways, and apoptotic or necrotic cell death [START_REF] Rumbaugh | Distinct synaptic and extrasynaptic NMDA receptors in developing cerebellar granule neurons[END_REF][START_REF] Tovar | The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro[END_REF]Hardingham and Bading 2002;Hardingham et al. 2002;[START_REF] Sinagra | Reelin, very-low-density lipoprotein receptor, and apolipoprotein E receptor 2 control somatic NMDA receptor composition during hippocampal maturation in vitro[END_REF][START_REF] Ivanov | Opposing role of synaptic and extrasynaptic NMDA receptors in regulation of the extracellular signal-regulated kinases (ERK) activity in cultured rat hippocampal neurons[END_REF]. Because forebrain NMDARs principally include NR2A and/or NR2B subunits [START_REF] Sheng | Changing subunit composition of heteromeric NMDA receptors during development of rat cortex[END_REF][START_REF] Takai | Distribution of N-methyl-D-aspartate receptors (NMDARs) in the developing rat brain[END_REF], the development of antagonists selective for the NR2B subunit has also been suggested [START_REF] Gill | Pharmacological characterization of Ro 63-1908 (1-[2-(4-hydroxy-phenoxy)-ethyl]-4-(4-methyl-benzyl)-piperidin-4-ol), a novel subtype-selective N-methyl-D-aspartate antagonist[END_REF]).

In the early eighties', [START_REF] Kamenka | Chemical synthesis and molecular pharmacology of hydroxylated 1-(1phenylcyclohexyl-piperidine derivatives[END_REF] reported the synthesis of a new NMDAR antagonist derived from the phencyclidine (PCP) structure: GK11. Pharmacological studies then revealed that this compound is a channel blocker the binding site of which overlaps that of the classical NMDAR uncompetitive antagonist MK801 [START_REF] Hamon | The search for TCP analogs binding to the low affinity PCP receptor sites in the rat cerebellum[END_REF]Hirbec et al. 2000a;Hirbec et al. 2000b). GK11 was shown to display a relative high affinity for telencephalic NMDARs with Kd in the nanomolar range (Hirbec et al. 2000a;Hirbec et al. 2000b). In agreement with these data, GK11 has potent neuroprotective properties both in vitro [START_REF] Drian | In vitro neuroprotection against glutamate toxicity provided by novel non-competitive N-methyl-D-aspartate antagonists [In Process Citation[END_REF]) and in vivo (Hirbec et al. 2001a). In either contusive or photochemical rat models of spinal cord injury (SCI), GK11 efficiently prevented both the functional and morphological impairments associated with SCI (Gaviria et al. 2000a;Gaviria et al. 2000b).

GK11 also displayed beneficial effects in brain trauma [START_REF] Smith | Effects of the novel NMDA receptor antagonist gacyclidine on recovery from medial frontal cortex contusion injury in rats[END_REF]. Very interestingly and unlike the other high affinity NMDAR antagonists, preliminary experiments have shown that GK11 is nearly devoid of any intrinsic neurotoxicity even at extremely high doses (20mg/kg i.v.) (Hirbec et al. 2001b). This was further confirmed in a much more extensive study aimed at comparing the intrinsic neurotoxicity of GK11 and MK801 using a wide range of behavioural and histological techniques (Hirbec et al. unpublished results). The safe pharmacological profile of GK11 was also confirmed by the results from phase I & IIb clinical trials in which GK11 was assessed for SCI treatment [START_REF] Lepeintre | Neuroprotective effect of gacyclidine. A multicenter double-blind pilot trial in patients with acute traumatic brain injury[END_REF]).

The present study was designed to determine whether the special properties of GK11, in particular its safer pharmacological profile compared with MK801, could be explained by different molecular mode of action of these two compounds at NMDARs. As a first step we therefore compared, on sister hippocampal cultures, both the neuroprotective and intrinsic neurotoxic effects of both drugs. By using patch-clamp techniques, we also compared their respective effects on native NMDARs expressed in hippocampal neurons. Then, to probe the selectivity of GK11 and MK801, we measured their effects on recombinant NR1a/NR2A and NR1a/NR2B receptors expressed in HEK293 cells and showed that in contrast with MK801, GK11 did not totally block the currents mediated through the NR2A-containing receptors.

Finally, we used specific electrophysiological approaches and calcium imaging on native neurons to understand the significance of GK11 subtype selectivity. Here we demonstrate that GK11 preferentially blocks extrasynaptic NMDARs while partially preserving synaptic communication. Our results show that although at first sight both drugs seem to have comparable effects on native NMDARs, their subunit selectivity may account, at least partly, for their differences in terms of pharmacological tolerance.

MATERIALS AND METHODS

PRIMARY CULTURE OF NEURONAL CELLS

Hippocampal culture

Hippocampal cultures were prepared as previously described [START_REF] Noel | Surface expression of AMPA receptors in hippocampal neurons is regulated by an NSF-dependent mechanism[END_REF]. Briefly, hippocampal neuronal cells were isolated from embryonic rats (E18) and plated on either poly-D-Lysine (Sigma-Aldrich, Saint Louis, MO, USA) and laminin (Invitrogen, Cergy Pontoise, France) treated plastic plates (electrophysiological studies) or poly-D-lysine and laminin treated glass coverslips (neuroprotection and neurotoxicity studies) using Neurobasal medium (Invitrogen) supplemented with 1% L-Glutamine (Invitrogen), 2% B27 (Invitrogen) and 3% foetal bovine serum (Invitrogen). Cultures were grown at 35°C in a humidified atmosphere containing 5% CO 2 , and from the third day in culture, the medium was supplemented with 5 µM cytosine-b-D-arabinofuranoside (ARAC; Sigma-Aldrich).

