Kaori Akashi

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.

Subtypes of GABAergic neurons project axons in the neocortex.

γ-aminobutyric acid (GABA)ergic neurons in the neocortex have been regarded as interneurons and speculated to modulate the activity of neurons locally. Recently, however, several experiments revealed that neuronal nitric oxide synthase (nNOS)-positive GABAergic neurons project cortico-cortically with long axons. In this study, we illustrate Golgi-like images of the nNOS-positive GABAergic neurons using a nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) reaction and follow the emanating axon branches in cat brain sections. These axon branches projected cortico-cortically with other non-labeled arcuate fibers, contra-laterally via the corpus callosum and anterior commissure. The labeled fibers were not limited to the neocortex but found also in the fimbria of the hippocampus. In order to have additional information on these GABAergic neuron projections, we investigated green fluorescent protein (GFP)-labeled GABAergic neurons in GAD67-Cre knock-in/GFP Cre-reporter mice. GFP-labeled axons emanate densely, especially in the fimbria, a small number in the anterior commissure, and very sparsely in the corpus callosum. These two different approaches confirm that not only nNOS-positive GABAergic neurons but also other subtypes of GABAergic neurons project long axons in the cerebral cortex and are in a position to be involved in information processing.

GABAergic neurons in the preoptic area send direct inhibitory projections to orexin neurons

Populations of neurons in the hypothalamic preoptic area (POA) fire rapidly during sleep, exhibiting sleep/waking state-dependent firing patterns that are the reciprocal of those observed in the arousal system. The majority of these preoptic “sleep-active” neurons contain the inhibitory neurotransmitter GABA. On the other hand, a population of neurons in the lateral hypothalamic area (LHA) contains orexins, which play an important role in the maintenance of wakefulness, and exhibit an excitatory influence on arousal-related neurons. It is important to know the anatomical and functional interactions between the POA sleep-active neurons and orexin neurons, both of which play important, but opposite roles in regulation of sleep/wakefulness states. In this study, we confirmed that specific pharmacogenetic stimulation of GABAergic neurons in the POA leads to an increase in the amount of non-rapid eye movement (NREM) sleep. We next examined direct connectivity between POA GABAergic neurons and orexin neurons using channelrhodopsin 2 (ChR2) as an anterograde tracer as well as an optogenetic tool. We expressed ChR2-eYFP selectively in GABAergic neurons in the POA by AAV-mediated gene transfer, and examined the projection sites of ChR2-eYFP-expressing axons, and the effect of optogenetic stimulation of ChR2-eYFP on the activity of orexin neurons. We found that these neurons send widespread projections to wakefulness-related areas in the hypothalamus and brain stem, including the LHA where these fibers make close appositions to orexin neurons. Optogenetic stimulation of these fibers resulted in rapid inhibition of orexin neurons. These observations suggest direct connectivity between POA GABAergic neurons and orexin neurons.

Tangential migration and proliferation of intermediate progenitors of GABAergic neurons in the mouse telencephalon

In the embryonic neocortex, neuronal precursors are generated in the ventricular zone (VZ) and accumulate in the cortical plate. Recently, the subventricular zone (SVZ) of the embryonic neocortex was recognized as an additional neurogenic site for both principal excitatory neurons and GABAergic inhibitory neurons. To gain insight into the neurogenesis of GABAergic neurons in the SVZ, we investigated the characteristics of intermediate progenitors of GABAergic neurons (IPGNs) in mouse neocortex by immunohistochemistry, immunocytochemistry, single-cell RT-PCR and single-cell array analysis. IPGNs were identified by their expression of some neuronal and cell cycle markers. Moreover, we investigated the origins of the neocortical IPGNs by Cre-loxP fate mapping in transgenic mice and the transduction of part of the telencephalic VZ by Cre-reporter plasmids, and found them in the medial and lateral ganglionic eminence. Therefore, they must migrate tangentially within the telencephalon to reach the neocortex. Cell-lineage analysis by simple-retrovirus transduction revealed that the neocortical IPGNs self-renew and give rise to a small number of neocortical GABAergic neurons and to a large number of granule and periglomerular cells in the olfactory bulb. IPGNs are maintained in the neocortex and may act as progenitors for adult neurogenesis.

