Rie Natsume

Impairment of suckling response, trigeminal neuronal pattern formation, and hippocampal LTD in NMDA receptor ε2 subunit mutant mice

Multiple epsilon subunits are major determinants of the NMDA receptor channel diversity. Based on their functional properties in vitro and distributions, we have proposed that the epsilon 1 and epsilon 2 subunits play a role in synaptic plasticity. To investigate the physiological significance of the NMDA receptor channel diversity, we generated mutant mice defective in the epsilon 2 subunit. These mice showed no suckling response and died shortly after birth but could survive by hand feeding. The mutation hindered the formation of the whisker-related neuronal barrelette structure and the clustering of primary sensory afferent terminals in the brainstem trigeminal nucleus. In the hippocampus of the mutant mice, synaptic NMDA responses and longterm depression were abolished. These results suggest that the epsilon 2 subunit plays an essential role in both neuronal pattern formation and synaptic plasticity.

Serine racemase is predominantly localized in neurons in mouse brain

D‐Serine is the endogenous ligand for the glycine binding site of the N‐methyl‐D‐aspartate (NMDA)‐type glutamate receptor (GluR) channel and is involved in the regulation of synaptic plasticity, neural network formation, and neurodegenerative disorders. D‐Serine is synthesized from L‐serine by serine racemase (SR), which was first reported to be localized in astrocytes. However, recently, SR mRNA and its protein have been detected in neurons. In this study, we examined the SR distribution in the brain during postnatal development and in cultured cells by using novel SR knockout mice as negative controls. We found that SR is predominantly localized in pyramidal neurons in the cerebral cortex and hippocampal CA1 region. Double immunofluorescence staining revealed that SR signals colocalized with those of the neuron‐specific nuclear protein, but not with the astrocytic markers glial fibrillary acid protein and 3‐phosphoglycerate dehydrogenase. In the striatum, we observed SR expression in γ‐aminobutyric acid (GABA)ergic medium‐spiny neurons. Furthermore, in the adult cerebellum, we detected weak but significant SR signals in GABAergic Purkinje cells. From these findings, we conclude that SR is expressed predominantly in many types of neuron in the brain and plays a key role in the regulation of brain functions under physiological and pathological conditions via the production of the neuromodulator D‐serine. J. Comp. Neurol. 510:641–654, 2008.

Abundant distribution of TARP γ-8 in synaptic and extrasynaptic surface of hippocampal neurons and its major role in AMPA receptor expression on spines and dendrites

Transmembrane α‐amino‐3‐hydroxyl‐5‐isoxazolepropionate (AMPA) receptor regulatory proteins (TARPs) play pivotal roles in AMPA receptor trafficking and gating. Here we examined cellular and subcellular distribution of TARP γ‐8 in the mouse brain. Immunoblot and immunofluorescence revealed the highest concentration of γ‐8 in the hippocampus. Immunogold electron microscopy demonstrated dense distribution of γ‐8 on the synaptic and extrasynaptic surface of hippocampal neurons with very low intracellular labeling. Of the neuronal surface, γ‐8 was distributed at the highest level on asymmetrical synapses of pyramidal cells and interneurons, whereas their symmetrical synapses selectively lacked immunogold labeling. Then, the role of γ‐8 in AMPA receptor expression was pursued in the hippocampus using mutant mice defective in the γ‐8 gene. In the mutant cornu ammonis (CA)1 region, synaptic and extrasynaptic AMPA receptors on dendrites and spines were severely reduced to 35–37% of control levels, whereas reduction was mild for extrasynaptic receptors on somata (74%) and no significant decrease was seen for intracellular receptors within spines. In the mutant CA3 region, synaptic AMPA receptors were reduced mildly at asymmetrical synapses in the stratum radiatum (67% of control level), and showed no significant decrease at mossy fiber–CA3 synapses. Therefore, γ‐8 is abundantly distributed on hippocampal excitatory synapses and extrasynaptic membranes, and plays an important role in increasing the number of synaptic and extrasynaptic AMPA receptors on dendrites and spines, particularly, in the CA1 region. Variable degrees of reduction further suggest that other TARPs may also mediate this function at different potencies depending on hippocampal subregions, input sources and neuronal compartments.