Hippocampal cultures were prepared either at low density (125 cell/mm 2 ) for electophysiological studies or high density (1000 cell/mm 2 ) for neuroprotection, intrinsic neurotoxicity studies and spontaneous activity recordings. The cultures were used for experiments 12-14 days after plating for all experiments except for spontaneous activity.

Indeed, for such experiment we observed that when cultures were kept for 19-21 days in vitro (DIV), the frequency of the spontaneous events was increased a fact that is likely to be associated with a better developed neuritic network.

Cortex culture

Primary cortical cultures were prepared from foetal mice at 15-16 days gestation as previously described [START_REF] Rose | Cytotoxicity in murine neocortical cell culture[END_REF]. Cerebral cortices were then dissected in DMEM (Sigma-Aldrich). Dissociated cortical cells were re-suspended in DMEM supplemented with 5% foetal bovine serum, 5% horse serum and 2 mM glutamine and plated in glass Petri dishes coated with poly-D-lysine and laminin. Cultures were kept at 37°C in a humidified atmosphere containing 5% CO 2 . Experiments of calcium recordings were performed after 14 DIV.

PHARMACOLOGICAL TREATMENTS

Neuroprotection studies

To assess the neuroprotective properties of the drugs, 12-14 DIV hippocampal cultures were challenged with Neurobasal medium devoid of serum and containing 500 µM glutamate (adapted from [START_REF] Drian | In vitro neuroprotection against glutamate toxicity provided by novel non-competitive N-methyl-D-aspartate antagonists [In Process Citation[END_REF]. After 5 min of incubation, the culture medium was replaced with Neurobasal containing the neuroprotective drug at the appropriate concentration.

Cultures were then returned to the incubator. The two drugs used in the present study were GK11 (gacyclidine) and MK801, were tested at concentrations ranging between 100 nM and 10 µM. After 24h, the cells were fixed for immunocytochemistry by immersion of the coverslips for 45 min at room temperature in 4% paraformaldehyde in 0.1 M phosphate buffer. Labelling of the surviving neurones was performed as followed: after 10 min treatment with 1% H 2 O 2 , cells were incubated overnight at 4°C with monoclonal anti-Map2 (clone HM-2, 1:1000; Sigma-Aldrich) antibody prepared in PBS containing 2% BSA and 0.1% Triton X-100. The secondary antibody was peroxidase-coupled anti-mouse antibody (1:500; Jackson ImmunoResearch Laboratories, West Grove, PA, USA), revelation was performed with 0.2% DAB in 0.1 M Tris Buffer.

The number of surviving neurones, as assessed by counting the density of Map 2 positive cells, was compared to both sham cultures (i.e. those submitted to the same number of medium changes as the experimental protocol), and to control cultures that were kept untouched. Counts were performed on at least 3 wells per condition and in 3 independent series of experiments, using the Samba image analysis software (Samba technologies, France). Results are means ± SEM and statistical analyses were performed using the oneway ANOVA statistical test.

Intrinsic neurotoxicity studies

To assess the potential adverse effects of GK11 and MK801 on hippocampal neuron viability, 12-14 DIV hippocampal cultures were challenged with 10 µM and 100 µM GK11 or MK801 by directly adding the compounds to the culture medium. Control cultures received an equivalent volume (50 µl) of water. Cultures were then returned to the incubator. After 24h, they were fixed for immunocytochemistry and further processed for Map 2 labelling as previously described. Neuronal viability was assessed by measuring the percentage of Map 2 immunopositive surface area with Samba image analysis software. Compared to neurone counting, this parametre allowed us to quantify not only neuronal survival but also to evaluate their health. A decrease in the percentage of immunolabelled surface may reveal either neuronal death (loss of neurones) or shrinkage and reorganization of the neuritic arborization which can be interpreted as a sign of neuronal suffering. Quantification was performed on at least 3 wells per condition and in 4 series of independent experiments. Results are means ± SEM and statistical analyses were performed using the one-way ANOVA statistical test.

ELECTROPHYSIOLOGICAL RECORDINGS

NMDA-induced current recordings

Whole cell patch recordings were performed at a -70 mV holding potential, using an Axopatch 200B amplifier (Molecular Devices Corp., Sunnyvale, CA, USA). Ionic currents were recorded using the following extracellular solution maintained at room temperature (mM): NaCl (140), KCl (5), CaCl 2 (2), Hepes (10), Glucose (20), glycine (0.01), pH = 7.35 (adjusted with NaOH). Mg 2+ was omitted to prevent voltage-dependent block of NMDAR operated channels [START_REF] Mayer | Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones[END_REF]. Voltage-activated Na + channels were blocked by adding to this solution 0.5 µM tetrodotoxin (TTX; Latoxan, Valence, France), AMPA/kainate glutamate receptors were inhibited by adding 1 µM NBQX (Sigma-Aldrich) and glycine receptors were inhibited by adding 1 µM strychnine (Sigma-Aldrich). Recording pipettes (2-4 MΩ) were pulled from microhematocrit tubes (Modulohm A/S, Herlev, Denmark) and filled with the following solution (mM): CsCl (136), Hepes (25), Glucose (10), EGTA (10), Mg-ATP (3), Na-GTP (1), pH = 7.35 (adjusted with CsOH). The osmolarity of all buffers used in this study was 310 mOsm. The recording chamber was continuously superfused with the extracellular solution using a custom-made perfusion system with an outflow of 250µl/min. NMDA-induced currents were elicited every 2 min by a standard 5 second application of 100 µM NMDA with an outflow of 500µl/min. Tested substances (GK11, MK801 and DAPV) were diluted in the perfusion medium. They were applied at the appropriate concentrations in presence of 100 µM NMDA by switching channels of the perfusion system.

