Genetically fused charged peptides induce rapid crystallization of proteins.

We utilized electrostatic interaction to induce rapid crystallization of streptavidin. Simply mixing streptavidins possessing either a positively or negatively charged peptide at their C-terminus generated diffraction-quality crystals in a few hours. We modified the streptavidin crystals with fluorescent molecules using biotin, demonstrating the concept of protein crystals as functional biomaterials.

The relationship between stroke severity and prior direct oral anticoagulant therapy in patients with acute ischaemic stroke and non-valvular atrial fibrillation.

BACKGROUND AND PURPOSE: Anticoagulant treatment with a vitamin K antagonist (VKA) has been reported to reduce stroke severity when patients with atrial fibrillation (AF) suffer acute ischaemic stroke (AIS). Direct oral anticoagulant (DOAC) therapy also has the potential to reduce the initial severity of AIS. However, the effect of DOAC therapy on the severity of AIS is not well known. The aim of the present study was to investigate the effect of DOACs on initial stroke severity in patients with AIS and non-valvular AF. METHODS: From March 2011 to July 2016, consecutive patients with AIS having non-valvular AF were recruited. The effects of prior DOAC treatment on severity were assessed by multivariate logistic regression analyses. RESULTS: A total of 484 patients [208 women; median age 79 (interquartile range, 71-85) years; National Institutes of Health Stroke Scale (NIHSS) score 9 (interquartile range, 3-20)] were enrolled. Of these, 352 (73%) were on no anticoagulant medication, 54 (11%) were undertreated with a VKA, 35 (7%) were sufficiently treated (admission prothrombin time-international normalized ratio: ≥2.0 for patients <70 years old and ≥1.6 for ≥70 years old) with a VKA and 43 (9%) were on a DOAC. The initial NIHSS score (median 10 in patients with no anticoagulation, 13 in undertreated VKA, 7 in sufficient VKA and 6 in DOAC, P = 0.018) was different among the groups. Multivariate analysis showed that DOAC was independently and negatively associated with severe (initial NIHSS score ≥ 10) stroke (odds ratio, 0.39; P = 0.041), compared with no anticoagulant therapy. CONCLUSIONS: Direct oral anticoagulant treatment prior to the event should reduce initial stroke severity in patients with AIS and non-valvular AF.

Loss of GluN2A-containing NMDA receptors impairs extra-dimensional set-shifting.

Glutamate neurotransmission via the N-methyl-D-aspartate receptor (NMDAR) is thought to mediate the synaptic plasticity underlying learning and memory formation. There is increasing evidence that deficits in NMDAR function are involved in the pathophysiology of cognitive dysfunction seen in neuropsychiatric disorders and addiction. NMDAR subunits confer different physiological properties to the receptor, interact with distinct intracellular postsynaptic scaffolding and signaling molecules, and are differentially expressed during development. Despite these known differences, the relative contribution of individual subunit composition to synaptic plasticity and learning is not fully elucidated. We have previously shown that constitutive deletion of GluN2A subunit in the mouse impairs discrimination and re-learning phase of reversal when exemplars are complex picture stimuli, but spares acquisition and extinction of non-discriminative visually cued instrumental response. To investigate the role of GluN2A containing NMDARs in executive control, we tested GluN2A knockout (GluN2A(KO) ), heterozygous (GluN2A(HET) ) and wild-type (WT) littermates on an attentional set-shifting task using species-specific stimulus dimensions. To further explore the nature of deficits in this model, mice were tested on a visual discrimination reversal paradigm using simplified rotational stimuli. GluN2A(KO) were not impaired on discrimination or reversal problems when tactile or olfactory stimuli were used, or when visual stimuli were sufficiently easy to discriminate. GluN2A(KO) showed a specific and significant impairment in ventromedial prefrontal cortex-mediated set-shifting. Together these results support a role for GluN2A containing NMDAR in modulating executive control that can be masked by overlapping deficits in attentional processes during high task demands.

