Animal paradigms to assess cognition with translation to humans.

Cognition is a complex brain function that represents processes such as learning and memory, attention, working memory, and executive functions amongst others. Impairments in cognition are prevalent in many neuropsychiatric and neurological disorders with few viable treatment options. The development of new therapies is challenging, and poor efficacy in clinical development continues to be one of the most consistent reasons compounds fail to advance, suggesting that traditional animal models are not predictive of human conditions and behavior. An effort to improve the construct validity of neuropsychological testing across species with the intent of facilitating therapeutic development has been strengthening over recent years. With an emphasis on understanding the underlying biology, optimizing the use of appropriate systems (e.g., transgenic animals) to model targeted disease states, and incorporating non-rodent species (e.g., non-human primates) that may enable a closer comparison to humans, an improvement in the translatability of the results will be possible. This chapter focuses on some promising translational cognitive paradigms for use in rodents, non-human primates, and humans.

Basmisanil, a highly selective GABAA-α5 negative allosteric modulator: preclinical pharmacology and demonstration of functional target engagement in man.

GABAA-α5 subunit-containing receptors have been shown to play a key modulatory role in cognition and represent a promising drug target for cognitive dysfunction, as well as other disorders. Here we report on the preclinical and early clinical profile of a novel GABAA-α5 selective negative allosteric modulator (NAM), basmisanil, which progressed into Phase II trials for intellectual disability in Down syndrome and cognitive impairment associated with schizophrenia. Preclinical pharmacology studies showed that basmisanil is the most selective GABAA-α5 receptor NAM described so far. Basmisanil bound to recombinant human GABAA-α5 receptors with 5 nM affinity and more than 90-fold selectivity versus α1, α2, and α3 subunit-containing receptors. Moreover, basmisanil inhibited GABA-induced currents at GABAA-α5 yet had little or no effect at the other receptor subtypes. An in vivo occupancy study in rats showed dose-dependent target engagement and was utilized to establish the plasma exposure to receptor occupancy relationship. At estimated receptor occupancies between 30 and 65% basmisanil attenuated diazepam-induced spatial learning impairment in rats (Morris water maze), improved executive function in non-human primates (object retrieval), without showing anxiogenic or proconvulsant effects in rats. During the Phase I open-label studies, basmisanil showed good safety and tolerability in healthy volunteers at maximum GABAA-α5 receptor occupancy as confirmed by PET analysis with the tracer [11C]-Ro 15-4513. An exploratory EEG study provided evidence for functional activity of basmisanil in human brain. Therefore, these preclinical and early clinical studies show that basmisanil has an ideal profile to investigate potential clinical benefits of GABAA-α5 receptor negative modulation.

Phosphorylation of Tau at S422 is enhanced by Abeta in TauPS2APP triple transgenic mice.

Amyloid beta peptides and microtubule-associated protein Tau are misfolded and form aggregates in brains of Alzheimer's disease patients. To examine their specific roles in the pathogenesis of Alzheimer's disease and their relevance in neurodegenerative processes, we have created TauPS2APP triple transgenic mice that express human mutated Amyloid Precursor Protein, presenilin 2 and Tau. We present a cross-sectional analysis of these mice at 4, 8, 12 and 16 months of age. By comparing with single transgenic Tau mice, we demonstrate that accumulation of Abeta in TauPS2APP triple transgenic mice impacts on Tau pathology by increasing the phosphorylation of Tau at serine 422, as determined by a novel immunodetection method that is able to reliably measure phospho-Tau species in transgenic mouse brains. The TauPS2APP triple transgenic mouse model will be very useful for studying the effect of new therapeutic paradigms on amyloid deposition and downstream neurofibrillary tangle development.

Synthesis and characterization of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives: Part 4. In vivo active potent and selective non-competitive metabotropic glutamate receptor 2/3 antagonists.

This study completes a series of papers devoted to the characterization of the non-competitive mGluR2/3 antagonist properties of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives with particular emphasis on derivatizations compatible with brain penetration and in vivo activity. Especially the compounds bearing a para-pyridine consistently showed in vivo activity in rat behavioral models after oral administration, for example, blockade of the mGluR2/3 agonist LY354740-induced hypoactivity and improvement of a working memory deficit induced either by LY354740 or scopolamine in the delayed match to position task (DMTP). Moreover, combination studies with a cholinesterase inhibitor show apparent synergistic effects on working memory impairment induced by scopolamine.