Signals were filtered at 5 kHz, digitized at 10 kHz, and stored on-line using pCLAMP 6 (Molecular Devices Corporation, Sunnyvale, CA, USA). Inhibition of NMDA-induced current by the antagonist at a given concentration was measured either as the total area (recordings on hippocampal neurones) or at the maximum value of the peak (recordings on HEK293 cells). It was expressed as percentage of the current elicited by NMDA only (mean of 3 successive evoked responses). Data reported are means ± SEM of the indicated number of individual determinations. IC 50s were calculated by fitting the data to either formula a (single binding site) or formula b (two binding sites) using SigmaPlot 10.0 software (Formula a: I(x) = Ax/(IC 50 +x); Formula b: I(x) = (A1x/(IC 50_1 +x)) +(A2x/(IC 50_2 +x)); where: I is the inhibition percentage of the NMDA-induced current, x is the concentration of the antagonist (mol.l -1 ), A is the maximum response and IC 50 is the half-maximal inhibition concentration. IC 50 were reported as means ± SE. To estimate whether the double sigmoidal fitting (two binding sites) was more probable that the single sigmoidal fitting (single binding site), we compared the square residues using a t-test.

Spontaneous excitatory postsynaptic current (sEPSC) recording

The compositions of the extracellular and intracellular medium used were the same as described above. Hippocampal neurones were voltage-clamped at -70mV and sEPSC recorded in the presence of 2 µM NBQX (Sigma-Aldrich) and 2 µM Gabazine (SR-95531; Sigma-Aldrich) to eliminate the AMPA-Kainate and GABA A -receptor mediated spontaneous currents respectively. EPSC were filtered at 5 kHz, sampled at 10 kHz. Drugs were applied for 80s via the perfusion system. For the calculation of the frequency and the amplitude of bursts, events were analysed for 40s before and for the last 40 seconds of the drug application.

Calcium Imaging

Primary cortical neurone cultures were loaded for 45 min at 37°C with 10 µM fura-2/AM and 0.2% pluronic acid (F-127; Invitrogen) and incubated for an additional 15 min at room temperature in HEPES and Bicarbonate -Buffered Saline Solution (HBBSS; Invitrogen) containing (in mM) 116 NaCl, 5.4 KCl, 1.8 CaCl 2 , 0.8 MgSO 4 , 1.3 NaH 2 PO 4 , 12 HEPES, 5.5 Glucose, 25 bicarbonates and 10 µM glycine at pH= 7.45. Experiments were performed at room temperature with continuous perfusion at 2 ml/min with a peristaltic pump, on the stage of an inverted Nikon Eclipse microscope equipped with a 100 W mercury lamp and oil immersion Nikon 40X objective with 1.4 numerical aperture. Fura-2 (excitation: 340/380nm, emission: 510 nm) ratio images were acquired every 2 seconds with a CCD camera (Princeton Instruments, Trenton, NJ, USA) and digitized (512 x 512 pixels) using Metafluor 6.3 (Universal Imaging Corporation, Chester, USA). Fluorescence ratio (340/380 nm) were converted to intracellular Ca 2+ concentrations using the following formula:

[Ca 2+ ] i = Kd[(R- Rmin)/(Rmax-R)] F 0 /F s ,
where R is the observed 340/380 fluorescence ratio, R min is the ratio for a Ca 2+ -free solution, R max is the ratio for a saturated Ca 2+ solution, Kd=135nmol/l (the dissociation constant for fura-2), F 0 is the intensity of a Ca 2+ -free solution at 380 nm, and F s is the intensity of a satured Ca 2+ solution at 380 nm. The recordings of selective synaptic or extrasynaptic activities are well described by Hardingham et al. [START_REF] Hardingham | Nuclear calcium signaling controls CREB-mediated gene expression triggered by synaptic activity[END_REF]Hardingham et al. 2002). In brief, the synaptic activity was induced by blocking Human embryonic kidney 293 cells (HEK293) were grown in Dublecco's medium (Invitrogen)

supplemented with 1% L-glutamine (Invitrogen), 1% penicillin/streptomycin (Sigma-Aldrich) and 10% foetal bovine serum (Invitrogen). Exponentially growing cells were plated on 12-mm glass coverslips (Marienfeld GmbH, Lauda-Königshofen, Germany) coated with poly-D-

Lysine and grown at 37°C in a humidified atmosphere containing 5% CO 2 . 24 hours after plating, cells were transiently transfected with a total of 0.5 µg DNA using 1.5 µl of FuGENE 6 transfection reagent (Roche, Meylan, France). DNA ratios were NR1a:NR2X (X=A or B) :GFP = 10:10:1. Co-transfection with GFP allowed ready recognition of the transfected cells.

Additionally, after transfection, cells were treated with 100 µM D-APV to prevent cell death.

Studies on the recombinant receptors were performed 24 hours after transfection.

Characterization of the recombinant NMDAR subunits

Characterization of our recombinant model showed that transfection efficiency for the three DNAs was about 11% (data not shown), and that 81% of the GFP-expressing cells mediated NMDA evoked responses indicating that functional ion channels had been expressed. When HEK293 cells were transfected with GFP or GFP/NR1a only, NMDA application did not elicit any current. In contrast, the maximum current peaks recorded on the cells expressing NR1a/NR2A and NR1a/NR2B recombinant receptors were 696±53 pA (n= 70) and 786±145 pA (n=16) respectively. These currents were totally blocked after addition of 500 µM D-APV to the extracellular medium (data not shown).

MATERIALS (drugs)

GK11 (cis(pip/me)-1-[1-(2-Thienyl)-2-methylcyclohexyl]piperidine, Gacyclidine) was a generous gift from Expansia (France). All other chemicals were obtained from commercial sources and were of the highest purity available (Sigma-Aldrich, Saint-Louis, MO, USA).