Phenotypic characterization of orofacial movement topography in mutants with disruption of amino acid mechanisms: glutamate N2A/B/D [GluRε1/2/4] subtypes and the GABA synthesizing enzyme GAD65.

To investigate the role of glutamate receptor subtypes and GABA in orofacial function, six individual topographies of orofacial movement, both spontaneous and induced by the dopamine D1-like receptor agonist [R/S]-3-methyl-6-chloro-7,8-dihydroxy-1-[3-methyl-phenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF 83959), were quantified in mutant mice with deletion of (a) GluN2A, B or D receptors, and (b) the GABA synthesizing enzyme, 65-kD isoform of glutamate decarboxylase (GAD65). In GluN2A mutants, habituation of head movements was disrupted and vibrissae movements were reduced, with an overall increase in locomotion; responsivity to SKF 83959 was unaltered. In GluN2B mutants, vertical and horizontal jaw movements and incisor chattering were increased, with an overall decrease in locomotion; under challenge with SKF 83959, head and vibrissae movements were reduced. In GluN2D mutants, horizontal jaw movements, incisor chattering and vibrissae movements were increased, with reduced tongue protrusions and no overall change in locomotion; under challenge with SKF 83959, horizontal jaw movements were increased. In GAD65 mutants, vertical jaw movements were increased, with disruption to habituation of locomotion; under challenge with SKF 83959, vertical and horizontal jaw movements and incisor chattering were decreased. Effects on orofacial movements differed from their effects on regulation of overall locomotor behavior. These findings (a) indicate novel, differential roles for GluN2A, B and D receptors and for GAD65-mediated GABA in the regulation of individual topographies of orofacial movement and (b) reveal how these roles differ from and/or interact with the established role of D1-like receptors in pattern generators and effectors for such movements.

Impact of polyunsaturated fatty acid consumption prior to ischemic stroke.

OBJECTIVE: The Japanese have higher levels of n-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in their diets. These facts may contribute to the lower rates of atherosclerosis in Japanese. The purposes of this study were to assess the PUFA levels in patients with subtypes of acute ischemic stroke and to assess the relationship between severity and PUFA levels. MATERIAL AND METHODS: We studied 75 patients with lacunar infarction (LI; n = 25), atherothrombotic infarction (AT; n = 32), and cardiogenic embolism (CE; n = 18). The patients underwent blood examinations in a fasting state next morning of hospitalization, including examination of low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), triglyceride (TG), blood glucose, hemoglobin A1c (HbA1c), uric acid, and fatty acid fractions of EPA, DHA, dihomo-γ-linolenic acid (DGLA), and arachidonic acid (AA). We used the modified Rankin Scale (mRS) to assess clinical severity at discharge. RESULTS: There was no significant difference in the EPA/AA and DHA/AA ratio among the three stroke subgroups, although the DGLA/AA ratio was significantly higher in patients with LI than in patients with CE. Considering the confounding factors, the mRS was negatively correlated with EPA/AA and positively correlated with age, DHA/AA, and blood glucose. CONCLUSIONS: High EPA/AA ratio was associated with good outcome in ischemic stroke. Our paper suggests that prestroke dietary habits affect the severity in patients with ischemic stroke.

Striatal Distribution of Dopamine Transporters and Dopamine D2 Receptors at Different Stages of Parkinson's Disease. A CFT and RAC PET Study.

