Searching for cognitive enhancement in the Morris water maze: better and worse performance in D‐amino acid oxidase knockout (Dao−/−) mice

A common strategy when searching for cognitive‐enhancing drugs has been to target the N‐methyl‐d ‐aspartate receptor (NMDAR), given its putative role in synaptic plasticity and learning. Evidence in favour of this approach has come primarily from studies with rodents using behavioural assays like the Morris water maze. D‐amino acid oxidase (DAO) degrades neutral D‐amino acids such as D‐serine, the primary endogenous co‐agonist acting at the glycine site of the synaptic NMDAR. Inhibiting DAO could therefore provide an effective and viable means of enhancing cognition, particularly in disorders like schizophrenia, in which NMDAR hypofunction is implicated. Indirect support for this notion comes from the enhanced hippocampal long‐term potentiation and facilitated water maze acquisition of ddY/Dao⁻ mice, which lack DAO activity due to a point mutation in the gene. Here, in Dao knockout (Dao^−/−) mice, we report both better and worse water maze performance, depending on the radial distance of the hidden platform from the side wall of the pool. Dao^−/− mice displayed an increased innate preference for swimming in the periphery of the maze (possibly due to heightened anxiety), which facilitated the discovery of a peripherally located platform, but delayed the discovery of a centrally located platform. By contrast, Dao^−/− mice exhibited normal performance in two alternative assays of long‐term spatial memory: the appetitive and aversive Y‐maze reference memory tasks. Taken together, these results question the proposed relationship between DAO inactivation and enhanced long‐term associative spatial memory. They also have generic implications for how Morris water maze studies are performed and interpreted.     

d‐amino acid oxidase knockout (Dao−/−) mice show enhanced short‐term memory performance and heightened anxiety,but no sleep or circadian rhythm disruption

d ‐amino acid oxidase (DAO, DAAO) is an enzyme that degrades d ‐serine, the primary endogenous co‐agonist of the synaptic N‐methyl‐d ‐aspartate receptor. Convergent evidence implicates DAO in the pathophysiology and potential treatment of schizophrenia. To better understand the functional role of DAO, we characterized the behaviour of the first genetically engineered Dao knockout (Dao^?/?) mouse. Our primary objective was to assess both spatial and non‐spatial short‐term memory performance. Relative to wildtype (Dao^+/+) littermate controls, Dao^?/? mice demonstrated enhanced spatial recognition memory performance, improved odour recognition memory performance, and enhanced spontaneous alternation in the T‐maze. In addition, Dao^?/? mice displayed increased anxiety‐like behaviour in five tests of approach/avoidance conflict: the open field test, elevated plus maze, successive alleys, light/dark box and novelty‐suppressed feeding. Despite evidence of a reciprocal relationship between anxiety and sleep and circadian function in rodents, we found no evidence of sleep or circadian rhythm disruption in Dao^?/? mice. Overall, our observations are consistent with, and extend, findings in the natural mutant ddY/Dao^? line. These data add to a growing body of preclinical evidence linking the inhibition, inactivation or deletion of DAO with enhanced cognitive performance. Our results have implications for the development of DAO inhibitors as therapeutic agents.     

Phenotypic characterization of mice heterozygous for a null mutation of glutamate carboxypeptidase II

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Disturbed glutamate signaling resulting in hypofunction of N‐methyl‐D ‐aspartate receptors (NMDAR) has been implicated in the pathophysiology of schizophrenia. Glutamate Carboxypeptidase II (GCP II) hydrolyzes N‐acetyl‐alpha L ‐aspartyl‐L ‐glutamate (NAAG) into glutamate and N‐acetyl‐aspartate. NAAG is a neuropeptide that is an NMDAR antagonist as well as an agonist for the metabotropic glutamate receptor‐3 (mGluR3), which inhibits glutamate release. The aggregate effect of NAAG is thus to attenuate NMDAR activation. To manipulate the expression of GCP II, LoxP sites were inserted flanking exons 1 and 2, which were excised by crossing with a Cre‐expressing mouse. The mice heterozygous for this deletion showed a 50% reduction in the expression level of protein and functional activity of GCP II in brain samples. Heterozygous mutant crosses did not yield any homozygous null animals at birth or as embryos (N > 200 live births and fetuses). These data are consistent with the previous report that GCP II homozygous mutant mice generated by removing exons 9 and 10 of GCP II gene were embryonically lethal and confirm our hypothesis that GCP II plays an essential role early in embryonic development. Heterozygous mice, however, developed normally to adulthood and exhibited increased locomotor activity, reduced social interaction, and a subtle cognitive deficit in working memory. Synapse 63:625–635, 2009. © 2009 Wiley‐Liss, Inc.     