RESULTS

Pharmacological properties of MK801 and GK11 on native NMDA receptors

In vitro neuroprotective properties of GK11 and MK801

Previous studies from our laboratory have demonstrated that GK11 and MK801 displayed very similar neuroprotective properties on cultured cortical neurones challenged with a toxic dose of glutamate [START_REF] Drian | In vitro neuroprotection against glutamate toxicity provided by novel non-competitive N-methyl-D-aspartate antagonists [In Process Citation[END_REF]). To better interpret our functional experiments (see below), here we investigated whether it was reproduced on primary hippocampal cultures.

Sister control cultures only submitted to medium changes showed cell death which was much more pronounced than on cortical cultures reaching about 90% (p<0.001; Fig. 1A).

Treatment with 500 µM glutamate induced additional cell death with only 1.2±0.1% of surviving neurones (p<0.001 compared to both sham and control cultures). Treatment of the glutamate-challenged cultures with either MK801 or GK11 improved the survival rate of the neurones in a concentration dependant manner. The neuroprotective effects were significant from the lowest concentration tested (p<0.01 for both GK11 and MK801 treatments at 100nM). At the highest concentrations (10 and 100 µM), treatment with NMDAR antagonists exhibited strong neuroprotective effects with increased density of surviving neurones compared to sham cultures, and thus suggesting that at these concentrations, GK11 and MK801 protected both against the specific effects of glutamate treatment and the toxic consequences associated with medium changes. At 1 µM, the proportions of surviving neurones were 30.8±2.1% for GK11 and 38.9±1.7% for MK801 compared to control cultures (p<0.001), whereas at 10 µM they were 54.6±2.3% and 44.3±2.0% respectively (p<0.001).

As expected, our results show that both compounds had similar neuroprotective effects on hippocampal neurones.

In vitro intrinsic neurotoxicity of MK801 and GK11

To optimize the evaluation of GK11 and MK801 intrinsic neurotoxicity, we developed an in vitro pharmacological approach based on the measurement of the Map 2 immunopositive surface area (see materials and methods) to detect neuronal suffering after incubation in the presence of the drugs. Fig. 1B shows that after 24h, treatment with 10 µM MK801 led to a very significant reduction in the Map 2 immunopositive surface (82.4±2.3% compared to control cultures, p<0.001). This decrease may reveal both neuronal cell death and adverse effects on the cell viability. MK801 deleterious effects were further increased at 100 µM with a percentage of Map 2 immunopositive surface reduced to 77.9±3.1% compared to control cultures (p<0.001). In contrast, treatment of the cultures with equivalent doses of GK11 had no significant effect on neuronal viability. Indeed, treatment with GK11 did not significantly affect the percentage of surface labeled with Map 2 (compared to control cultures: 96.3±3.0% at 10 µM and 90.6±2.4% at 100 µM, p<0.001). When the cultures were challenged for longer time (i.e. 48h) with the drugs, the intrinsic neurotoxicity of MK801 was markedly increased.

Under these conditions and compared to control cultures, the percentage of surface area labeled with Map2 was reduced to 56.4±2.4% at 10 µM and 44.6±1.7% at 100 µM (Fig. 1C, p<0.001). Under light microscopy, examinations of the MK801-treated cultures revealed alterations in the neuritic field organization, with neurites either packed in coarse cables extending in between cell clumps (10 µM) or retracted around clumps (100 µM) (Fig. 1D).

Additionally, some strongly stained tightly packed pericarya revealed alterations in the neurons morphology. In comparison, extending the exposure time to GK11 had only minor effect on the neuritic network. Compared to control cultures, the surface area labeled with Map2 was only reduced to 83.0±2.7% at 10 µM and 70.8±2.1% at 100 µM, p<0.001).

Additionally, the effects of GK11 were always significantly lower than those of MK801 (p<0.01 whatever the concentration and the duration of exposure to the drug). GK11-treated cultures also appeared to be much healthier (Fig. 1D), with neurites being either well dispersed on the dish with a few lighty stained pericarya (10 µM) or slightly bundled inbetween flattened groups of moderately stained pericarya (100 µM). Interestingly, at 100 µM GK11 had significantly less adverse effects than 10 µM MK801 (p<0.05 and p<0.001 after 24h and 48h treatment respectively). The present data thus reveal that at the optimal neuroprotective concentration, MK801 exhibits significant intrinsic neurotoxicity while GK11

only weakly affected the cell viability.

Effects of MK801 and GK11 on NMDA-induced currents in hippocampal neurones

Using the patch-clamp technique in the whole-cell configuration, we recorded NMDA-evoked currents elicited by application of 100 µM NMDA and 10 µM glycine on 12-14 DIV hippocampal neurones. In the absence of NMDAR antagonists, the recordings displayed the typical pattern of NMDA-induced responses (Fig. 2), the maximum peak currents measured were 1146±49 pA (n=80). Responses were stable for at least 15 min (Fig. 3a). They were completely blocked in the presence of 500 µM D-APV (data not shown) and partially blocked in the presence of either 10 µM GK11 or MK801 (Figs 2a andb).

MK801 is known to act as an irreversible blocker of the NMDAR associated channel. To test whether GK11 displayed the same properties, we evaluated the recovery rate of the NMDAinduced responses after blockade with 10 µM GK11. As for MK801, GK11 mediated blockade persisted when the cell was washed with control solution for 5 min and then tested with subsequent applications of NMDA (Figs 2a andb). Under our experimental conditions, recovery of either MK801 or GK11 blockade was not faster when NMDA was applied continuously to the cell (Figs 2c andd). As compared to -70mV, when the cells were held at +30 mV, the blockade was decreased by about 2 fold, demonstrating some voltagedependant block for both compounds. Indeed, at -70 mV the NMDA-induced currents were blocked by 65.6±8.5% with MK801 and 45.1±4.5% with GK11 whereas at +30 mV they were blocked by 36.0±5.7% and 22.2±5.3% respectively (Figs 2e andf). However, whatever the holding potential or the antagonist, the blockade remained stable throughout the NMDA application (continuous perfusion). Thus our results show that as for MK801, GK11 remained trapped inside the channel and could be considered as a permanent channel blocker. As a result of this property, we used only one cell per concentration to build up the dose-response curves of GK11 and MK801.