We investigated the alteration of dopaminergic system in striata of Parkinson's disease (PD) at different stages using positron emission tomography (PET), [(11)C]2ß-carbomethoxy-3ß-(4-fluorophenyl)tropane (CFT) for dopamine transporter (DAT), and [(11)C]raclopride (RAC) for dopamine D2 receptor (D2R). We studied eight elderly healthy volunteers (Group A), 13 drug naïve patients with PD (Group B), and seven advanced PD patients with mild dyskinesia (Group D). Six patients in Group B were re-examined after antiparkinsonian therapy (Group C). Regions of interest were drawn on the cerebellar hemisphere, head of the caudate nucleus (CN), and anterior (AP) and posterior putamen (PP) in the PET images. We calculated uptake ratio index (URI), asymmetry index (AI) and presynapse-to-postsynapse ratio (PPR) to evaluate dopaminergic function. DAT was smaller in the three PD groups than the Group A. URI of RAC in the PP was significantly larger in Group B than in Groups A and C. AI of CFT in the putamen was larger in the PD groups than in normal subjects, and AI of RAC in the PP was the largest in the Group B. PPRs in the AP and PP were smaller in the three PD groups than in Group A. DAT decreased with disease progression in patients with PD. Binding of RAC was largest in the putamen of drug-naïve PD patients, but the enhanced binding could not be detected in the therapeutic patients with PD because of weak D2R affinity of RAC.

Influence of parallel fiber-Purkinje cell synapse formation on postnatal development of climbing fiber-Purkinje cell synapses in the cerebellum.

The climbing fiber (CF) to Purkinje cell (PC) synapse in the cerebellum provides an ideal model for the study of developmental rearrangements of neural circuits. At birth, each PC is innervated by multiple CFs. These surplus CFs are eliminated during postnatal development, and mono innervation is attained by postnatal day 20 (P20) in mice. Earlier studies on spontaneous mutant mice and animals with "hypogranular" cerebella indicate that regression of surplus CFs requires normal generation of granule cells and their axons, parallel fibers (PFs), and normal formation of PF-PC synapses. Our understanding of how PF-PC synapse formation affects development of CF-PC synapse has been greatly advanced by analyses of mutant mice deficient in glutamate receptor delta2 subunit (GluRdelta2), an orphan receptor expressed selectively in PCs. Deletion of GluRdelta2 results in impairment of PF-PC synapse formation, which leads to defects in development of CF-PC synapses. In this article, we review how impaired PF-PC synapse formation affects wiring of CFs to PCs based mostly on our data on GluRdelta2 knockout mice. We propose a new scheme that CF-PC synapses are shaped by the three consecutive events, namely functional differentiation of multiple CFs into one strong and a few weak inputs from P3 to P7, "early phase" of CF synapse elimination from P7 to around P11, and "late phase" of CF synapse elimination from around P12. Normal PF-PC synapse formation is required for the "late phase" of CF synapse elimination.

Short-term plasticity of central benzodiazepine receptors in status epilepticus: case report.

(123)I-iomazenil SPECT is of value in determining an epileptogenic focus, however, transient uptake change has been rarely reported in epileptic disorders. A 78-year-old woman diagnosed as status epilepticus (SE) showed transient reduction in (123)I-iomazenil uptake within the epileptic foci on SPECT images during a couple of weeks. It suggests a seizure-related 'short-term' plasticity in the central benzodiazepine receptors and dynamic change in the regulatory mechanisms of inhibitory neurotransmitter system within the epileptic foci in patients with SE.

[Fusion proteins encoded by orf 129L of ectromelia and orf A30L of smallpox viruses cross-react with neutralizing monoclonal antibodies].

Open reading frame (orf) 129L of ectromelia (EV) and orf A30L of smallpox viruses (SPV) encoding fusion proteins were cloned and expressed in E. coli cells. The recombinant polypeptides (prA30L H pr129L) were purified from cell lysates by Ni-NTA chromatography. Recombinant polypeptides were able to form trimers in buffered saline and they destroyed under treatment with SDS and 2-mercaptoethanol. Reactivity of prA30L, pr129L and orthopoxvirus proteins was analyzed by ELISA and Western blotting with panel of 22 monoclonal antibodies (MAbs) against orthopoxviruses (19 against EV, 2 MAbs against vaccinia virus and 1 Mabs against cowpox virus). This data allowed us to conclude that there are 12 EV-specific epitopes of pr129L and EV fusion proteins, ten orthopox-specific epitopes of EV, VV, CPV fusion proteins, from them 9 orthopox-specific epitopes of prA30L and SPV fusion proteins. Five Mabs, which cross-reacted with orthopox-specific epitopes, were able to neutralize the VV on Vero cells and from them two MAbs has neutralizing activity against smallpox virus. Our findings demonstrate that 129L fusion protein have EV-specific epitopes, that EV 129L and SPV A30L fusion proteins have a several orthopox-specific epitopes to induce a neutralizing antibodies against human pathogenic orthopoxviruses.