Effects of extracellular pH on the dynorphin A inhibition of N-methyl-D-aspartate receptors expressed in Xenopus oocytes

Dynorphin A (Dyn A) (1-17), the postulated endogenous ligand for the kappa-opioid receptor, inhibits N-methyl-D-aspartate (NMDA) receptor-mediated currents in neuronal preparations and in Xenopus oocytes expressing recombinant NMDA receptors. Although direct interactions of Dyn A with the NMDA receptor have been reported, the mechanisms mediating the inhibitory actions of Dyn A are unknown. Extracellular pH is a crucial factor regulating NMDA receptor function. To date, however, the influence of pH on the inhibitory actions of Dyn A has not been examined. In the present study we used voltage-clamp recording techniques in Xenopus oocytes expressing recombinant NR1A/2A receptors to address this issue. We report that decreasing the pH of the external solution from 7.5 to 6.7 significantly enhances Dyn A inhibition of NMDA receptor-mediated currents. On the contrary, increasing the pH of the external solution to 9.2 prevents the inhibitory action of Dyn A. The influence of external pH was independent of membrane potential and the potentiation of inhibition with decreasing pH was not associated with alterations in the charge of the Dyn A molecule. These findings demonstrate that Dyn A inhibition of the NMDA receptor current is pH-dependent. They further suggest that the efficacy of neuronally released Dyn A in inhibiting NMDA receptor function may be increased in response to nerve injury and other conditions associated with decreased extracellular pH.     

Enhanced prefrontal serotonin 2A receptor signaling in the subchronic phencyclidine mouse model of schizophrenia

Prefrontal serotonin 2A receptors (5‐HT_2ARs) have been linked to the pathogenesis and treatment of schizophrenia. Many antipsychotics fully occupy 5‐HT_2AR at clinical relevant doses, and activation of 5‐HT_2A receptors by lysergic acid diethylamide (LSD) and LSD‐like drugs induces a schizophrenia‐like psychosis in humans. Subchronic phencyclidine (PCP) administration is a well‐established model for schizophrenia‐like symptoms in rodents. The aim of the present study was to investigate whether subchronic PCP administration changes expression, binding, or functionality of cortical 5‐HT_2ARs. As a measure of 5‐HT_2AR functionality, we used the 5‐HT_2AR agonist 2,5‐dimethoxy‐4‐iodoamphetamine (DOI)‐induced head‐twitch response (HTR) and mRNA expression of the immediate‐early genes (IEGs) activity‐related cytoskeletal associated‐protein (Arc), c‐fos, and early growth response protein 2 (egr‐2) in the frontal cortex. Mice were treated with PCP (10 mg/kg) or saline for 10 days, followed by a 5‐day washout period. The PCP pretreatment increased the overall induction of HTR and frontal cortex IEG mRNA expression following a single challenge with DOI. These functional changes were not associated with changes in 5‐HT_2AR binding. Also, binding of the 5‐HT_1AR and the 5‐HT transporter was unaffected. Finally, basal mRNA level of Arc was increased in the prefrontal cortex after subchronic PCP administration as revealed with in situ hybridization. Together these findings indicate that PCP administration produces changes in the brain that result in an increase in the absolute effect of DOI. Therefore, neurotransmission involving the 5‐HT_2AR could contribute to the behavioral deficits observed after PCP treatment. © 2013 Wiley Periodicals, Inc.     