As shown in Fig. 3b, increasing MK801 and GK11 concentrations reduced NMDA steady state currents in a dose-dependent manner. Both MK801 and GK11 presented doseresponse curves (Fig. 3c) that were statistically best fitted using a two site interaction model (p<0.0001, t-test). This suggested the presence of at least two different NMDAR populations on 2 week cultured hippocampal neurones: a high affinity (site 1) and a low affinity site (site 2) (Table 1). Analysis of the fitting parametres revealed that GK11 had a 5 fold lower affinity than MK801 on the low affinity site and only a two-fold weaker affinity on the high affinity site (Table 1). On the low affinity site, IC 50 values were 55.9±3.9 µM and 197± 31 µM for MK801

and GK11 respectively, whereas on the high affinity site, they were respectively of 105±54 nM and 262±23 nM. In addition, MK801 and GK11 blocks were complete at concentrations higher than 1mM. The present results thus confirm that both MK801 and GK11 are potent uncompetitive blockers of the NMDAR associated channel.

Pharmacological properties of MK801 and GK11 on recombinant NMDARs

Effects of MK801 and GK11 on NMDA-induced currents in recombinant NMDARs

To investigate whether MK801 and GK11 differences in terms of intrinsic neurotoxicity could be explained by subunit selectivity of these two compounds, we tested the effect of GK11 on NMDA-induced currents elicited on HEK293 cells transiently transfected with the NR1a/NR2A or the NR1a/NR2B NMDAR subtypes, and compared its dose-response curves to those of MK801. Whatever the subunit composition, increasing the concentrations of both NMDAR antagonists blocked NMDA-induced currents in a dose dependent manner. Doseresponse curves were statistically best fitted using a single site interaction model. However, Fig. 4 shows that the effects of two compounds differed according to the subunit composition. On NR1a/NR2B containing receptors, for concentrations above 1mM, both antagonists were able to totally inhibit NMDA-induced currents (Fig. 4b). On this receptor subtype, GK11 had a 6 fold weaker affinity than MK801 and IC 50s were 5.8±1.3 µM for MK801 and 37.4±8.7 µM for GK11 (Fig. 4b). On NR1a/NR2A containing receptors, both antagonists presented close affinities with IC 50 equal to 2.23±0.30 µM and 5.25±1.4 µM for MK801 and GK11 respectively (Fig. 4d). However, very interestingly on this type of recombinant NMDAR, the two compounds greatly differ in terms of maximal inhibitory effect.

Indeed, for the two NMDAR antagonists, the maximal inhibitory effect was achieved for concentration above 50 µM. However, at these concentrations, MK801 totally blocked NMDA-induced currents, whereas GK11 inhibited them only by 52.3±2.6%. Taken together, these results show that MK801 and GK11 are significantly different in terms of blockade of the different NMDAR subtypes.

Functional consequences of MK801 and GK11 blockade

Taking into account the proposed subcellular localization of both NMDAR subunits, namely NR2A preferentially synaptic and NR2B preferentially extrasynaptic and our results on recombinant NMDARs, we speculated that GK11 in contrast with MK801 will at least partially preserve the normal synaptic transmission.

Effects of MK801 and GK11 on synaptic & extrasynaptic evoked NMDA-mediated responses

To test this hypothesis, we used the experimental protocol developed by Hardingham et al. [START_REF] Hardingham | Nuclear calcium signaling controls CREB-mediated gene expression triggered by synaptic activity[END_REF] to specifically activate either the SynNMDARs or the ExNMDARs and compared the blocking effects of GK11 and MK801. As shown in Fig. 5, at 10 µM GK11 prevented 34.5±7.1% of the calcium load mediated through the SynNMDARs and 52.6±5.5%

of that mediated through the ExNMDARs (Figs 5b andc). At 100 µM GK11, the blockade of ExNMDARs was nearly total (94.3±1.6%) whereas the calcium load through SynNMDARs was only blocked by 56.4±4.3%. Similar experiments performed with MK801 showed that it was more potent than GK11 at SynNMDARs. Indeed, at 1 µM, it was as potent as 10 µM GK11 at preventing calcium load (about 35% of blockade), and at 10 µM it was more potent than 100 µM GK11 (61.3±5.3% and 56.4±4.3% blockade with MK801 and GK11 respectively). Interestingly, this is not the case for ExNMDARs. Indeed, at 10 µM MK801 was only slightly more potent than 10 µM GK11, preventing 62.9% of the calcium load compared to 51.6% for the latter compound.

Effects of MK801 and GK11 on spontaneous activity

To obtain further insight on the physiological consequences of treatment with either GK11 or MK801, we compared the effects of both drugs on spontaneous neuronal activity (Fig. 6). To obtain sufficient baseline events, recordings were performed on more mature (19-21 DIV)

dense hippocampal neuronal cultures. As indicated in materials and methods, sEPSCs were isolated in the presence of AMPA-and GABA A -receptor antagonists and in the absence Mg 2+ . Under these conditions, sEPSC occur as bursts with frequency ranging from 1.9 to 7.8

Hz. These bursts were fully blocked after application of the competitive NMDAR antagonist D-APV (data not shown). Perfusion of 10 µM MK801 significantly reduced the spontaneous activity, decreasing by about 50% the frequency and the amplitude of the sEPSCs. At 100 µM, these effects were further increased with frequency and amplitude blocked by about 80% (Fig. 6a). In contrast, application of 10 µM GK11 had no specific effect on either the frequency or the amplitude of the sEPSCs. At 100 µM, there was a small reduction (37%) in the amplitude of the NMDAR-mediated events (Fig. 6b). Notably, the effect of 100 µM GK11 perfusion on sEPSCs tended to be smaller than that measured with 10 µM MK801.