Rational design of novel pyrrolidine derivatives as orally active neurokinin-3 receptor antagonists.

The rational design of a novel series of pyrrolidine derivatives as neurokinin-3 receptor antagonists is reported starting from a selective neurokinin-1 receptor antagonist. Typical representatives in this series showed in vivo efficacy after oral administration in a NK3 mediated functional assay. This series of NK3 antagonists shows promise to deliver a novel antipsychotic.

Discovery of potent, balanced and orally active dual NK1/NK3 receptor ligands.

During a program directed at selective NK(1) receptor antagonists, we serendipitously discovered an NK(1) receptor ligand with additional affinity for the NK(3) receptor. Recognising an opportunity for a drug discovery program aiming for dual NK(1)/NK(3) receptor antagonists, we prepared a series of analogues from a novel, versatile building block. From this series emerged compounds with high and balanced affinities for the NK(1) and the NK(3) receptors. Typical representatives of this series were active in the gerbil foot tapping assay after oral administration.

Hippocampal-dependent spatial memory functions might be lateralized in rats: An approach combining gene expression profiling and reversible inactivation.

The hippocampus is involved in spatial memory processes, as established in a variety of species such as birds and mammals including humans. In humans, some hippocampal-dependent memory functions may be lateralized, the right hippocampus being predominantly involved in spatial navigation. In rodents, the question of possible lateralization remains open. Therefore, we first microdissected the CA1 subregion of the left and right dorsal hippocampi for analysis of mRNA expression using microarrays in rats having learnt a reference memory task in the Morris water-maze. Relative to untrained controls, 623 genes were differentially expressed in the right hippocampus, against only 74 in the left hippocampus, in the rats that had learnt the hidden platform location. Thus, in the right hippocampus, 299 genes were induced, 324 were repressed, and about half of them participate in signaling and transport, metabolism, and nervous system functions. In addition, most differentially expressed genes associated with spatial learning have been previously related to synaptic plasticity and memory. We then subjected rats to unilateral (left or right) or bilateral reversible functional inactivations in the dorsal hippocampus; lidocaine was infused either before each acquisition session or before retrieval of a reference spatial memory in the Morris water maze. We found that after drug-free acquisition, right or bilateral lidocaine inactivation (vs. left, or bilateral phosphate buffered saline (PBS) infusions) of the dorsal hippocampus just before a delayed (24 h) probe trial impaired performance. Conversely, left or bilateral hippocampus inactivation (vs. right, or bilateral PBS infusions) before each acquisition session weakened performance during a delayed, drug-free probe trial. Our data confirm a functional association between transcriptional activity within the dorsal hippocampus and spatial memory in the rat. Further, they suggest that there could be a leftward bias of hippocampal functions in engram formation or information transfer, and a rightward bias in spatial memory storage/retrieval processes.

Imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepines as potent and highly selective GABAA alpha5 inverse agonists with potential for the treatment of cognitive dysfunction.

In a search for GABAA alpha5 ligands that combine high subtype binding selectivity with a marked inverse agonism imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepines were identified as a promising class. A short tandem reaction allowed rapid access to this chemical series, thereby facilitating rapid SAR generation which guided the optimization process. Two compounds (10e and 11f) were found to be active in an in vivo paradigm for cognitive improvement.

The discovery and unique pharmacological profile of RO4938581 and RO4882224 as potent and selective GABAA alpha5 inverse agonists for the treatment of cognitive dysfunction.

Lead optimisation of the imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepine class led to the identification of two clinical leads [RO4882224 (11) and RO4938581 (44)] functioning as novel potent and selective GABAA alpha5 inverse agonists. The unique pharmacological profiles and optimal pharmacokinetic profiles resulted in in vivo activity in selected cognition models.

Discovery of the imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepine scaffold as a novel, potent and selective GABA(A) alpha5 inverse agonist series.