NMDA Receptor GluN2B (GluRε2/NR2B) Subunit Is Crucial for Channel Function, Postsynaptic Macromolecular Organization, and Actin Cytoskeleton at Hippocampal CA3 Synapses

GluN2B (GluRε2/NR2B) subunit is involved in synapse development, synaptic plasticity, and cognitive function. However, its roles in synaptic expression and function of NMDA receptors (NMDARs) in the brain remain mostly unknown because of the neonatal lethality of global knock-out mice. To address this, we generated conditional knock-out mice, in which GluN2B was ablated exclusively in hippocampal CA3 pyramidal cells. By immunohistochemistry, GluN2B disappeared and GluN1 (GluRζ1/NR1) was moderately reduced, whereas GluN2A (GluRε1/NR2A) and postsynaptic density-95 (PSD-95) were unaltered in the mutant CA3. This was consistent with protein contents in the CA3 crude fraction: 9.6% of control level for GluN2B, 47.7% for GluN1, 90.6% for GluN2A, and 98.0% for PSD-95. Despite the remaining NMDARs, NMDAR-mediated currents and long-term potentiation were virtually lost at various CA3 synapses. Then, we compared synaptic NMDARs by postembedding immunogold electron microscopy and immunoblot using the PSD fraction. In the mutant CA3, GluN1 was severely reduced in both immunogold (20.6-23.6%) and immunoblot (24.6%), whereas GluN2A and PSD-95 were unchanged in immunogold but markedly reduced in the PSD fraction (51.4 and 36.5%, respectively), indicating increased detergent solubility of PSD molecules. No such increased solubility was observed for GluN2B in the CA3 of GluN2A-knock-out mice. Furthermore, significant decreases were found in the ratio of filamentous to globular actin (49.5%) and in the density of dendritic spines (76.2%). These findings suggest that GluN2B is critically involved in NMDAR channel function, organization of postsynaptic macromolecular complexes, formation or maintenance of dendritic spines, and regulation of the actin cytoskeleton.

TARPs γ-2 and γ-7 are essential for AMPA receptor expression in the cerebellum

The α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)‐type glutamate receptors require auxiliary subunits termed transmembrane AMPA receptor regulatory proteins (TARPs), which promote receptor trafficking to the cell surface and synapses and modulate channel pharmacology and gating. Of six TARPs, γ‐2 and γ‐7 are the two major TARPs expressed in the cerebellum. In the present study, we pursued their roles in synaptic expression of cerebellar AMPA receptors. In the cerebellar cortex, γ‐2 and γ‐7 were preferentially localized at various asymmetrical synapses. Using quantitative Western blot and immunofluorescence, we found severe reductions in GluA2 and GluA3 and mild reduction in GluA4 in γ‐2‐knockout (KO) cerebellum, whereas GluA1 and GluA4 were moderately reduced in γ‐7‐KO cerebellum. GluA2, GluA3 and GluA4 were further reduced in γ‐2/γ‐7 double‐KO (DKO) cerebellum. The large losses of GluA2 and GluA3 in γ‐2‐KO mice and further reductions in DKO mice were confirmed at all asymmetrical synapses examined with postembedding immunogold. Most notably, the GluA2 level in the postsynaptic density fraction, GluA2 labeling density at parallel fiber–Purkinje cell synapses, and AMPA receptor‐mediated currents at climbing fiber–Purkinje cell synapses were all reduced to approximately 10% of the wild‐type levels in DKO mice. On the other hand, the reduction in GluA4 in γ‐7‐KO granular layer reflected its loss at mossy fiber–granule cell synapses, whereas that of GluA1 and GluA4 in γ‐7‐KO molecular layer was caused, at least partly, by their loss in Bergmann glia. Therefore, γ‐2 and γ‐7 cooperatively promote synaptic expression of cerebellar AMPA receptors, and the latter also promotes glial expression.