DISCUSSION

Over-activation of NMDARs occurs in many different pathological situations and can lead to cell death and severe impairment of important neurological functions [START_REF] Arundine | Molecular mechanisms of calcium-dependent neurodegeneration in excitotoxicity[END_REF]. Thus, blocking excitotoxicity with NMDAR antagonists would be a rational approach for treatment. Despite considerable efforts to develop NMDA antagonists for the treatment of acute or chronic disorders, very few molecules with a good therapeutic index have been produced. Several potent and selective NMDA antagonists failed in clinical trials principally because of their side effects [START_REF] Ikonomidou | Why did NMDA receptor antagonists fail clinical trials for stroke and traumatic brain injury?[END_REF][START_REF] Hoyte | The rise and fall of NMDA antagonists for ischemic stroke[END_REF]).

Over the last decade, our laboratory has developed therapeutic strategies for acute degenerative diseases and in particular SCI (Gimenez y Ribotta and [START_REF] Gimenez Y Ribotta | Biological interventions for spinal cord injury[END_REF][START_REF] Gimenez Y Ribotta | Strategies for regeneration and repair in spinal cord traumatic injury[END_REF][START_REF] Privat | Pathophysiology and treatment of spinal cord injury][END_REF]. One approach, which consisted in developing neuroprotective molecules based on the structure of the PCP, led us to identify GK11 as a potential new drug candidate (Hirbec et al. 2001a). Initial pharmacological studies showed that GK11 is a potent NMDAR antagonist, with affinity for NMDARs close to that of MK801, the reference antagonist (Hirbec et al. 2000b). Interestingly, further experimental studies revealed that, whether in animal models (Hirbec et al. 2001a) or in man [START_REF] Tadie | early care and treatment with a neuroprotective drug, gacyclidine, in patients with acute spinal cord injury[END_REF][START_REF] Lepeintre | Neuroprotective effect of gacyclidine. A multicenter double-blind pilot trial in patients with acute traumatic brain injury[END_REF]), GK11 did not exhibit the adverse side effects that are normally observed with classical high affinity NMDAR antagonists and led us to investigate whether the safer profile of GK11 could be ascribed to specific pharmacological properties on NMDARs.

Our study shows that, as already demonstrated for MK801 [START_REF] Huettner | Block of N-methyl-D-aspartate-activated current by the anticonvulsant MK-801: selective binding to open channels[END_REF], GK11

reduces NMDA-activated currents in primary hippocampal neurones and acts as a permanent blocker of the NMDAR associated channels. Consistent with the binding of both MK801 and GK11 inside the channel, we found that blockade of the NMDA-induced currents were voltage dependent. The voltage dependence of GK11 blockade is consistent with previous results obtained for ketamine and phencyclidine, two other PCP related compounds [START_REF] Honey | Ketamine and phencyclidine cause a voltagedependent block of responses to L-aspartic acid[END_REF][START_REF] Macdonald | Use-dependent block of excitatory amino acid currents in cultured neurons by ketamine[END_REF]. The antagonistic effects elicited by the two compounds were dose dependent and computations of the dose-response curves revealed that both compounds interacted with two independent sites. Affinities on the high and low affinity sites were respectively in the low nanomolar and micromolar range. GK11 appeared less potent at preventing NMDAR induced currents, with IC 50 values for both sites 2-5 times higher than those calculated for MK801. These results differ slightly from our previous experiments on tissue homogenates, which showed that GK11 and MK801 exhibited similar Kd values for NMDARs (Hirbec et al. 2000a;Hirbec et al. 2000b). However the divergence between the two studies is not considerable and is probably due to the experimental conditions. Indeed electrophysiological recordings in neurones represent a condition closer to the physiological functioning of the receptor. Thus, better integrity of the cell membranes as well as preserved downstream scaffolding signalling molecules may affect the conformation of the channel and are likely to influence the binding parametres. In slight contrast with the electrophysiological recordings but in agreement with the binding results, GK11 and MK801 displayed similar neuroprotective effects on glutamate-induced neuronal death. Notably, at low concentrations, MK801 was slightly more protective than GK11, a result which is in agreement with its higher affinity for NMDARs. Compared to the initial study from our laboratory [START_REF] Drian | In vitro neuroprotection against glutamate toxicity provided by novel non-competitive N-methyl-D-aspartate antagonists [In Process Citation[END_REF], this effect could be measured even when the neuroprotective treatment was administered after the insult.

In vitro determination of the intrinsic neurotoxicity of the tested compounds revealed that the decrease of the percentage of Map 2 immunopositive surface area measured was highly significant, with maximal effect reaching about 60% decrease after 48h exposure to the drug.

This relatively low apparent toxic effect may be attributed to different factors: (i) complexing of the drug with the serum contained in the medium; (ii) presence of unknown neuroprotective substances in Neurobasal (commercial product with undisclosed detailed formula); (iii) lastly, as compared to the in vivo situation, it should be remembered that neuronal cultures consist of isolated cells the connections of which in vitro are not identical to their in situ counterparts. Nevertheless, using the quantitative parametres employed here, neuronal cultures were shown to represent an adequate model to assess the potential intrinsic neurotoxicity of the molecules. In agreement with published in vivo data [START_REF] Olney | Pathological changes induced in cerebrocortical neurons by phencyclidine and related drugs[END_REF], our results reveal that cultures treated with neuroprotective concentrations of MK801 (i.e. 10 µM) exhibit neuronal suffering. In cultures, this was characterized by disruption in the neuritic network organization and increase in the Map 2 labelling in the cell bodies. Neuron viability was further reduced when the concentration or the duration of exposure to the drug was further increased. In contrast with MK801, our results show that at optimal concentrations for neuroprotection, GK11 did not elicit any neuronal suffering after 24h exposure and only very low intrinsic neurotoxicity after 48h. At 10-fold higher concentrations signs of neuronal suffering slightly increased. Interestingly, the neurotoxic effect of GK11 was at least 10 times lower than that of MK801, a fact that should be compared with our neuroprotection data, which show that both compounds displayed very similar profiles. The reduced intrinsic neurotoxicity of GK11 confirms our preliminary in vivo findings (Hirbec et al. 2001b) and is supported by ex-vivo experiments performed in Dr A. Ring's laboratory (Norwegian Defence Research Establishment). The latter have shown that, in contrast with other NMDAR antagonists, no significant increase in propidium iodide incorporation was detected in acute hippocampal slices treated for up-to 96h with GK11 (personal communication). Together, these results suggested that GK11 is likely to have an original mode of action.

