Takayasu Mikuni

Retrograde semaphorin signaling regulates synapse elimination in the developing mouse brain

Making and breaking neuronal synapses As the brain develops, early synapse formation is exuberant and haphazard. But as development progresses, connections are refined into functional networks. In that process, many synapses get eliminated. Uesaka et al. now show that molecules already known for axon guidance are functional later on when they regulate the synaptic pruning needed to refine the circuits connected during axon guidance. Science, this issue p. 1020 Elimination of redundant synapses and formation of functional circuits in developing brain involves axon guidance molecules. Neural circuits are shaped by elimination of early-formed redundant synapses during postnatal development. Retrograde signaling from postsynaptic cells regulates synapse elimination. In this work, we identified semaphorins, a family of versatile cell recognition molecules, as retrograde signals for elimination of redundant climbing fiber to Purkinje cell synapses in developing mouse cerebellum. Knockdown of Sema3A, a secreted semaphorin, in Purkinje cells or its receptor in climbing fibers accelerated synapse elimination during postnatal day 8 (P8) to P18. Conversely, knockdown of Sema7A, a membrane-anchored semaphorin, in Purkinje cells or either of its two receptors in climbing fibers impaired synapse elimination after P15. The effect of Sema7A involves signaling by metabotropic glutamate receptor 1, a canonical pathway for climbing fiber synapse elimination. These findings define how semaphorins retrogradely regulate multiple processes of synapse elimination.

Arc/Arg3.1 Is a Postsynaptic Mediator of Activity-Dependent Synapse Elimination in the Developing Cerebellum

Neural circuits are shaped by activity-dependent elimination of redundant synapses during postnatal development. In many systems, postsynaptic activity is known to be crucial, but the precise mechanisms remain elusive. Here, we report that the immediate early gene Arc/Arg3.1 mediates elimination of surplus climbing fiber (CF) to Purkinje cell (PC) synapses in the developing cerebellum. CF synapse elimination was accelerated when activity of channelrhodopsin-2-expressing PCs was elevated by 2-day photostimulation. This acceleration was suppressed by PC-specific knockdown of either the P/Q-type voltage-dependent Ca(2+) channels (VDCCs) or Arc. PC-specific Arc knockdown had no appreciable effect until around postnatal day 11 but significantly impaired CF synapse elimination thereafter, leaving redundant CF terminals on PC somata. The effect of Arc knockdown was occluded by simultaneous knockdown of P/Q-type VDCCs in PCs. We conclude that Arc mediates the final stage of CF synapse elimination downstream of P/Q-type VDCCs by removing CF synapses from PC somata.

Synapse type-independent degradation of the endocannabinoid 2-arachidonoylglycerol after retrograde synaptic suppression

The endocannabinoid 2-arachidonoylglycerol (2-AG) mediates retrograde synaptic suppression. Although the mechanisms of 2-AG production are well characterized, how 2-AG is degraded is less clearly understood. Here we found that expression of the 2-AG hydrolyzing enzyme monoacylglycerol lipase (MGL) was highly heterogeneous in the cerebellum, being rich within parallel fiber (PF) terminals, weak in Bergman glia (BG), and absent in other synaptic terminals. Despite this highly selective MGL expression pattern, 2-AG–mediated retrograde suppression was significantly prolonged at not only PF-Purkinje cell (PC) synapses but also climbing fiber-PC synapses in granule cell-specific MGL knockout (MGL-KO) mice whose cerebellar MGL expression was confined to the BG. Virus-mediated expression of MGL into the BG of global MGL-KO mice significantly shortened 2-AG–mediated retrograde suppression at PF-PC synapses. Furthermore, contribution of MGL to termination of 2-AG signaling depended on the distance from MGL-rich PFs to inhibitory synaptic terminals. Thus, 2-AG is degraded in a synapse-type independent manner by MGL present in PFs and the BG. The results of the present study strongly suggest that MGL regulates 2-AG signaling rather broadly within a certain range of neural tissue, although MGL expression is heterogeneous and limited to a subset of nerve terminals and astrocytes.

Organotypic Coculture Preparation for the Study of Developmental Synapse Elimination in Mammalian Brain

We developed an organotypic coculture preparation allowing fast and efficient identification of molecules that regulate developmental synapse elimination in the mammalian brain. This coculture consists of a cerebellar slice obtained from rat or mouse at postnatal day 9 (P9) or P10 and a medullary explant containing the inferior olive dissected from rat at embryonic day 15. We verified that climbing fibers (CFs), the axons of inferior olivary neurons, formed functional synapses onto Purkinje cells (PCs) in the cerebellum of cocultures. PCs were initially reinnervated by multiple CFs with similar strengths. Surplus CFs were eliminated subsequently, and the remaining CFs became stronger. These changes are similar to those occurring in developing cerebellum in vivo. Importantly, the changes in CF innervations in cocultures involved the same molecules required for CF synapse elimination in vivo, including NMDA receptor, type 1 metabotropic glutamate receptor and glutamate receptor δ2 (GluRδ2). We demonstrate that gain- and loss-of-function analyses can be efficiently performed by lentiviral-mediated overexpression and RNAi-induced knockdown of GluRδ2. Using this approach, we identified neuroligin-2 as a novel molecule that promotes CF synapse elimination in postsynaptic PCs. Thus, our coculture preparation will greatly facilitate the elucidation of molecular mechanisms of synapse elimination.