Glutamate Receptor δ2 Is Essential for Input Pathway-Dependent Regulation of Synaptic AMPAR Contents in Cerebellar Purkinje Cells

The number of synaptic AMPA receptors (AMPARs) is the major determinant of synaptic strength and is differently regulated in input pathway-dependent and target cell type-dependent manners. In cerebellar Purkinje cells (PCs), the density of synaptic AMPARs is approximately five times lower at parallel fiber (PF) synapses than at climbing fiber (CF) synapses. However, molecular mechanisms underlying this biased synaptic distribution remain unclear. As a candidate molecule, we focused on glutamate receptor δ2 (GluRδ2 or GluD2), which is known to be efficiently trafficked to and selectively expressed at PF synapses in PCs. We applied postembedding immunogold electron microscopy to GluRδ2 knock-out (KO) and control mice, and measured labeling density for GluA1-4 at three excitatory synapses in the cerebellar molecular layer. In both control and GluRδ2-KO mice, GluA1-3 were localized at PF and CF synapses in PCs, while GluA2-4 were at PF synapses in interneurons. In control mice, labeling density for each of GluA1-3 was four to six times lower at PF-PC synapses than at CF-PC synapses. In GluRδ2-KO mice, however, their labeling density displayed a three- to fivefold increase at PF synapses, but not at CF synapses, thus effectively eliminating input pathway-dependent disparity between the two PC synapses. Furthermore, we found an unexpected twofold increase in labeling density for GluA2 and GluA3, but not GluA4, at PF-interneuron synapses, where we identified low but significant expression of GluRδ2. These results suggest that GluRδ2 is involved in a common mechanism that restricts the number of synaptic AMPARs at PF synapses in PCs and molecular layer interneurons.

TARPs gamma-2 and gamma-7 are essential for AMPA receptor expression in the cerebellum.

The α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)‐type glutamate receptors require auxiliary subunits termed transmembrane AMPA receptor regulatory proteins (TARPs), which promote receptor trafficking to the cell surface and synapses and modulate channel pharmacology and gating. Of six TARPs, γ‐2 and γ‐7 are the two major TARPs expressed in the cerebellum. In the present study, we pursued their roles in synaptic expression of cerebellar AMPA receptors. In the cerebellar cortex, γ‐2 and γ‐7 were preferentially localized at various asymmetrical synapses. Using quantitative Western blot and immunofluorescence, we found severe reductions in GluA2 and GluA3 and mild reduction in GluA4 in γ‐2‐knockout (KO) cerebellum, whereas GluA1 and GluA4 were moderately reduced in γ‐7‐KO cerebellum. GluA2, GluA3 and GluA4 were further reduced in γ‐2/γ‐7 double‐KO (DKO) cerebellum. The large losses of GluA2 and GluA3 in γ‐2‐KO mice and further reductions in DKO mice were confirmed at all asymmetrical synapses examined with postembedding immunogold. Most notably, the GluA2 level in the postsynaptic density fraction, GluA2 labeling density at parallel fiber–Purkinje cell synapses, and AMPA receptor‐mediated currents at climbing fiber–Purkinje cell synapses were all reduced to approximately 10% of the wild‐type levels in DKO mice. On the other hand, the reduction in GluA4 in γ‐7‐KO granular layer reflected its loss at mossy fiber–granule cell synapses, whereas that of GluA1 and GluA4 in γ‐7‐KO molecular layer was caused, at least partly, by their loss in Bergmann glia. Therefore, γ‐2 and γ‐7 cooperatively promote synaptic expression of cerebellar AMPA receptors, and the latter also promotes glial expression.

