Takayuki Yamashita

Membrane Potential Dynamics of Neocortical Projection Neurons Driving Target-Specific Signals

Primary sensory cortex discriminates incoming sensory information and generates multiple processing streams toward other cortical areas. However, the underlying cellular mechanisms remain unknown. Here, by making whole-cell recordings in primary somatosensory barrel cortex (S1) of behaving mice, we show that S1 neurons projecting to primary motor cortex (M1) and those projecting to secondary somatosensory cortex (S2) have distinct intrinsic membrane properties and exhibit markedly different membrane potential dynamics during behavior. Passive tactile stimulation evoked faster and larger postsynaptic potentials (PSPs) in M1-projecting neurons, rapidly driving phasic action potential firing, well-suited for stimulus detection. Repetitive active touch evoked strongly depressing PSPs and only transient firing in M1-projecting neurons. In contrast, PSP summation allowed S2-projecting neurons to robustly signal sensory information accumulated during repetitive touch, useful for encoding object features. Thus, target-specific transformation of sensory-evoked synaptic potentials by S1 projection neurons generates functionally distinct output signals for sensorimotor coordination and sensory perception.

Developmental shift to a mechanism of synaptic vesicle endocytosis requiring nanodomain Ca2

Ca2+ is thought to be essential for the exocytosis and endocytosis of synaptic vesicles. However, the manner in which Ca2+ coordinates these processes remains unclear, particularly at mature synapses. Using membrane capacitance measurements from calyx of Held nerve terminals in rats, we found that vesicle endocytosis is initiated primarily in Ca2+ nanodomains around Ca2+ channels, where exocytosis is triggered. Bulk Ca2+ outside of the domain could also be involved in endocytosis at immature synapses, although only after extensive exocytosis at more mature synapses. This bulk Ca2+-dependent endocytosis required calmodulin and calcineurin activation at immature synapses, but not at more mature synapses. Similarly, GTP-independent endocytosis, which occurred after extensive exocytosis at immature synapses, became negligible after maturation. We propose that nanodomain Ca2+ simultaneously triggers exocytosis and endocytosis of synaptic vesicles and that the molecular mechanisms underlying Ca2+-dependent endocytosis undergo major developmental changes at this fast central synapse.

Ca2+-dependent regulation of synaptic vesicle endocytosis.

Action potentials, when arriving at presynaptic terminals, elicit Ca(2+) influx through voltage-gated Ca(2+) channels. Intracellular [Ca(2+)] elevation around the channels subsequently triggers synaptic vesicle exocytosis and also induces various protein reactions that regulate vesicle endocytosis and recycling to provide for long-term sustainability of synaptic transmission. Recent studies using membrane capacitance measurements, as well as high-resolution optical imaging, have revealed that the dominant type of synaptic vesicle endocytosis at central nervous system synapses is mediated by clathrin and dynamin. Furthermore, Ca(2+)-dependent mechanisms regulating endocytosis may operate in different ways depending on the distance from Ca(2+) channels: (1) intracellular Ca(2+) in the immediate vicinity of a Ca(2+) channel plays an essential role in triggering endocytosis, and (2) intracellular Ca(2+) traveling far from the channels has a modulatory effect on endocytosis at the periactive zone. Here, I integrate the latest progress in this field to propose a compartmental model for regulation of vesicle endocytosis at synapses and discuss the possible roles of presynaptic Ca(2+)-binding proteins including calmodulin, calcineurin and synaptotagmin.

Involvement of AMPA receptor desensitization in short‐term synaptic depression at the calyx of Held in developing rats

Paired‐pulse facilitation (PPF) and depression (PPD) are forms of short‐term plasticity that are generally thought to reflect changes in transmitter release probability. However, desensitization of postsynaptic AMPA receptors (AMPARs) significantly contributes to PPD at many glutamatergic synapses. To clarify the involvement of AMPAR desensitization in synaptic PPD, we compared PPD with AMPAR desensitization, induced by paired‐pulse glutamate application in patches excised from postsynaptic cells at the calyx of Held synapse of developing rats. We found that AMPAR desensitization contributed significantly to PPD before the onset of hearing (P10–12), but that its contribution became negligible after hearing onset. During postnatal development (P7–21) the recovery of AMPARs from desensitization became faster. Concomitantly, glutamate sensitivity of AMPAR desensitization declined. Single‐cell reverse transcription‐polymerase chain reaction (RT‐PCR) analysis indicated a developmental decline of GluR1 expression that correlated with speeding of the recovery of AMPARs from desensitization. Transmitter release probability declined during the second postnatal week (P7–14). Manipulation of the extracellular Ca2+/Mg2+ ratio, to match release probability at P7–8 and P13–15 synapses, revealed that the release probability is also an important factor determining the involvement of AMPAR desensitization in PPD. We conclude that the extent of involvement of AMPAR desensitization in short‐term synaptic depression is determined by both pre‐ and postsynaptic mechanisms.