Through iterative design cycles we have discovered a number of novel new classes where the imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepine was deemed the most promising GABA(A) alpha5 inverse agonist class with potential for cognitive enhancement. This class combines a modest subtype binding selectivity with inverse agonism and has the most favourable molecular properties for further lead optimisation towards a central nervous system (CNS) acting medicine.

Oxytocin attenuates phencyclidine hyperactivity and increases social interaction and nucleus accumben dopamine release in rats.

The pituitary neuropeptide oxytocin promotes social behavior, and is a potential adjunct therapy for social deficits in schizophrenia and autism. Oxytocin may mediate pro-social effects by modulating monoamine release in limbic and cortical areas, which was investigated herein using in vivo microdialysis, after establishing a dose that did not produce accompanying sedative or thermoregulatory effects that could concomitantly influence behavior. The effects of oxytocin (0.03-0.3 mg/kg subcutaneous) on locomotor activity, core body temperature, and social behavior (social interaction and ultrasonic vocalizations) were examined in adult male Lister-hooded rats, using selective antagonists to determine the role of oxytocin and vasopressin V1a receptors. Dopamine and serotonin efflux in the prefrontal cortex and nucleus accumbens of conscious rats were assessed using microdialysis. 0.3 mg/kg oxytocin modestly reduced activity and caused hypothermia but only the latter was attenuated by the V1a receptor antagonist, SR49059 (1 mg/kg intraperitoneal). Oxytocin at 0.1 mg/kg, which did not alter activity and had little effect on temperature, significantly attenuated phencyclidine-induced hyperactivity and increased social interaction between unfamiliar rats without altering the number or pattern of ultrasonic vocalizations. In the same rats, oxytocin (0.1 mg/kg) selectively elevated dopamine overflow in the nucleus accumbens, but not prefrontal cortex, without influencing serotonin efflux. Systemic oxytocin administration attenuated phencyclidine-induced hyperactivity and increased pro-social behavior without decreasing core body temperature and selectively enhanced nucleus accumbens dopamine release, consistent with activation of mesocorticolimbic circuits regulating associative/reward behavior being involved. This highlights the therapeutic potential of oxytocin to treat social behavioral deficits seen in psychiatric disorders such as schizophrenia.

Synthesis and characterization of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives: Part 3. New potent non-competitive metabotropic glutamate receptor 2/3 antagonists.

A series of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives was evaluated as non-competitive mGluR2/3 antagonists. Replacement of the (2-aryl)-ethynyl-moiety in 8-position with smaller less lipophilic substituents produced compounds inhibiting the binding of [3H]-LY354740 to rat mGluR2 with low nanomolar affinity and consistent functional effect at both mGluR2 and mGluR3. These compounds were able to reverse LY354740-mediated inhibition of field excitatory postsynaptic potentials in the rat dentate gyrus and in vivo activity could be demonstrated by reversal of the LY354740-induced hypoactivity in mice after oral administration.

A schizophrenia relevant 5-Choice Serial Reaction Time Task for mice assessing broad monitoring, distractibility and impulsivity.

The 5-Choice Serial Reaction Time Task (5-CSRTT) is an automated test for rodents allowing the assessment of multiple cognitive measures. Originally designed to assess cognitive deficits relevant to attention deficit hyperactivity disorder, it has been widely used in the investigation of neural systems of attention. In the current study, we have set up a modified version, which reduced the training phase to only 8-9 days with minimal food deprivation and without single-housing. Furthermore, based on evidence that patients with schizophrenia are more impaired in broad monitoring abilities than in sustained attention, we successfully developed a protocol replicating the Spatial Attentional Resource Allocation Task (SARAT), used in humans to assess broad monitoring. During this task, when the target appeared at a single pre-cued location, mice selectively responded faster. Instead, increasing the number of validly cued locations proportionately decreased accuracy. We then validated a protocol which is relevant for neuropsychiatric disorders in which additional irrelevant pre-cue lights selectively disrupted attention (distractibility). Finally, we improved previously used protocols changing inter-trial intervals from 5 to 7 s by randomly presenting this shift only in 20% of the trials. This resulted in a selective effect on premature responses (impulsivity), with important implications for schizophrenia as well as for other mental disorders. Therefore, this revised 5-CSRTT reduced training and stress on the animals while selectively measuring different cognitive functions with translational validity to schizophrenia and other psychiatric disorders.