Developmental expression of N-methyl-D-aspartate glutamate receptor 1 splice variants in the chick retina

Glutamate is the major excitatory neurotransmitter in the vertebrate retina. The N-methyl-D-aspartate glutamate receptor (NMDAR) is assembled as a tetramer containing NR1 and NR2, and possibly NR3 subunits, NR1 being essential for the formation of the ion channel. The NMDAR1 (NR1) gene encodes for mRNAs that generate at least eight functional variants by alternative splicing of exon 5 (cassette N1), 21 (cassette C1), or 22 (cassettes C2 or C2'). NR1 splice variants were identified in the mature chick retina, and their variation during embryonic development (ED) was analyzed. NR1 was shown to lack N1 in early ED, shifting to N1-containing variants in the mature retina, which could contribute to explaining the distinct biochemical properties of retinal NMDARs compared with the CNS. Sequence analysis of C-terminal variants containing C1 and C2 cassettes suggests a membrane-targeting mechanism for avian NMDARs distinct from that in mammals. An NR1 variant containing a novel alternative C-terminal splice exon named C3 was found, which encodes six amino acids containing a predicted casein kinase II phosphorylation site. This new variant is expressed in the retina during a restricted period of ED, coincident with the generation of spontaneous calcium activity waves, which precedes synapse formation in the retina, suggesting its participation in this process.     

Glutamate agonists for treating schizophrenia have affinity for dopamine D2High and D3 receptors

Although the glutamate agonist LY 404,039 has been used to treat schizophrenia, its closest congener LY 379,268 has an affinity for both glutamate and dopamine (DA) D2^High receptors. Considering that all antipsychotics act on dopamine receptors, and considering that another laboratory reported that LY 379,268 did not have any affinity for the D2^High receptor, it was necessary to examine whether such glutamate agonists have an affinity for D2 and D3 dopamine receptors in vitro. The present data show that 50–200 nM LY 379,268 inhibited the binding of [³H]domperidone and [³H](+)PHNO to cloned dopamine D2 receptors consistently and reproducibly by 16% with dissociation constants of 2.1 and 2.5 nM at D2^High, respectively. In addition, LY 379,268 inhibited the binding of [³H]domperidone and [³H](+)PHNO to cloned dopamine D3 receptors with dissociation constants of 130 and 10 nM, respectively. LY 379,268 also inhibited the binding of [³H]domperidone to rat striata with a dissociation constant of 22 nM, predicting a clinical antipsychotic dose of 80–100 mg/day. LY 379,268 appears to act as an agonist at D2^High and as an antagonist at D3, because guanine nucleotide eliminated the competition at D2^High but had no effect on the competition at D3. The findings indicate that this type of glutamate agonist, LY 379,268, has a significant affinity for D2^High and D3 receptors. Synapse 63:705–709, 2009. © 2009 Wiley‐Liss, Inc.     

局灶性脑缺血后海马各区NMDAR表达的实验研究

目的：观察局灶性脑缺血对N－甲基－D－天门冬氨酸受体（NMDAR）及NMDARmRNA表达的影响。方法：采用线栓法制作可复流脑中动脉闭塞大鼠模型。30只雄性大鼠随机分为3组;正常组、假手术组和大脑中动脉闭塞（MCAO）组。MCADO后第5天处死动物,取脑组织进行NR1免疫组化及NR1mRNA原们杂交检测。结果（1）正常组海马各区均有NR1阳性细胞伯分布,其中海马CA3区、DG区分布较多;MCAO组NR1阳性细胞在海马CA1区、CA3区及DG区的数密度、光密度较正常组高;（2）正常组海马各区均有极少量的NR1mRNA阳性细胞分布,MCAO组NR1mRNA阳性细胞在海马CA1区、CA3区的数密度、光密度均较正常组有显著性增高。结论：大鼠海马各区都存在着NR1及NR1mRNA不同程度的表达,MCAO后NR1及NR1mRNA的表达水平上调。     

Diversity in anti-N-methyl-D-aspartate receptor encephalitis: case-based evidence

Antibodies against N-methyl-D-aspartate receptor (NMDAR) are identified in the form of immune-mediated encephalitis in which typical manifestations include neuropsychiatric symptoms, seizures, abnormal movements, dysautonomia and hypoventilation. The authors report two cases of anti-NMDAR encephalitis with different presentations and patterns of progression. The first patient presented with status epilepticus and later developed psychosis, pyramidal signs and diffuse encephalopathy. The second patient presented with acute psychosis followed a week later by seizures, dystonia, rigidity, oromandibular dyskinesias and dysautonomia. Possible mechanisms responsible for the clinical manifestations of this disease are discussed in light of recently described additional clinical and laboratory findings.     