N-methyl-D-aspartate receptors play important roles in acquisition and expression of the eyeblink conditioned response in glutamate receptor subunit delta2 mutant mice.

Classical eyeblink conditioning has been known to depend critically on the cerebellum. Apparently consistent with this, glutamate receptor subunit delta2 null mutant mice, which have serious morphological and functional deficiencies in the cerebellar cortex, are severely impaired in delay paradigm. However, these mutant mice successfully learn in trace paradigm, even in '0-trace paradigm,' in which the unconditioned stimulus starts just after the conditioned stimulus terminates. Our previous studies revealed that the hippocampus and the muscarinic acetylcholine receptors play crucial roles in 0-trace paradigm in glutamate receptor subunit delta2 null mutant mice unlike in wild-type mice, suggesting a large contribution of the forebrain to 0-trace conditioning in this type of mutant mice. In the present study, we investigated the role of N-methyl-D-aspartate receptors in 0-trace eyeblink conditioning in glutamate receptor subunit delta2 null mutant mice. Mice were injected intraperitoneally with the noncompetitive N-methyl-d-aspartate receptor antagonist (+)MK-801 (0.1mg/kg) or saline, and conditioned with 350-ms tone conditioned stimulus followed by 100-ms periorbital shock unconditioned stimulus. Glutamate receptor subunit delta2 null mutant mice that received (+)MK-801 injection exhibited a severe impairment in acquisition of the conditioned response, compared with the saline-injected glutamate receptor subunit delta2 null mutant mice. In contrast, wild-type mice were not impaired in acquisition of 0-trace conditioned response by (+)MK-801 injection. After the injection solution was changed from (+)MK-801 to saline, glutamate receptor subunit delta2 null mutant mice showed a rapid and partial recovery of performance of the conditioned response. On the other hand, when the injection solution was changed from saline to (+)MK-801, glutamate receptor subunit delta2 null mutant mice showed a marked impairment in expression of the pre-acquired conditioned response, whereas impairment of the expression was small in wild-type mice. Injection of (+)MK-801 had no significant effects on spontaneous eyeblink frequency or startle eyeblink frequency to the tone conditioned stimulus in either glutamate receptor subunit delta2 null mutant mice or wild-type mice. These results suggest that N-methyl-D-aspartate receptors play critical roles both in acquisition and expression of the conditioned response in 0-trace eyeblink conditioning in glutamate receptor subunit delta2 null mutant mice.

[Species-specific detection of Proteus vulgaris and Proteus mirabilis by the polymerase chain reaction].

Sets of primers for the species-specific detection of P. mirabilis and P. vulgaris by the polymerase chain reaction (PCR) were developed. As targets for these primers beta-lactamase and 16S rRNA gene fragments were chosen on the basis of the multiple leveling of the sequences of the DNA of all known P. mirabilis and P. vulgaris isolates. For differential detection oligonucleotides were selected in such a way that primers, specific for P. vulgaris, contained the non-paired nucleotide for P. mirabilis isolate at the 3'-end, and all other nucleotides were complementary to the beta-lactamase gene fragment. Primers, specific for gene 16S rRNA of P. mirabilis, contained the non-paired nucleotide for P. vulgaris isolates at the 3'-end. Standard PCR was carried out for 6 P. mirabilis and P. vulgaris strains. The use of PCR species-specific primers to P. vulgaris DNA made it possible to amplify the DNA fragment of the expected length only for P. vulgaris isolates, while the result of PCR for P. mirabilis was negative. PCR with primers specific to P. mirabilis permitted the detection of amplicon sized 101 nucleotides pairs only for P. mirabilis strains. These primers were optimized so as to use them in the specific differentiation of closely related P. mirabilis and P. vulgaris species by multiplex PCR. Genus-specific primers permitted the detection of bacterial gyrB gene of the genus Proteus were developed also.