Enhancing effects of nicotine and impairing effects of scopolamine on distinct aspects of performance in computerized attention and working memory tasks in marmoset monkeys.

With the CAmbridge Neuropsychological Test Automated Battery (CANTAB), computerized neuropsychological tasks can be presented on a touch-sensitive computer screen, and this system has been used to assess cognitive processes in neuropsychiatric patients, healthy volunteers, and species of non-human primate, primarily the rhesus macaque and common marmoset. Recently, we reported that the common marmoset, a small-bodied primate, can be trained to a high and stable level of performance on the CANTAB five-choice serial reaction time (5-CSRT) task of attention, and a novel task of working memory, the concurrent delayed match-to-position (CDMP) task. Here, in order to increase understanding of the specific cognitive demands of these tasks and the importance of acetylcholine to their performance, the effects of systemic delivery of the muscarinic receptor antagonist scopolamine and the nicotinic receptor agonist nicotine were studied. In the 5-CSRT task, nicotine enhanced performance in terms of increased sustained attention, whilst scopolamine led to increased omissions despite a high level of orientation to the correct stimulus location. In the CDMP task, scopolamine impaired performance at two stages of the task that differ moderately in terms of memory retention load but both of which are likely to require working memory, including interference-coping, abilities. Nicotine tended to enhance performance at the long-delay stage specifically but only against a background of relatively low baseline performance. These data are consistent with a dissociation of the roles of muscarinic and nicotinic cholinergic receptors in the regulation of both sustained attention and working memory in primates.

The effects of serotonin reuptake inhibitors on locomotor activity in gerbils.

In the current study we examined the effects of serotonin reuptake inhibitors on the locomotor activity of gerbils, and undertook experiments to understand the mechanisms involved in their effects. The selective serotonin reuptake inhibitors (SSRIs) fluoxetine (1-30 mg/kg, i.p.) and escitalopram (0.03-10 mg/kg, i.p.) dose-dependently increased locomotor activity, whereas the serotonin and noradrenaline reuptake inhibitor duloxetine (0.3-30 mg/kg, i.p.) did not. The noradrenaline reuptake inhibitor (NRI) reboxetine, which alone did not significantly affect locomotion (1-30 mg/kg, i.p.), markedly reduced the effects of escitalopram. The locomotor effects of fluoxetine and escitalopram were dependent on novelty since both compounds showed rapid habituation in novel cages and were inactive when tested in home cages. Both diazepam (0.3-10 mg/kg, i.p.) and d-amphetamine (0.3-10 mg/kg, s.c.) increased locomotor activity but only the effects of diazepam were novelty-dependent. The finding that SSRIs increased locomotion, with a negative modulatory role for NRI, in a novelty-dependent manner, similar to diazepam, suggests that anxiety plays an important role in the present paradigm. The increase in locomotion as observed in our test conditions can be readily used as a selective and sensitive in vivo assay for serotonin transport inhibition in gerbils.

Severe impairment of NMDA receptor function in mice carrying targeted point mutations in the glycine binding site results in drug-resistant nonhabituating hyperactivity.

NMDA receptor hypofunction has been implicated in the pathophysiology of schizophrenia, and pharmacological and genetic approaches have been used to model such dysfunction. We previously have described two mouse lines carrying point mutations in the NMDA receptor glycine binding site, Grin1(D481N) and Grin1(K483Q), which exhibit 5- and 86-fold reductions in receptor glycine affinity, respectively. Grin1(D481N) animals exhibit a relatively mild phenotype compatible with a moderate reduction in NMDA receptor function, whereas Grin1(K483Q) animals die shortly after birth. In this study we have characterized compound heterozygote Grin1(D481N/K483Q) mice, which are viable and exhibited biphasic NMDA receptor glycine affinities compatible with the presence of each of the two mutated alleles. Grin1(D481N/K483Q) mice exhibited a marked NMDA receptor hypofunction revealed by deficits in hippocampal long-term potentiation, which were rescued by the glycine site agonist d-serine, which also facilitated NMDA synaptic currents in mutant, but not in wild-type, mice. Analysis of striatal monoamine levels revealed an apparent dopaminergic and serotonergic hyperfunction. Behaviorally, Grin1(D481N/K483Q) mice were insensitive to acute dizocilpine pretreatment and exhibited increased startle response but normal prepulse inhibition. Most strikingly, mutant mice exhibited a sustained, nonhabituating hyperactivity and increased stereotyped behavior that were resistant to suppression by antipsychotics and the benzodiazepine site agonist Zolpidem. They also displayed a disruption of nest building behavior and were unable to perform a cued learning paradigm in the Morris water maze. We speculate that the severity of NMDA receptor hypofunction in these mice may account for their profound behavioral phenotype and insensitivity to antipsychotics.