Serial EEG findings in anti‐NMDA receptor encephalitis: correlation between clinical course and EEG

Anti‐NMDA receptor encephalitis is a paraneoplastic encephalitis characterised by psychiatric features, involuntary movement, and autonomic instability. Various EEG findings in patients with anti‐NMDA receptor encephalitis have been reported, however, the correlation between the EEG findings and clinical course of anti‐NMDA receptor encephalitis remains unclear. We describe a patient with anti‐NMDA receptor encephalitis with a focus on EEG findings, which included: status epilepticus, generalised rhythmic delta activity, excess beta activity, extreme delta brush, and paroxysmal alpha activity upon arousal from sleep, which we term“arousal alpha pattern”. Initially, status epilepticus was observed on the EEG when the patient was comatose with conjugate deviation. The EEG then indicated excess beta activity, followed by the emergence of continuous slow activity, including generalised rhythmic delta activity and extreme delta brush, in the most severe phase. Slow activity gradually faded in parallel with clinical amelioration. Excess beta activity persisted, even after the patient became almost independent in daily activities, and finally disappeared with full recovery. In summary, our patient with anti‐NMDA receptor encephalitis demonstrated slow activity on the EEG, including extreme delta brush during the most severe phase, which gradually faded in parallel with clinical amelioration, with excess beta activity persisting into the recovery phase.     

Glutamate agonist LY404,039 for treating schizophrenia has affinity for the dopamine D2High receptor

The glutamate agonist LY404,039 has been used to treat schizophrenia. Because all currently used antipsychotics act on dopamine receptors, it was decided to examine whether this glutamate agonist also had an affinity for dopamine D2 receptors in vitro. The present data show that LY404,039 inhibited the binding of [³H]domperidone and [³H](+)PHNO by 15.5 ± 1.5% to the high‐affinity state, D2^High, of cloned dopamine D2_Long receptors and rat striatal tissue with dissociation constants of between 8.2 and 12.6 nM. This high‐affinity component of LY404,039 on the binding of [³H]domperidone was inhibited by the presence of guanine nucleotide, indicating an agonist action of the drug at D2^High. LY404,039 also stimulated the incorporation of [³⁵S]GTP‐γ‐S into D2_Long receptors (EC50% = 80 ± 15 nM) over the same range of concentrations as occurred for the inhibition of [³H]domperidone by LY404,039 at D2^High (IC50%^High = 50 ± 10 nM). A possible clinical antipsychotic action of LY404,039 may depend on the combined stimulation of glutamate receptors and a partial dopamine agonist action that would interfere with neurotransmission at D2^High receptors. Synapse 63:935–939, 2009. © 2009 Wiley‐Liss, Inc.     

Properties of 4 Hz stimulation‐induced parallel fiber–Purkinje cell presynaptic long‐term plasticity in mouse cerebellar cortex in vivo

Cerebellar parallel fiber–Purkinje cell (PF–PC) long‐term synaptic plasticity is important for the formation and stability of cerebellar neuronal circuits, and provides substrates for motor learning and memory. We previously reported both presynaptic long‐term potentiation (LTP) and long‐term depression (LTD) in cerebellar PF–PC synapses in vitro. However, the expression and mechanisms of cerebellar PF–PC synaptic plasticity in the cerebellar cortex in vivo are poorly understood. In the present study, we studied the properties of 4 Hz stimulation‐induced PF–PC presynaptic long‐term plasticity using in vivo the whole‐cell patch‐clamp recording technique and pharmacological methods in urethane‐anesthetised mice. Our results demonstrated that 4 Hz PF stimulation induced presynaptic LTD of PF–PC synaptic transmission in the intact cerebellar cortex in living mice. The PF–PC presynaptic LTD was attenuated by either the N‐methyl‐D‐aspartate receptor antagonist, D‐aminophosphonovaleric acid, or the group 1 metabotropic glutamate receptor antagonist, JNJ16259685, and was abolished by combined D‐aminophosphonovaleric acid and JNJ16259685, but enhanced by inhibition of nitric oxide synthase. Blockade of cannabinoid type 1 receptor activity abolished the PF–PC LTD and revealed a presynaptic PF–PC LTP. These data indicate that both endocannabinoids and nitric oxide synthase are involved in the 4 Hz stimulation‐induced PF–PC presynaptic plasticity, but the endocannabinoid‐dependent PF–PC presynaptic LTD masked the nitric oxide‐mediated PF–PC presynaptic LTP in the cerebellar cortex in urethane‐anesthetised mice.     