Function of sigma1 receptors in Parkinson's disease.

OBJECTIVE: The objective of this study was to investigate the mapping of sigma1 receptors in Parkinson's disease (PD) using [11C]SA4503 and positron emission tomography (PET), and to assess whether sigma1 receptors are involved in the damaged dopaminergic system in PD patients. MATERIALS AND METHODS: We studied seven normal volunteers and six PD patients. The low density of dopamine transporters and the normal or high density of dopamine receptors were confirmed in the putamen of all patients using [11C]CFT and [11C]RAC PET. A dynamic series of PET data acquisition was performed with arterial blood sampling. We computed the binding potential (BP) of [11C]SA4503. RESULTS: In PD patients, the BP was significantly lower on the more affected than the less affected side of the anterior putamen, although there was no significant difference with respect to the BP between patients and controls. CONCLUSIONS: Release of dopamine is reduced asymmetrically in the putamen of early PD. [11C]SA4503 PET is an indicator of presynaptic dopaminergic damage in PD.

Midbrain hypometabolism as early diagnostic sign for progressive supranuclear palsy.

OBJECTIVE: Progressive supranuclear palsy (PSP) is often misdiagnosed in early phase. The purpose of this study is to investigate the feature of [(18)F]fluoro-2-deoxy-d-glucose (FDG) positron emission tomography images for the early diagnosis of PSP. METHODS: We studied 15 patients with PSP and 16 normal subjects. Using SPM99 and analysis of covariance to eliminate the effect of aging, the differences between PSP and normals were displayed as a statistical map. In the PSP, we also investigated the correlation with duration and with the subscores of Unified Parkinson's Disease Rating Scale. RESULTS: The glucose metabolism of midbrain was significantly lower in PSP than in normals. However, correlation was not found between the metabolism of midbrain and clinical deterioration. CONCLUSIONS: The statistical map clearly demonstrated the hypometabolism of midbrain in PSP, which is independent of the clinical deterioration. The hypometabolism of midbrain is one of the most promising sign for early diagnosis of PSP.

Chronopharmacological studies of ketamine in normal and NMDA epsilon1 receptor knockout mice.

BACKGROUND: The effectiveness and toxicity of many drugs depends on the dosing-time schedule, relative to the circadian rhythms of biochemical, physiological, and behavioural processes. Previous studies have found chronopharmacology of ketamine, which is a N-methyl-d-aspartate (NMDA) receptor antagonist. The in vivo contribution of the NMDA receptor epsilon1 subunit (NR2A) in this effect is unclear. METHODS: In the present study, daily variations in the hypnotic effect of ketamine were determined in wild-type mice and NMDA epsilon1 knockout (KO) mice. RESULTS: The effect of ketamine had a definite daily variation in wild-type mice. No significant difference in blood concentration was observed at different dosing times (10:00 and 22:00). In NMDA receptor epsilon1 KO mice, the hypnotic effect of ketamine was weaker than in wild-type mice and there was no dependence on the time of administration. Significant pharmacokinetic differences were not observed between wild-type and KO mice. CONCLUSIONS: The enhanced hypnotic effect in the active phase of the circadian cycle is likely a result of changes with the time of day in the susceptibility of the central nervous system to ketamine. Knockout of the NMDA receptor epsilon1 subunit gene markedly reduced the effect of ketamine, and eliminated the time-dependent sensitivity to ketamine.

Autoantibodies to NMDA receptor in patients with chronic forms of epilepsia partialis continua.