Retrograde Signaling for Climbing Fiber Synapse Elimination

Neurons form exuberant synapses with target cells early in development. Then, necessary synapses are selectively strengthened whereas unnecessary connections are weakened and eventually eliminated during postnatal development. This process is known as synapse elimination and is a crucial step for shaping immature neural circuits into functionally mature versions. Accumulating evidence suggests that retrograde signaling from postsynaptic cells regulates synapse elimination, but the underlying mechanisms remain unknown. Here, we show that semaphorin3A (Sema3A) and semaphorin7A (Sema7A) mediate retrograde signals for elimination of redundant climbing fiber (CF) to Purkinje cell (PC) synapses in the developing cerebellum, a representative model of synapse elimination in the central nervous system. We picked up candidate retrograde signaling molecules that are expressed in PCs during the period of CF synapse elimination and the receptors of these candidate molecules that are present in CFs. We then assessed the effects of lentivirus-mediated RNAi-knockdown of these molecules on CF synapse elimination. By this systematic screening, we found that knockdown of Sema3A in PCs or its co-receptor, plexinA4 (PlxnA4), in CFs accelerated CF synapse elimination and decreased CF-mediated synaptic inputs. Conversely, knockdown of Sema7A in PCs or either of the two receptors for Sema7A, plexinC1 (PlxnC1) and integrinB1 (ItgB1), in CFs impaired CF synapse elimination. Importantly, the effect of Sema7A involves signaling by type 1 metabotropic glutamate receptor (mGluR1), a canonical pathway in PCs for the final stage of CF synapse elimination. These results demonstrate that specific semaphorins act as retrograde signaling molecules and regulate distinct processes of CF synapse elimination during postnatal cerebellar development.

Alternative splicing in the C-terminal tail of Cav2.1 is essential for preventing a neurological disease in mice

Alternative splicing (AS) that occurs at the final coding exon (exon 47) of the Cav2.1 voltage-gated calcium channel (VGCC) gene produces two major isoforms in the brain, MPI and MPc. These isoforms differ in their splice acceptor sites; human MPI is translated into a polyglutamine tract associated with spinocerebellar ataxia type 6 (SCA6), whereas MPc splices to an immediate stop codon, resulting in a shorter cytoplasmic tail. To gain insight into the functional role of the AS in vivo and whether modulating the splice patterns at this locus can be a potential therapeutic strategy for SCA6, here we created knockin mice that exclusively express MPc by inserting the splice-site mutation. The resultant Cacna1aCtmKO/CtmKO mice developed non-progressive neurological phenotypes, featuring early-onset ataxia and absence seizure without significant alterations in the basic properties of the channel. Interactions of Cav2.1 with Cavβ4 and Rimbp2 were significantly reduced while those with GABAB2 were enhanced in the cerebellum of Cacna1aCtmKO/CtmKO mice. Treatment with the GABAB antagonist CGP35348 partially rescued the motor impairments seen in Cacna1aCtmKO/CtmKO mice. These results suggest that the carboxyl-terminal domain of Cav2.1 is not essential for maintaining the basic properties of the channel in the cerebellar Purkinje neurons but is involved in multiple interactions of Cav2.1 with other proteins, and plays an essential role in preventing a complex neurological disease.

Synapse “non-specific” degradation of the endocannabinoid 2-arachidonoylglycerol mediating depolarization-induced retrograde synaptic suppression in cerebellar Purkinje cells

O2-G-1-1 Synapse “non-specific” degradation of the endocannabinoid 2-arachidonoylglycerol mediating depolarization-induced retrograde synaptic suppression in cerebellar Purkinje cells Asami Tanimura 1 , Maya Yamazaki 2, Motokazu Uchigashima 3, Naofumi Uesaka 1, Takayasu Mikuni 1, Kouichi Hashimoto 1,4,5, Masahiko Watanabe 3, Kenji Sakimura 2, Masanobu Kano 1 1 Dep. of Neurophysiol, Grad. Sch. of Med, Univ. of Tokyo, Tokyo, Japan 2 Dept. Cell. Neurobiol., Brain Res. Inst., Niigata Univ. Japan 3 Dept. Anat., Grad. Sch. Med., Hokkaido Univ. Japan 4 Dept. Neurophysiol., Grad. Sch. Biomed. Sci., Hiroshima Univ. Japan 5 PRESTO, JST Saitama, Japan

Gene transfer techniques for CNS organotypic cultures

We have developed a novel experimental system for introduction of genetically encoded tools by an adenovirus-mediated gene transfer technique. Here we tested the validity of the system by analyzing the expression pattern of introduced fluorescent proteins. We found that fluorescent cells are pyramidal cells in the cerebral cortex (NeuN-positive, GABA-negative, and GFAP-negative) and Purkinje cells in the cerebellum (IP3R1-positive). Interestingly, the expression pattern in the cortex showed a specific pattern depending on the time when the adenovirus-injection was performed: the injection at embryonic day (E) 12.5 led to the preferential expression in cerebral layer 5/6 neurons (∼90% of expressing neurons), whereas the injection at E14.5 led to the preferential expression in layer 2/3 neurons (∼70% of expressing neurons). Our novel experimental system enables us to introduce genetically encoded tools in specific subtypes of neurons and thus would be a promising way to perform optical recording/manipulation of neural activities in vivo.