Etsuko Tarusawa

Number and density of AMPA receptors in single synapses in immature cerebellum.

The number of ionotropic receptors in synapses is an essential factor for determining the efficacy of fast transmission. We estimated the number of functional AMPA receptors at single postsynaptic sites by a combination of two-photon uncaging of glutamate and the nonstationary fluctuation analysis in immature rat Purkinje cells (PCs), which receive a single type of excitatory input from climbing fibers. Areas of postsynaptic membrane specialization at the recorded synapses were measured by reconstruction of serial ultrathin sections. The number of functional AMPA receptors was proportional to the synaptic area with a density of ∼1280 receptors/μm2. Moreover, highly sensitive freeze-fracture replica labeling revealed a homogeneous density of immunogold particles for AMPA receptors in synaptic sites (910 ± 36 particles/μm2) and much lower density in extrasynaptic sites (19 ± 2 particles/μm2) in the immature PCs. Our results indicate that in this developing synapse, the efficacy of transmission is determined by the synaptic area.

Number and Density of AMPA Receptors in Individual Synapses in the Rat Cerebellum as Revealed by SDS-Digested Freeze-Fracture Replica Labeling

The number of AMPA receptor (AMPAR) is the major determinant of synaptic strength at glutamatergic synapses, but little is known about the absolute number and density of AMPARs in individual synapses. Using SDS-digested freeze-fracture replica labeling, which has high detection efficiency comparable with electrophysiological noise analysis for functional AMPAR, we analyzed three kinds of excitatory synapses in the molecular layer of the adult rat cerebellum. In parallel fiber (PF)–Purkinje cell (PC) synapses, we found large variability in the number (38.1 ± 34.4 particles per synapse, mean ± SD; range, 2–178 particles per synapse) and density (437 ± 277 particles/μm2; range, 48–1210 particles/μm2) of immunogold-labeled AMPARs. Two-dimensional view and high sensitivity of this method revealed irregular-shaped small AMPAR clusters within synapses. Climbing fiber (CF)–PC synapses had higher number of AMPAR labeling (68.6 ± 34.5 particles per synapse) than PF–PC and PF–interneuron synapses (36.8 ± 14.4 particles per synapse). Furthermore, AMPAR density at CF–PC and PF–interneuron synapses was approximately five times higher and more uniform than that at PF–PC synapses. These results suggest input- and target-dependent regulation of AMPAR-mediated synaptic strength.

CTCF Is Required for Neural Development and Stochastic Expression of Clustered Pcdh Genes in Neurons

The CCCTC-binding factor (CTCF) is a key molecule for chromatin conformational changes that promote cellular diversity, but nothing is known about its role in neurons. Here, we produced mice with a conditional knockout (cKO) of CTCF in postmitotic projection neurons, mostly in the dorsal telencephalon. The CTCF-cKO mice exhibited postnatal growth retardation and abnormal behavior and had defects in functional somatosensory mapping in the brain. In terms of gene expression, 390 transcripts were expressed at significantly different levels between CTCF-deficient and control cortex and hippocampus. In particular, the levels of 53 isoforms of the clustered protocadherin (Pcdh) genes, which are stochastically expressed in each neuron, declined markedly. Each CTCF-deficient neuron showed defects in dendritic arborization and spine density during brain development. Their excitatory postsynaptic currents showed normal amplitude but occurred with low frequency. Our results indicate that CTCF regulates functional neural development and neuronal diversity by controlling clustered Pcdh expression.

Input-specific intrasynaptic arrangements of ionotropic glutamate receptors and their impact on postsynaptic responses

To examine the intrasynaptic arrangement of postsynaptic receptors in relation to the functional role of the synapse, we quantitatively analyzed the two-dimensional distribution of AMPA and NMDA receptors (AMPARs and NMDARs, respectively) using SDS-digested freeze-fracture replica labeling (SDS-FRL) and assessed the implication of distribution differences on the postsynaptic responses by simulation. In the dorsal lateral geniculate nucleus, corticogeniculate (CG) synapses were twice as large as retinogeniculate (RG) synapses but expressed similar numbers of AMPARs. Two-dimensional views of replicas revealed that AMPARs form microclusters in both synapses to a similar extent, resulting in larger AMPAR-lacking areas in the CG synapses. Despite the broad difference in the AMPAR distribution within a synapse, our simulations based on the actual receptor distributions suggested that the AMPAR quantal response at individual RG synapses is only slightly larger in amplitude, less variable, and faster in kinetics than that at CG synapses having a similar number of the receptors. NMDARs at the CG synapses were expressed twice as many as those in the RG synapses. Electrophysiological recordings confirmed a larger contribution of NMDAR relative to AMPAR-mediated responses in CG synapses. We conclude that synapse size and the density and distribution of receptors have minor influences on quantal responses and that the number of receptors acts as a predominant postsynaptic determinant of the synaptic strength mediated by both the AMPARs and NMDARs.