The subcellular localization of the different NMDAR subunits has been a matter of debate for several years, but recent in-vitro studies have demonstrated that both NR2A-and NR2Bcontaining NMDARs can be located in either synaptic or extrasynaptic compartments [START_REF] Thomas | Synaptic and extrasynaptic NMDA receptor NR2 subunits in cultured hippocampal neurons[END_REF][START_REF] Liu | NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo[END_REF]). Recordings on hippocampal slices have also evidenced that although the vast majority of synaptic receptors are NR2A-containing, a small proportion of functional NR1/NR2B receptors are expressed at the synapse [START_REF] Liu | Switching of NMDA receptor 2A and 2B subunits at thalamic and cortical synapses during early postnatal development[END_REF][START_REF] Wong | Expression and characterization of soluble amino-terminal domain of NR2B subunit of Nmethyl-D-aspartate receptor[END_REF]). Interestingly using cortical neuronal cultures, [START_REF] Liu | NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo[END_REF] have demonstrated that NR2B-containing receptors account for about 1/3 of the synaptic currents and that about 25% of the total currents gated by extrasynaptic NMDARs. Our work on recombinant NMDARs shows that MK801 is a potent inhibitor of both NR2A-and NR2B-containing receptors and does not display significant subunit specificity. GK11 is also able to totally prevent NMDA-induced currents at NR2B-containing NMDARs but appeared about 6-7 times less potent than MK801. Very interestingly, we also demonstrated that GK11, even at very high concentrations (up to 1 mM), fails to totally block the NMDA-elicited currents at the NR2A-containing receptors. The molecular mechanism involved in this unique property is currently not known and needs further investigation. However, taking into account the results from [START_REF] Liu | NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo[END_REF], this finding suggests that both compounds would block the activation of the extrasynaptic receptors that contains majority of NR2B-containing NMDARs whereas, MK801, but not GK11, will be an efficient blocker at synaptic NR2A enriched receptors.

Using a similar protocol to that developed by Hardingham et al. (2002) to selectively activate SynNMDARs or ExNMDARs; we have shown that, as expected, MK801 is able to efficiently block both receptor subtypes. It even appears slightly more potent at SynNMDARs.

Interestingly, and in agreement with our data on recombinant receptors, here we show that GK11 is much more potent at blocking ExNMDARs versus SynNMDARs. The maximal blocking effect of GK11 at SynNMDARs was 56%, a value that is close to the maximal blocking effect observed on NR2A-containing recombinant receptors. Moreover at this concentration, GK11 blocked activation of the ExNMDARs by about 95 %. In agreement with these results on evoked NMDA-mediated responses we have shown that, compared to MK801, GK11 better preserves the spontaneous activity of the neurones mediated through NMDARs. These latter results are in good agreement with the reported in vitro (i.e. present study) and in vivo [START_REF] Olney | Pathological changes induced in cerebrocortical neurons by phencyclidine and related drugs[END_REF]) and Hirbec et al., in preparation) intrinsic neurotoxicity of GK11 and MK801 respectively. Indeeed in vivo blockade of NMDARs induces neuronal apoptosis in many regions of the developing brain [START_REF] Olney | NMDA antagonist neurotoxicity: mechanism and prevention[END_REF][START_REF] Sharp | MK-801 and ketamine induce heat shock protein HSP72 in injured neurons in posterior cingulate and retrosplenial cortex[END_REF], and studies on cultured neurones suggest a neuroprotective role for SynNMDARs [START_REF] Brenneman | Multi-determinate regulation of neuronal survival: neuropeptides, excitatory amino acids and bioelectric activity[END_REF]) that may be attributed to specific activation of signalling cascades such as the ERK signalling pathways [START_REF] Ivanov | Opposing role of synaptic and extrasynaptic NMDA receptors in regulation of the extracellular signal-regulated kinases (ERK) activity in cultured rat hippocampal neurons[END_REF]). Thus, better preservation of normal synaptic function under GK11 treatment would prevent NMDAR hypofunction mediated neuronal death.

In their study [START_REF] Liu | NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo[END_REF] reported that activation of either synaptic or extrasynaptic NR2B-containing receptors results in excitotoxicity, whereas activation of either synaptic or extrasynaptic NR2A-containing receptors promotes neuronal survival. In contrast, in their work [START_REF] Ivanov | Opposing role of synaptic and extrasynaptic NMDA receptors in regulation of the extracellular signal-regulated kinases (ERK) activity in cultured rat hippocampal neurons[END_REF] concluded that the NR2B subunit exerts a dual role in the regulation of the ERK signalling cascade with synaptic NR2B activating survival pathways and extrasynaptic NR2B leading to ERK inactivation. These findings suggest that the safer profile of GK11 may either be related to (i) its lower blocking effect at NR2A-containing NMDARs and in that case irrespective of its subcellular localization, or (ii) its lower blocking effects at synaptic NMDARs.