BACKGROUND: Antibody-mediated and cytotoxic T cell-mediated pathogenicity have been implicated as the autoimmune pathophysiologic mechanisms in Rasmussen's encephalitis. METHODS: The authors investigated autoantibodies against the NMDA glutamate receptor (GluR) epsilon2 subunit and their epitopes in serum and CSF samples from 15 patients with chronic epilepsia partialis continua (EPC), 17 with West syndrome, 10 with Lennox-Gastaut syndrome, and 11 control subjects. RESULTS: In 15 patients with chronic EPC, we detected NMDA-type GluR epsilon2 autoantibodies in histologically proven Rasmussen's encephalitis (3/3 patients), clinical Rasmussen's encephalitis (6/7 patients), acute encephalitis/encephalopathy (2/3 patients), and nonprogressive EPC (2/2 patients). Serum IgM autoantibodies were found in the early phase of EPC and became negative later in four patients. The autoantibodies were not detected in West syndrome, Lennox-Gastaut syndrome, or controls. Among 10 patients with histologically proven or clinical Rasmussen's encephalitis, epitope analyses showed that the autoantibodies were predominantly against C-terminal epitopes and rarely against N-terminal epitope, with inconsistency in profile during the courses of disease. Epitope recognition spectrum of autoantibodies was broader in CSF than in serum, and the serum or CSF profile showed an increase in number of epitopes as disease progressed in some patients. CONCLUSIONS: The presence of autoantibodies against NMDA GluR epsilon2 suggests autoimmune pathologic mechanisms but is not a hallmark of Rasmussen's encephalitis. Patients with Rasmussen's encephalitis may have autoantibodies against several neural molecules, and these autoantibodies may be produced in the CNS after cytotoxic T cell-mediated neuronal damage.

Enhancement of neurogenesis by running wheel exercises is suppressed in mice lacking NMDA receptor epsilon 1 subunit.

Neurogenesis continues throughout adulthood in the dentate gyrus in mice, and is regulated by environmental, endocrine, and pharmacological stimuli. Although running wheel exercises have been reported to enhance neurogenesis, details of molecule mechanisms of the enhancement are not well understood. We report here that the hippocampal neurogenesis is enhanced when wild-type mice are raised in cages with running wheels for 3 weeks, but the wheel exercise does not enhance the neurogenesis in mice lacking the NMDA receptor epsilon1 subunit. Brain-derived neurotrophic factor (BDNF) has been reported to affect neuronal cell proliferation and survival. We examined the BDNF levels in the hippocampi of wild-type and epsilon1 knockout mice, and found that the BDNF level was increased through wheel exercises in the wild-type but not in the knockout mice. The enhancement of neurogenesis by the wheel exercise was also found to be reversible: when the exercise-stimulated wild-type mice were returned to the environment without running wheels for 3 weeks, the neurogenesis was the same as that in the mice which had never experienced the exercise. These results suggest that the wheel exercise may activate NMDA receptors in the hippocampus, which in turn may enhance BDNF production and neurogenesis.

Developmental loss of miniature N-methyl-D-aspartate receptor currents in NR2A knockout mice.

The N-methyl-d-aspartate (NMDA) glutamate receptor (NMDAR), long implicated in developmental plasticity, shows decay time kinetics that shorten postnatally as NR2A subunits are added to the receptor. Neither the mechanism nor immediate effect of this change is known. We studied developing NMDAR currents by using visual neurons in slices from NR2A knockout (NR2AKO) and WT mice. Both strains show increased dendritic levels of synaptic density scaffolding protein PSD-95 with age. Dendritic levels of NR2A increased at the same time in WT and immunoprecipitated with PSD-95. PSD-95NMDAR binding was significantly decreased in the NR2AKO. Moreover, NMDAR miniature currents (minis) were lost and rise times of NMDAR evoked currents increased in mutant mice. Age-matched WT cells showed NR2A-rich receptors predominating in minis, yet slow NR2B mediated currents persisted in evoked currents. Disrupting photoreceptor activation of retinal ganglion cells eliminated increases in PSD-95 and NR2A in superior collicular dendrites of WT mice and slowed the loss of miniature NMDAR currents in NR2AKOs. These data demonstrate that NMDARs that respond to single quantal events mature faster during development by expressing the NR2A subunit earlier than NMDARs that respond to evoked release. We hypothesize that NR2A-rich NMDARs may be localized to the center of developing synapses by an activity-dependent process that involves the targeting of PSD-95 to the postsynaptic density. Neonatal receptors become restricted to perisynpatic or extrasynaptic sites, where they participate primarily in evoked currents.