Schwann cells exhibit excitotoxicity consistent with release of NMDA receptor agonists

Neurodegenerative effects of Schwann cells transplanted into the central nervous system have been observed previously. We report here that conditioned medium from Schwann cell cultures exhibit degenerative influences on hippocampal neurons. Aliquots of Schwann cell-conditioned medium compromised the morphologic integrity of the neurons, markedly elevated their intracellular calcium concentrations, and decreased their viability. The degenerative effects of Schwann cell medium on neuronal morphology and viability were blocked by N-methyl-D-aspartate (NMDA) receptor antagonists D-(-)-2-amino-5-phosphonopentanoic acid (D-APV) and 5,7-dicholorokynurenic acid (DCKA). Glutamate was detected in Schwann cell-conditioned medium at a concentration on the order of 10(-5) M. D-Amino acid oxidase (DAAOx) also attenuated the neurotoxicity exhibited by Schwann cells. These data suggest that Schwann cells release biologically relevant concentrations of excitotoxins that include glutamate and D-serine.     

Glutamate and aspartate do not exhibit the same changes in their extracellular concentrations in the rat striatum after N-methyl-D-aspartate local administration

To determine whether glutamate (Glu) and aspartate (Asp) undergo a similar regulation of their extracellular levels, Glu and Asp were simultaneously monitored in the striatum of anesthetized rats after local N-methyl-D-aspartate (NMDA) receptor stimulation, using 1-min in vivo microdialysis coupled to capillary electrophoresis with laser-induced fluorescence detection. Application of NMDA (10 min, 10(-3) M) through the dialysis probe induced 1) an increase (+50%) in Asp during the NMDA administration and 2) a surprising biphasic effect on Glu, with a rapid increase (+30%) and a return to baseline before the end of NMDA application, followed by a second increase (+40%) occurring after and linked to the end of NMDA administration. When studied in the presence of 10 microM tetrodotoxin (TTX) or 0.1 mM Ca(2+), the increase in Asp was partially TTX-dependent, and the early increase in Glu appeared to be partially TTX and Ca(2+) dependent, whereas the second increase in Glu was not. The second increase in Glu level was still present when NMDA antagonists (AP5 or MK-801) were administered at the end of NMDA application. Finally, only extracellular Asp was increased through application of lower NMDA concentrations (10(-4) M, 10(-5) M), whereas extracellular Glu was not affected. In conclusion, these results suggest a differential control of Glu and Asp extracellular levels in rat striatum by distinct mechanisms linked to NMDA receptors and involving neuronal or nonneuronal release.     

Immunohistological and electrophysiological evidence that N‐acetylaspartylglutamate is a co‐transmitter at the vertebrate neuromuscular junction

Immunohistochemical studies previously revealed the presence of the peptide transmitter N‐acetylaspartylglutamate (NAAG) in spinal motor neurons, axons and presumptive neuromuscular junctions (NMJs). At synapses in the central nervous system, NAAG has been shown to activate the type 3 metabotropic glutamate receptor (mGluR3) and is inactivated by an extracellular peptidase, glutamate carboxypeptidase II. The present study tested the hypothesis that NAAG meets the criteria for classification as a co‐transmitter at the vertebrate NMJ. Confocal microscopy confirmed the presence of NAAG immunoreactivity and extended the resolution of the peptide's location in the lizard (Anolis carolinensis) NMJ. NAAG was localised to a presynaptic region immediately adjacent to postsynaptic acetylcholine receptors. NAAG was depleted by potassium‐induced depolarisation and by electrical stimulation of motor axons. The NAAG receptor, mGluR3, was localised to the presynaptic terminal consistent with NAAG's demonstrated role as a regulator of synaptic release at central synapses. In contrast, glutamate receptors, type 2 metabotropic glutamate receptor (mGluR2) and N‐methyl‐d ‐aspartate, were closely associated with acetylcholine receptors in the postsynaptic membrane. Glutamate carboxypeptidase II, the NAAG‐inactivating enzyme, was identified exclusively in perisynaptic glial cells. This localisation was confirmed by the loss of immunoreactivity when these cells were selectively eliminated. Finally, electrophysiological studies showed that exogenous NAAG inhibited evoked neurotransmitter release by activating a group II metabotropic glutamate receptor (mGluR2 or mGluR3). Collectively, these data support the conclusion that NAAG is a co‐transmitter at the vertebrate NMJ.