Determination of kainate receptor subunit ratios in mouse brain using novel chimeric protein standards

Kainate‐type glutamate receptors (KARs) are tetrameric channels assembled from GluK1‐5. GluK1‐3 are low‐affinity subunits that form homomeric and heteromeric KARs, while GluK4 and GluK5 are high‐affinity subunits that require co‐assembly with GluK1‐3 for functional expression. Although the subunit composition is thought to be highly heterogeneous in the brain, the distribution of KAR subunits at the protein level and their relative abundance in given regions of the brain remain largely unknown. In the present study, we titrated C‐terminal antibodies to each KAR subunit using chimeric GluA2‐GluK fusion proteins, and measured their relative abundance in the P2 and post‐synaptic density (PSD) fractions of the adult mouse hippocampus and cerebellum. Analytical western blots showed that GluK2 and GluK3 were the major KAR subunits, with additional expression of GluK5 in the hippocampus and cerebellum. In both regions, GluK4 was very low and GluK1 was below the detection threshold. The relative amount of low‐affinity subunits (GluK2 plus GluK3) was several times higher than that of high‐affinity subunits (GluK4 plus GluK5) in both regions. Of note, the highest ratio of high‐affinity subunits to low‐affinity subunits was found in the hippocampal PSD fraction (0.32), suggesting that heteromeric receptors consisting of high‐ and low‐affinity subunits highly accumulate at hippocampal synapses. In comparison, this ratio was decreased to 0.15 in the cerebellar PSD fraction, suggesting that KARs consisting of low‐affinity subunits are more prevalent in the cerebellum. Therefore, low‐affinity KAR subunits are predominant in the brain, with distinct subunit combinations between the hippocampus and cerebellum.

Immunohistochemical localization of kainate receptors, GluK2/3 (GluR6/7) and GluK5 (KA2), in the mouse hippocampus

The maintenance of synaptic functions is essential for reliable information transfer and storage. However cellular mechanisms underlying synaptic maintenance in the adult brain are not fully understood. In this study, we evaluated the involvement of inositol 1,4,5-trisphosphate (IP3) signaling in synaptic maintenance in the cerebral cortex. Metabotropic glutamate receptor (mGluR) or IP3 signaling in the neocortical pyramidal neurons was chronically inhibited in vivo by intraperitoneal injection of mGluR antagonists or expressing in these neurons exogenous IP3 5-phosphatase, which selectively hydrolyzes IP3. This chronic inhibition in postsynaptic pyramidal neurons significantly increased the value of the paired pulse ratio at glutamatergic synapses, indicating the reduction of presynaptic release probability. In contrast, the chronic inhibition of mGluR-IP3 signaling did not alter the amplitude of quantal synaptic responses, indicating that postsynaptic responsiveness was unchanged. These results suggest that an IP3-dependent retrograde signaling mechanism is involved in the maintenance of excitatory synapses in the cerebral cortex. Recently, we reported a similar IP3-dependent maintenance mechanism at parallel fiber-Purkinje cell synapses in the cerebellum. Thus, these studies provide new insights into the signaling mechanism underlying synaptic maintenance in the adult brain.

Synapse- and age-dependent reduction of NMDA receptor activities in mice lacking NMDA receptor ϵ 1 subunit

Extracellular application of either quisqualic acid (QA) or Phe-Met-Arg-Phe-NH1 (FMRFamide) to the identified neurons of Aplysia ganglion elicits a slow K’-current response under voltage clamp. The QA-induced K’-current response was markedly depressed in the presence of CNQX, an antagonist for non-NMDA receptors. On the other hand, application of kainate and AMPA, agonists for non-NMDA receptors, did not induce any significant response in the same neurons. QA-induced Kf-current response was not depressed at all by intracellular injection of guanosine S--O-(2-thiodiphosphate) (GDP@), while the FMRFamide-induced response was completely blocked by GDP-OS in the same cell. However. both the QAand FMRFamide-induced K’-current responses decreased markedly when the temperature was lowered to 15 “C from 22 “C. These results indicate that the QA-induced Kt-current response does not conform in its pharmacological characteristics to any of the known glutamate-induced responses in vertebrate.