Hippocampal Damage Disrupts Eyeblink Conditioning in Mice Lacking Glutamate Receptor Subunit δ2.

Cerebellar long-term depression (LTD) at the parallel fiber-Purkinje cell synapses has been proposed to be a neural substrate for classical eyeblink conditioning. Mutant mice lacking the glutamate receptor subunit δ2 (GluRδ2), in which the cerebellar LTD is disrupted, exhibited a severe impairment in the delay eyeblink conditioning with a temporal overlap of CS and US. However, they learned normally trace and delay conditioning without CS-US overlap, suggesting a learning mechanism which does not require the cerebellar LTD.In the present study, we tested possible involvement of the hippocampus in this cerebellar LTD-independent learning. We examined effects of scopolamine and hippocampal lesion on the delay conditioning without CS-US overlap. TheGluRδ2 mutant mice that received scopolamine or aspiration of the dorsalhippocampus together with its overlying cortex exhibited a severe impairment in learning, while the control mutant mice that received saline or aspiration of the overlying cortex learned normally. In contrast, wild-type mice that received either treatment learned as normally as the control wild-type mice. These results suggest that the hippocampus is essential in the cerebellar LTD-independent learning in the GluRδ2 mutant mice, indicating a newrole of hippocampus in the paradigm with a short trace interval.

Roles of glutamate receptor delta 2 subunit (GluRdelta 2) and metabotropic glutamate receptor subtype 1 (mGluR1) in climbing fiber synapse elimination during postnatal cerebellar development.

Climbing fiber (CF) synapse formation onto cerebellar Purkinje cells (PCs) is critically dependent on the synaptogenesis from parallel fibers (PFs), the other input to PCs. Previous studies revealed that deletion of the glutamate receptor delta2 subunit (GluRdelta2) gene results in persistent multiple CF innervation of PCs with impaired PF synaptogenesis, whereas mutation of the metabotropic glutamate receptor subtype 1 (mGluR1) gene causes multiple CF innervation with normal PF synaptogenesis. We demonstrate that atypical CF-mediated EPSCs (CF-EPSCs) with slow rise times and small amplitudes coexisted with typical CF-EPSCs with fast rise times and large amplitudes in PCs from GluRdelta2 mutant cerebellar slices. CF-EPSCs in mGluR1 mutant and wild-type PCs had fast rise times. Atypical slow CF responses of GluRdelta2 mutant PCs were associated with voltage-dependent Ca(2+) signals that were confined to PC distal dendrites. In the wild-type and mGluR1 mutant PCs, CF-induced Ca(2+) signals involved both proximal and distal dendrites. Morphologically, CFs of GluRdelta2 mutant mice extended to the superficial regions of the molecular layer, whereas those of wild-type and mGluR1 mutant mice did not innervate the superficial one-fifth of the molecular layer. It is therefore likely that surplus CFs of GluRdelta2 mutant mice form ectopic synapses onto distal dendrites, whereas those of wild-type and mGluR1 mutant mice innervate proximal dendrites. These findings suggest that GluRdelta2 is required for consolidating PF synapses and restricting CF synapses to the proximal dendrites, whereas the mGluR1-signaling pathway does not affect PF synaptogenesis but is involved in eliminating surplus CF synapses at the proximal dendrites.