Evidence for improved performance in cognitive tasks following selective NR2B NMDA receptor antagonist pre-treatment in the rat.

RATIONALE: We previously reported that the NR2B subunit-selective N-methyl-D-aspartate (NMDA) antagonist Ro 63-1908 produced a marked deficit in response control in the five-choice serial reaction time task (5-CSRTT). OBJECTIVES: The present studies were designed to investigate this further by studying the NR2B NMDA antagonists, ifenprodil, traxoprodil (CP101,606), Ro 25-6981 as well as Ro 63-1908 in this test. METHODS: Following training in the 5-CSRTT, separate groups of rats were either tested under (1) standard test conditions [5 s inter-trial interval (ITI), 0.5 s stimulus duration, 100 trials], (2) high (3 s ITI) and low (10 s ITI) event rate of stimulus presentation and (3) a 250-trial protocol in aged 2-year-old rats. In a final study, the effects of traxoprodil were investigated in an operant delayed match to position (DMTP) task, a test of working memory, and compared to dizocilpine and Ro 63-1908. RESULTS: Similar to Ro 63-1908, both traxoprodil (1-10 mg/kg) and Ro 25-6981 (3--30 mg/kg) increased premature responding but also increased response speed with no error trade-off. Conversely, ifenprodil (1--10 mg/kg) slowed response speed and increased omissions with no effect on premature responding. Tested under a variable ITI, Ro 63--1908 (1 mg/kg) increased premature responding at all ITIs, but this change was proportional to controls. At short ITI (3 s), Ro 63-1908 reliably improved performance both in terms of response speed and accuracy (percent correct). In a 250-trial protocol in aged rats, both Ro 63-1908 (0.1-0.3 mg/kg) and, particularly, traxoprodil (1--3 mg/kg) improved performance-increasing response speed and increasing the number of rewards earned during test. Finally, traxoprodil (1--10 mg/kg) improved accuracy and increased response speed in the DMTP task. CONCLUSIONS: The present studies support the view that selective NR2B NMDA antagonists promote impulsive-type responding in the 5-CSRTT; however, under certain test conditions, drugs of this class-notably traxoprodil-may also improve task performance.

The effect of the mGlu5 receptor antagonist MPEP in rodent tests of anxiety and cognition: a comparison.

RATIONALE: Antagonists at the metabotropic glutamate 5 (mGlu5) receptor produce robust anxiolytic effects in a number of rat tests. However, there is evidence that mGlu5 receptor antagonists may also impair working memory and spatial learning following intracerebroventricular administration. OBJECTIVES: The aim of this study is to compare the effect of the potent and selective mGlu5 receptor antagonist, 2-methyl-6-(phenylethynyl)-piperidine (MPEP), administered systemically on rodent tests of cognition and anxiety. METHODS: MPEP was assessed in the following rodent tests, 60 min following oral administration: Geller--Seifter conflict, conditioned emotional response (CER), Vogel conflict, delayed match to position (DMTP) and Morris water maze. Diazepam was also tested as a comparator. RESULTS: MPEP had a significant anxiolytic effect, comparable in magnitude to diazepam, at 10--30 mg/kg in the two conflict and CER tasks. There was no effect of MPEP up to 30 mg/kg on working memory in the DMTP task, but at 100 mg/kg, there was a significant reduction in choice accuracy at the longest delay interval (24 s). MPEP (3--30 mg/kg) did not significantly impair spatial learning in the Morris water maze, although during the last probe trial, 30-mg/kg-treated rats were significantly less accurate than controls. In contrast, diazepam significantly impaired performance in both the DMTP and Morris water maze tests. Assessment of plasma and brain concentration of MPEP approximately 75 min following oral administration showed a dose linearity from 3 to 30 mg/kg and good brain penetration, i.e. a brain/plasma ratio of 3.1. CONCLUSIONS: Oral administration of the selective mGlu5 receptor antagonist MPEP induces a robust anxiolytic-like effect in rat conflict tests comparable to that seen with diazepam, but in contrast to diazepam, MPEP does not impair working memory or spatial learning at anxiolytic doses.