Roles of the glutamate receptor ε2 and δ2 subunits in the potentiation and prepulse inhibition of the acoustic startle reflex

We examined the regulation of the acoustic startle response in mutant mice of the N‐methyl‐d‐aspartate (NMDA)‐ and δ‐subtypes of the glutamate receptor (GluR) channel, which play important roles in neural plasticity in the forebrain and the cerebellum, respectively. Heterozygous mutant mice with reduced GluRε2 subunits of the NMDA receptor showed strongly enhanced startle responses to acoustic stimuli. On the other hand, heterozygous and homozygous mutation of the other NMDA receptor GluRε subunits exerted no, or only small effects on acoustic startle responses. The threshold of the auditory brainstem response of the GluRε2‐mutant mice was comparable to that of the wild‐type littermates. The primary circuit of the acoustic startle response is a relatively simple oligosynaptic pathway located in the lower brainstem, whilst the expression of GluRε2 is restricted to the forebrain. We thus suggest that the NMDA receptor GluRε2 subunit plays a role in the regulation of the startle reflex. Ablation of the cerebellar Purkinje cell‐specific δ2 subunit of the GluR channel exerted little effect on the acoustic startle response but resulted in the enhancement of prepulse inhibition of the reflex. Because inhibition of the acoustic startle response by a weak prepulse is a measure of sensorimotor gating, the process by which an organism filters sensory information, these observations indicate the involvement of the cerebellum in the modulation of sensorimotor gating.

Chondroitin sulfate N-acetylgalactosaminyltransferase-1 is required for normal cartilage development

CS (chondroitin sulfate) is a glycosaminoglycan species that is widely distributed in the extracellular matrix. To understand the physiological roles of enzymes involved in CS synthesis, we produced CSGalNAcT1 (CS N-acetylgalactosaminyltransferase 1)-null mice. CS production was reduced by approximately half in CSGalNAcT1-null mice, and the amount of short-chain CS was also reduced. Moreover, the cartilage of the null mice was significantly smaller than that of wild-type mice. Additionally, type-II collagen fibres in developing cartilage were abnormally aggregated and disarranged in the homozygous mutant mice. These results suggest that CSGalNAcT1 is required for normal CS production in developing cartilage.

Opposing Role of NMDA Receptor GluN2B and GluN2D in Somatosensory Development and Maturation

Development of correct topographical connections between peripheral receptors and central somatosensory stations requires activity-dependent synapse refinement, in which the NMDA type of glutamate receptors plays a key role. Here we compared functional roles of GluN2B (GluRε2 or NR2B) and GluN2D (GluRε4 or NR2D), two major regulatory subunits of neonatal NMDA receptors, in development of whisker-related patterning at trigeminal relay stations. Compared with control littermates, both the appearance of whisker-related patterning and the termination of the critical period, as assessed by unilateral infraorbital nerve transection, were delayed by nearly a day in the somatosensory cortex of GluN2B+/− mice but advanced by nearly a day in GluN2D−/− mice. Similar temporal shifts were found at subcortical relay stations in the thalamus and brainstem of GluN2B+/− and GluN2D−/− mice. In comparison, the magnitude of lesion-induced critical period plasticity in the somatosensory cortex, as assessed following row-C whisker removal, was normal in both mutants. Thus, GluN2B and GluN2D play counteractive roles in temporal development and maturation of somatosensory maps without affecting the magnitude of critical period plasticity. To understand the opposing action, we then examined neuronal and synaptic expressions of the two subunits along the trigeminal pathway. At each trigeminal station, GluN2B was predominant at asymmetrical synapses of non-GABAergic neurons, whereas GluN2D was selective to asymmetrical synapses of GABAergic neurons. Together, our findings suggest that GluN2B expressed at glutamatergic synapses on glutamatergic projection neurons facilitates refinement of ascending pathway synapses directly, whereas GluN2D expressed at glutamatergic synapses on GABAergic interneurons delays it indirectly.