The N-methyl-d-aspartate receptor (NMDAR) plays various physiological and pathological roles in neural development, synaptic plasticity and neuronal cell death. It is composed of two GluN1 and two GluN2 subunits and, in the neonatal hippocampus, most synaptic NMDARs are GluN2B-containing receptors, which are gradually replaced with GluN2A-containing receptors during development. Here, we examined whether GluN2A could be substituted for GluN2B in neural development and functions by analysing knock-in (KI) mice in which GluN2B is replaced with GluN2A. The KI mutation was neonatally lethal, although GluN2A-containing receptors were transported to the postsynaptic membrane even without GluN2B and functional at synapses of acute hippocampal slices of postnatal day 0, indicating that GluN2A-containing NMDARs could not be substituted for GluN2B-containing NMDARs. Importantly, the synaptic ?-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit GluA1 was increased, and the transmembrane AMPAR regulatory protein, which is involved in AMPAR synaptic trafficking, was increased in KI mice. Although the regulation of AMPARs by GluN2B has been reported in cultured neurons, we showed here that AMPAR-mediated synaptic responses were increased in acute KI slices, suggesting differential roles of GluN2A and GluN2B in AMPAR expression and trafficking in vivo. Taken together, our results suggest that GluN2B is essential for the survival of animals, and that the GluN2B-GluN2A switching plays a critical role in synaptic integration of AMPARs through regulation of GluA1 in the whole animal.
Thiamine (vitamin B1) deficiency (TD) leads to focal brain necrosis in particular brain regions in humans and in experimental animal models. The precise mechanism of the selective topographic vulnerability triggered by TD still remains unclear. We examined the distribution pattern of cell death in the brains of mice in an experimental model of TD using anti-single-strand DNA immunohistochemistry and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling methods. We found that interneurons in the olfactory bulb were sensitive to TD. The morphologic aspects of cell death in the olfactory bulb resembled those of cell death in thalamic neurons, which have previously been examined in detail. Furthermore, cell death in the olfactory bulb was partly relieved by the administration of an N-methyl-D-aspartate receptor antagonist, as was the case in thalamic lesions by TD. The superficial part of the olfactory granule cell layer seemed to be the most sensitive to TD, suggesting that differences in the afferents between superficial and deep granule cells may influence the sensitivity of these cells to TD. Our results indicate that the olfactory bulb should be considered as one of the vulnerable regions to TD.
Although the apoptotic role of caspases has been largely understood, accumulating evidence in Drosophila suggests that caspases also control other processes than apoptotic cell death. However, how caspases contribute to the development of the mammalian nervous system remains obscure. Here, we provide unique evidence that Apaf-1/caspase-9-mediated caspase signaling regulates the development of olfactory sensory neurons (OSNs), which includes axonal projection, synapse formation, and maturation of these neurons. This caspase signaling leads to a cleavage of Semaphorin 7A, a membrane-anchored semaphorin that is required for the proper axonal projection. Mutant mice deficient for apaf-1 or caspase-9 exhibit misrouted axons, impaired synaptic formation, and defects in the maturation of OSNs without affecting the number of these cells. Our findings suggest that Apaf-1/caspase-9-mediated nonapoptotic caspase signaling is required for the proper neural network formation during olfactory development.
Caspases are essential in multicellular organisms for inducing cell death during normal development and in the immune system. However, caspases can also trigger the degenerative process under certain conditions such as pathophysiological conditions and aging. Here, we identified Semaphorin 7A (Sema7A) as a novel substrate for caspase-9 that can be used to monitor caspase-9 activity in mice, and found nonapoptotic caspase-9 activation in the aged olfactory bulb (OB). Immunostaining of the OB for the caspase-9-cleaved form of Sema7A revealed abundant caspase-9-activated cells in 2-year-old (aged) but not in 2-month-old (young) mice. In fact, various regions of the aged brain, including the OB, exhibited an increased level of caspase-9 activity. However, the number of dying cells in the aged OB was, intriguingly, much lower (<20%) than in the OB of young mice. Furthermore, we found that the lower number dying cells in the aged OB was accompanied by a decreased expression of procaspase-3. These results suggest a survival strategy for aged OB neurons, which can no longer regenerate, in which the central apoptotic machinery downstream of caspase-9 is inactivated.