Distinct cerebellar engrams in short-term and long-term motor learning.

Significance Long-term depression (LTD) of parallel fiber (PF) to Purkinje cell (PC) synapses has been postulated to cause cerebellar motor learning and extensively studied using in vitro preparations. However, there has been no in vivo evidence showing its occurrence after physiological learning, and much controversy on its role has persisted. We demonstrate that LTD as a form of AMPA receptor decrease does occur in PF–PC synapses after adaptation of horizontal optokinetic response. However, it lasted less than a day and was followed by elimination of these synapses. Our findings indicate distinct in vivo engrams for short-term and long-term memory in cerebellar motor learning, and open new mechanistic investigations of how the short-term memory is stabilized through structural reorganization of synaptic connections. Cerebellar motor learning is suggested to be caused by long-term plasticity of excitatory parallel fiber-Purkinje cell (PF–PC) synapses associated with changes in the number of synaptic AMPA-type glutamate receptors (AMPARs). However, whether the AMPARs decrease or increase in individual PF–PC synapses occurs in physiological motor learning and accounts for memory that lasts over days remains elusive. We combined quantitative SDS-digested freeze-fracture replica labeling for AMPAR and physical dissector electron microscopy with a simple model of cerebellar motor learning, adaptation of horizontal optokinetic response (HOKR) in mouse. After 1-h training of HOKR, short-term adaptation (STA) was accompanied with transient decrease in AMPARs by 28% in target PF–PC synapses. STA was well correlated with AMPAR decrease in individual animals and both STA and AMPAR decrease recovered to basal levels within 24 h. Surprisingly, long-term adaptation (LTA) after five consecutive daily trainings of 1-h HOKR did not alter the number of AMPARs in PF–PC synapses but caused gradual and persistent synapse elimination by 45%, with corresponding PC spine loss by the fifth training day. Furthermore, recovery of LTA after 2 wk was well correlated with increase of PF–PC synapses to the control level. Our findings indicate that the AMPARs decrease in PF–PC synapses and the elimination of these synapses are in vivo engrams in short- and long-term motor learning, respectively, showing a unique type of synaptic plasticity that may contribute to memory consolidation.

Establishment of high reciprocal connectivity between clonal cortical neurons is regulated by the Dnmt3b DNA methyltransferase and clustered protocadherins

BackgroundThe specificity of synaptic connections is fundamental for proper neural circuit function. Specific neuronal connections that underlie information processing in the sensory cortex are initially established without sensory experiences to a considerable extent, and then the connections are individually refined through sensory experiences. Excitatory neurons arising from the same single progenitor cell are preferentially connected in the postnatal cortex, suggesting that cell lineage contributes to the initial wiring of neurons. However, the postnatal developmental process of lineage-dependent connection specificity is not known, nor how clonal neurons, which are derived from the same neural stem cell, are stamped with the identity of their common neural stem cell and guided to form synaptic connections.ResultsWe show that cortical excitatory neurons that arise from the same neural stem cell and reside within the same layer preferentially establish reciprocal synaptic connections in the mouse barrel cortex. We observed a transient increase in synaptic connections between clonal but not nonclonal neuron pairs during postnatal development, followed by selective stabilization of the reciprocal connections between clonal neuron pairs. Furthermore, we demonstrate that selective stabilization of the reciprocal connections between clonal neuron pairs is impaired by the deficiency of DNA methyltransferase 3b (Dnmt3b), which determines DNA-methylation patterns of genes in stem cells during early corticogenesis. Dnmt3b regulates the postnatal expression of clustered protocadherin (cPcdh) isoforms, a family of adhesion molecules. We found that cPcdh deficiency in clonal neuron pairs impairs the whole process of the formation and stabilization of connections to establish lineage-specific connection reciprocity.ConclusionsOur results demonstrate that local, reciprocal neural connections are selectively formed and retained between clonal neurons in layer 4 of the barrel cortex during postnatal development, and that Dnmt3b and cPcdhs are required for the establishment of lineage-specific reciprocal connections. These findings indicate that lineage-specific connection reciprocity is predetermined by Dnmt3b during embryonic development, and that the cPcdhs contribute to postnatal cortical neuron identification to guide lineage-dependent synaptic connections in the neocortex.