Effects of the mGluR2/3 agonist LY354740 on computerized tasks of attention and working memory in marmoset monkeys.

RATIONALE: LY354740 is a recently developed metabotropic glutamatergic receptor 2 and 3 (mGluR2/3) agonist. A high density of mGluR2 has been reported in terminal fields of the perforant path in rodents and humans, suggesting its involvement in cognitive functions mediated by the temporal lobe, including memory. A small number of in vivo studies in rodents have assessed the effects of LY354740 on memory tasks, reporting the induction of impaired memory for spatial orientation in a water maze task and for delayed match and non-match to position in an operant version of these tasks. OBJECTIVE: In the present primate study, we used radioautography to describe the distribution and intensity of (3)H-LY354740 binding in the hippocampal formation of the common marmoset monkey (Callithrix jacchus) relative to the rat. In the major, in vivo part of the study, the effects of systemic LY354740 on computerized tasks of attention and memory were investigated. METHODS: Adult common marmosets were trained to perform a five-choice serial reaction time (5-CSRT) task and a concurrent delayed match-to-position (CDMP) task from the Cambridge Neuropsychological Automated test Battery (CANTAB). Filter tests of LY354740 effects on motor dexterity and motivation for reward revealed high inter-individual variation in sensitivity; therefore, on the 5-CSRT, subjects were tested at a dose range of 3--10 mg/kg, and on the CDMP, subjects were tested at 1--3 or 3--10 mg/kg. RESULTS: Radioautography revealed a relatively low level of (3)H-LY354740 binding in the marmoset hippocampal formation compared to the rat. Despite low binding, LY354740 reduced sustained-attention accuracy in the 5-CSRT, and reduced accuracy in two stages of the CDMP. CONCLUSIONS: The current study provides novel evidence for the importance of mGluR2/3 in the regulation of primate cognitive functioning.

The subchronic phencyclidine rat model: relevance for the assessment of novel therapeutics for cognitive impairment associated with schizophrenia.

RATIONALE: Current treatments for schizophrenia have modest, if any, efficacy on cognitive dysfunction, creating a need for novel therapies. Their development requires predictive animal models. The N-methyl-D-aspartate (NMDA) hypothesis of schizophrenia indicates the use of NMDA antagonists, like subchronic phencyclidine (scPCP) to model cognitive dysfunction in adult animals. OBJECTIVES: The objective of this study was to assess the scPCP model by (1) reviewing published findings of scPCP-induced neurochemical changes and effects on cognitive tasks in adult rats and (2) comparing findings from a multi-site study to determine scPCP effects on standard and touchscreen cognitive tasks. METHODS: Across four research sites, the effects of scPCP (typically 5 mg/kg twice daily for 7 days, followed by at least 7-day washout) in adult male Lister Hooded rats were studied on novel object recognition (NOR) with 1-h delay, acquisition and reversal learning in Morris water maze and touchscreen-based visual discrimination. RESULTS: Literature findings showed that scPCP impaired attentional set-shifting (ASST) and NOR in several labs and induced a variety of neurochemical changes across different labs. In the multi-site study, scPCP impaired NOR, but not acquisition or reversal learning in touchscreen or water maze. Yet, this treatment regimen induced locomotor hypersensitivity to acute PCP until 13-week post-cessation. CONCLUSIONS: The multi-site study confirmed that scPCP impaired NOR and ASST only and demonstrated the reproducibility and usefulness of the touchscreen approach. Our recommendation, prior to testing novel therapeutics in the scPCP model, is to be aware that further work is required to understand the neurochemical changes and specificity of the cognitive deficits.