Serotonergic axons from the raphe nuclei in the brainstem project to every region of the brain, where they make connections through their extensive terminal arborizations. This serotonergic innervation contributes to various normal behaviors and psychiatric disorders. The protocadherin-alpha (Pcdha) family of clustered protocadherins consists of 14 cadherin-related molecules generated from a single gene cluster. We found that the Pcdhas were strongly expressed in the serotonergic neurons. To elucidate their roles, we examined serotonergic fibers in a mouse mutant (Pcdha(Delta CR/Delta CR)) lacking the Pcdha cytoplasmic region-encoding exons, which are common to the gene cluster. In the first week after birth, the distribution pattern of serotonergic fibers in Pcdha(Delta CR/Delta CR) mice was similar to wild-type, but by 3 weeks of age, when the serotonergic axonal termini complete their arborizations, the distribution of the projections was abnormal. In some target regions, notably the globus pallidus and substantia nigra, the normally even distribution of serotonin axonal terminals was, in the mutants, dense at the periphery of each region, but sparse in the center. In the stratum lacunosum-molecular of the hippocampus, the mutants showed denser serotonergic innervation than in wild-type, and in the dentate gyrus of the hippocampus and the caudate-putamen, the innervation was sparser. Together, the abnormalities suggested that Pcdha proteins are important in the late-stage maturation of serotonergic projections. Further examination of alternatively spliced exons encoding the cytoplasmic tail showed that the A-type (but not the B-type) cytoplasmic tail was essential for the normal development of serotonergic projections.
Fyn, a Src-family kinase, is highly expressed in brain tissue and blood cells. In the mouse brain, Fyn participates in brain development, synaptic transmission through the phosphorylation of N-methyl-d-aspartate (NMDA) receptor subunits, and the regulation of emotional behavior. Recently, we found that Fyn is required for the signal transduction in striatal neurons that is initiated by haloperidol, an antipsychotic drug. To determine whether Fyn abnormalities are present in patients with schizophrenia, we analyzed Fyn expression in platelet samples from 110 patients with schizophrenia, 75 of the patients first-degree relatives, and 130 control subjects. A Western blot analysis revealed significantly lower levels of Fyn protein among the patients with schizophrenia and their relatives, compared with the level in the control group. At the mRNA level, the splicing patterns of fyn were altered in the patients and their relatives; specifically, the ratio of fynDelta7, in which exon 7 is absent, was elevated. An expression study in HEK293T cells revealed that FynDelta7 had a dominant-negative effect on the phosphorylation of Fyns substrate. These results suggest novel deficits in Fyn function, manifested as the downregulation of Fyn protein or the altered transcription of the fyn gene, in patients with schizophrenia.
The GLW-amide family is a neuropeptide family found in cnidarian species and is characterized by the C-terminal amino acid sequence -Gly-Leu-Trp-NH(2). To detect mammalian peptides structurally related to the GLW-amide family, we examined rat brain by immunohistochemistry with an anti-GLW-amide antibody. GLW-amide-like immunoreactivity (GLW-amide-LI) was observed in thin varicose fibers in some regions of the brain. Most neurons showing GLW-amide-LI were observed in the laterodorsal tegmental nucleus, pedunculopontine tegmental nucleus, and trigeminal/spinal ganglia. These results strongly suggest that the rat nervous system contains as yet unidentified GLW-amide-like peptides, and that GLW-amide-LI in the brain is a good marker for ascending projections from mesopontine cholinergic neurons.
Olfactory sensory neuron (OSN) axons coalesce into specific glomeruli in the olfactory bulb (OB) according to their odorant receptor (OR) expression. Several guidance molecules enhance the coalescence of homotypic OSN projections, in an OR-specific- and neural-activity-dependent manner. However, the mechanism by which homotypic OSN axons are organized into glomeruli is unsolved. We previously reported that the clustered protocadherin-? (Pcdh-?) family of diverse cadherin-related molecules plays roles in the coalescence and elimination of homotypic OSN axons throughout development. Here we showed that the elimination of small ectopic homotypic glomeruli required the constitutive expression of a Pcdh-? isoform and Pcdh-?s cytoplasmic region, but not OR specificity or neural activity. These results suggest that Pcdh-? proteins provide a cytoplasmic signal to regulate repulsive activity for homotypic OSN axons independently of OR expression and neural activity. The counterbalancing effect of Pcdh-? proteins for the axonal coalescence mechanisms mediated by other olfactory guidance molecules indicate a possible mechanism for the organization of homotypic OSN axons into glomeruli during development.
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