Dendritic Ih ensures high-fidelity dendritic spike responses of motion-sensitive neurons in rat superior colliculus.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that generate I(h) currents are widely distributed in the brain and have been shown to contribute to various neuronal functions. In the present study, we investigated the functions of I(h) in the motion-sensitive projection neurons [wide field vertical (WFV) cells] of the superior colliculus, a pivotal visual center for detection of and orientating to salient objects. Combination of whole cell recordings and immunohistochemical investigations suggested that HCN1 channels dominantly contribute to the I(h) in WFV cells among HCN isoforms expressed in the superficial superior colliculus and mainly located on their expansive dendritic trees. We found that blocking I(h) suppressed the initiation of short- and fixed-latency dendritic spike responses and led instead to long- and fluctuating-latency somatic spike responses to optic fiber stimulations. These results suggest that the dendritic I(h) facilitates the dendritic initiation and/or propagation of action potentials and ensures that WFV cells generate spike responses to distal synaptic inputs in a sensitive and robustly time-locked manner, probably by acting as continuous depolarizing drive and fixing dendritic membrane potentials close to the spike threshold. These functions are different from known functions of dendritic I(h) revealed in hippocampal and neocortical pyramidal cells, where they spatiotemporally limit the propagations of synaptic inputs along the apical dendrites by reducing dendritic membrane resistance. Thus we have revealed new functional aspects of I(h), and these dendritic properties are likely critical for visual motion processing in these neurons.

Clustered Protocadherins Are Required for Building Functional Neural Circuits

Neuronal identity is generated by the cell-surface expression of clustered protocadherin (Pcdh) isoforms. In mice, 58 isoforms from three gene clusters, Pcdhα, Pcdhβ, and Pcdhγ, are differentially expressed in neurons. Since cis-heteromeric Pcdh oligomers on the cell surface interact homophilically with that in other neurons in trans, it has been thought that the Pcdh isoform repertoire determines the binding specificity of synapses. We previously described the cooperative functions of isoforms from all three Pcdh gene clusters in neuronal survival and synapse formation in the spinal cord. However, the neuronal loss and the following neonatal lethality prevented an analysis of the postnatal development and characteristics of the clustered-Pcdh-null (Δαβγ) neural circuits. Here, we used two methods, one to generate the chimeric mice that have transplanted Δαβγ neurons into mouse embryos, and the other to generate double mutant mice harboring null alleles of both the Pcdh gene and the proapoptotic gene Bax to prevent neuronal loss. First, our results showed that the surviving chimeric mice that had a high contribution of Δαβγ cells exhibited paralysis and died in the postnatal period. An analysis of neuronal survival in postnatally developing brain regions of chimeric mice clarified that many Δαβγ neurons in the forebrain were spared from apoptosis, unlike those in the reticular formation of the brainstem. Second, in Δαβγ/Bax null double mutants, the central pattern generator (CPG) for locomotion failed to create a left-right alternating pattern even in the absence of neurodegeneraton. Third, calcium imaging of cultured hippocampal neurons showed that the network activity of Δαβγ neurons tended to be more synchronized and lost the variability in the number of simultaneously active neurons observed in the control network. Lastly, a comparative analysis for trans-homophilic interactions of the exogenously introduced single Pcdh-γA3 isoforms between the control and the Δαβγ neurons suggested that the isoform-specific trans-homophilic interactions require a complete match of the expressed isoform repertoire at the contacting sites between interactive neurons. These results suggested that combinations of clustered Pcdh isoforms are required for building appropriate neural circuits.

Ni2+-sensitive T-type Ca2+ channel currents are regulated in parallel with synaptic and visual response plasticity in visual cortex

Visual cortical neurons undergo depression and potentiation of their visual responses to stimulation of the deprived and non-deprived eyes, respectively, after monocular deprivation. This modification occurs predominantly during an early postnatal period in normal development, and this critical period is postponed until adulthood in animals reared in darkness from birth. We have proposed that Ni(2+)-sensitive T-type Ca(2+) channel-dependent long-term potentiation (T-LTP) mediates the potentiation of non-deprived eye responses. In this study, to investigate the development of Ni(2+)-sensitive T-type Ca(2+) channels, presumed CaV3.2 channels, we performed whole-cell recordings from layer 2/3 pyramidal neurons in rat visual cortical slices. T-type Ca(2+) channel currents were activated by voltage steps from -100mV to -40mV under a pharmacological blockade of Na(+) and K(+) channels. We estimated presumed CaV3.2 currents from the currents obtained after subtraction of the currents in the presence of Ni(2+) (50μM) from those in control solution. The estimated currents were very small before eye opening, peaked during the critical period and then returned to a small value by adulthood. Dark rearing prevented the developmental decline in these currents until adulthood. These results suggest that the regulation of CaV3.2 currents underlies the developmental changes in T-LTP and ocular dominance plasticity.

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.