In the past few years, different NMDAR antagonists with improved therapeutic safety have been developed. A first class of compounds includes Memantine, and the better safety profile of these types of compounds has been ascribed to either their moderate potency, strong voltage dependence and fast blocking/unblocking kinetics (for review see [START_REF] Lipton | Paradigm shift in neuroprotection by NMDA receptor blockade: memantine and beyond[END_REF]. Over these compounds GK11 has the advantage of presenting a greater safety margin (Hirbec et al., unpublished results). Another class of interesting compounds are those which are selective for NR2B-containing receptors, such as Ifenprodil [START_REF] Kemp | NMDA receptor pathways as drug targets[END_REF].

Compared to this latter compound, GK11 appeared about 50 times less potent at NR1a/NR2B recombinant receptors expressed in HEK293 cells (0.59±0.06 µM, Harvey-Girard and Dunn 2003) and can prevent 50% of the current mediated through NR1a/NR2A

recombinant receptors with a relative high affinity. On the contrary, Ifenprodil presents an about 400-fold lower affinity for NR2A-containing receptors [START_REF] Williams | Developmental switch in the expression of NMDA receptors occurs in vivo and in vitro[END_REF]). These differences are likely to be due to differences in mechanisms mode of action. Indeed, Ifenprodil binds to the extracellular domain of the NR2B subunit (for review see Williams 2001) whereas GK11 binding site is located inside the channel (Hirbec et al. 2000a). When compared with Ifenprodil and "Ifenprodil-like" compounds, GK11 seems to present a better pharmacokinetic behaviour and less off-target activities. In particular, it was shown that GK11

has no potent interaction with adrenergic α1 receptors (IPSEN-Beaufour, personal communication). Thus the specific features of GK11 will allow the use of lower doses and will thus minimize the risk of adverse side effects by interaction with other less-specific molecular targets.

All together the results from the present study provide the first link between the molecular action of GK11 and its safer pharmacological profile. Indeed, one can speculate that as for the other high affinity NMDAR antagonists, GK11 can exert good neuroprotective properties by efficiently blocking the extrasynaptic receptors that are activated by the glutamate overload. However, it will at least partially preserve the normal synaptic transmission principally mediated through NR2A-containing receptors and will thus exhibit a much safer profile. This is particularly relevant in regions that are not affected by the glutamate overload.

Nevertheless, the unique mode of action of GK11 at NMDARs might not be the only explanation for its original pharmacological profile. Indeed, we have previously shown that, in addition to NMDARs, GK11 also interacts at relative high affinity with another molecular target, the so-called "non-NMDA" binding sites which we speculated may play a role in the modulation of the NMDAR complex (Hirbec et al. 2001b).

  GABA A receptors with 50 µM biccuculine (Sigma-Aldrich) and the presence of 2.5 mM 4-AP (Sigma-Aldrich). Under these conditions, cortical neurones fired bursts of action potential which resulted in a calcium plateau visualized by videomicroscopy. This calcium increase was fully blocked by the co-application of 10 µM MK801, 100 µM D,L-amino-5phosphovalerate (D-APV, Sigma-Aldrich) or 0.5 µM TTX (data not shown). To selectively activate extrasynaptic NMDARs (ExNMDARs), synaptic NMDAR (SynNMDAR) responses were first inactivated by exposing neurones to 10 µM MK801 during a biccuculine and 4-AP treatment. Then, the extrasynaptic activity was activated by an application of 50 µM NMDA. At least 3 independent experiments and 120 to 130 individual neurones were analyzed. Results are expressed as means ± SEM. Statistical analysis was performed with StatView (Abacus, Berkeley, CA, USA) by one-way ANOVA followed by a PLSD Fisher test (*: p< 0rat full length sequences of NR1a, NR2A and NR2B cloned in pcDNA I were a generous gift from Dr. Jeremy Henley (University of Bristol, UK). The peGFP(C2) plasmid was obtained from Clonetech.

Fig. 1

 1 Fig. 1 Pharmacological properties of MK801 and GK11 on native NMDA receptors. (A)

Fig. 2

 2 Fig. 2 Recovery from blockade by 10 µM GK11 or 10 µM MK801 on NMDA-induced currents

Fig. 3 Fig. 4

 34 Fig. 3 Inhibition effects of MK801 and GK11 on NMDA-induced currents in rat hippocampal

Fig. 5

 5 Fig. 5 Effects of MK801 and GK11 on synaptic versus extrasynaptic NMDARs on mouse

Fig. 6

 6 Fig. 6 Modulation of spontaneous EPSCs on rat hippocampal neurones (DIV 19-21). (a) and

Figure 1

 1 Figure 1 Vandame D., Desmadryl G., Becerril O., Teigell M., Crouzin N., Buisson A., Privat A. and Hirbec H. J. Neurochem

Table 1

 1 Summary of the fitting parametres of the dose-response curves which illustrate the inhibitory

	properties of MK801 and GK11 on cultured hippocampal neurones and HEK cells transfected
	with either NR1a/NR2A or NR1a/NR2B subunits.
		MK801		GK11
		max ± SE	IC50 ± SE	max ± SE	IC50 ± SE
	Neurones
	Site 1	54.1 ± 8.9 %	105 ± 54 nM	26.9 ± 9.7 %	262 ± 23 nM
	Site 2	103 ± 4.6 %	55.9 ± 3.9 µM	111 ± 5.6 %	197 ± 31 µM
	HEK
	NR1a/NR2A	104 ± 2.4 %	2.23 ± 0.3 µM	52.3 ± 2.6 %	5.25 ± 1.4 µM
	NR1a/NR2B	95.9 ± 4.6 %	5.80 ± 1.3 µM	96.6 ± 5.4 %	37.4 ± 8.7 µM

Acknowledgments

We thank Dr. Keith Langley (Inserm U583, France) for critical reading of the text. This study was funded by the region Languedoc-Roussillon (France), the foundation IRME (France) and the spin-off NEUREVA. Technical support was provided by the French national institute INSERM.