Developmental changes in calcium/calmodulin-dependent inactivation of calcium currents at the rat calyx of Held

Ca2+‐binding to calmodulin (CaM) causes facilitation and/or inactivation of recombinant Ca2+ channels. At the rat calyx of Held, before hearing onset, presynaptic Ca2+ currents (IpCa) undergo Ca2+/CaM‐dependent inactivation during repetitive activation at around 1 Hz, implying that this may be a major cause of short‐term synaptic depression. However, it remains open whether the Ca2+/CaM‐dependent inactivation of IpCa persists in more mature animals. To address this question, we tested the effect of CaM inhibitors on the activity‐dependent modulation of IpCa in calyces, before (postnatal day (P) 7–9) and after (P13–15) hearing onset. Our results indicate that the CaM‐dependent IpCa inactivation during low‐frequency stimulation, and the ensuing synaptic depression, occur only at calyces in the prehearing period. However, CaM immunoreactivity in P8 and P14 calyces was equally strong. Even at P13–15, high frequency stimulation (200–500 Hz) could induce IpCa inactivation, which was attenuated by EGTA (10 mm) or a CaM inhibitor peptide loaded into the terminal. Furthermore, the CaM inhibitor peptide attenuated a transient facilitation of IpCa preceding inactivation observed at 500 Hz stimulation, whereas it had no effect on sustained IpCa facilitations during trains of 50–200 Hz stimulation. These results suggest that the Ca2+/CaM‐dependent IpCa modulation requires a high intraterminal Ca2+ concentration, which can be attained at immature calyces during low frequency stimulation, but only during unusually high frequency stimulation at calyceal terminals in the posthearing period.

Involvement of Ca2+ Channel Synprint Site in Synaptic Vesicle Endocytosis

The synaptic protein interaction (synprint) site of the voltage-gated Ca2+ channel (VGCC) α1 subunit can interact with proteins involved in exocytosis, and it is therefore thought to be essential for exocytosis of synaptic vesicles. Here we report that the synprint site can also directly bind the μ subunit of AP-2, an adaptor protein for clathrin-mediated endocytosis, in competition with the synaptotagmin 1 (Syt 1) C2B domain. In brain lysates, the AP-2–synprint interaction occurred over a wide range of Ca2+ concentrations but was inhibited at high Ca2+ concentrations, in which Syt 1 interacted with synprint site. At the calyx of Held synapse in rat brainstem slices, direct presynaptic loading of the synprint fragment peptide blocked endocytic, but not exocytic, membrane capacitance changes. We propose that the VGCC synprint site is involved in synaptic vesicle endocytosis, rather than exocytosis, in the nerve terminal, via Ca2+-dependent interactions with AP-2 and Syt.

Vesicular glutamate filling and AMPA receptor occupancy at the calyx of Held synapse of immature rats

At central glutamatergic synapses, neurotransmitter often saturates postsynaptic AMPA receptors (AMPARs), thereby restricting the dynamic range of synaptic efficacy. Here, using simultaneous pre‐ and postsynaptic whole‐cell recordings, at the calyx of Held synapse of immature rats, we have investigated the mechanism by which transmitter glutamate saturates postsynaptic AMPARs. When we loaded l‐glutamate (1–100 mm) into presynaptic terminals, the quantal EPSC (qEPSC) amplitude changed in a concentration‐dependent manner. At physiological temperature (36–37°C), the qEPSC amplitude increased when intraterminal l‐glutamate concentration was elevated from 1 mm to 10 mm, but it reached a plateau at 10 mm. This plateau persisted after bath‐application of the low affinity AMPAR antagonist kynurenate, suggesting that it was caused by saturation of vesicular filling with glutamate rather than by saturation of postsynaptic AMPARs. In contrast to qEPSCs, action potential‐evoked EPSCs remained unchanged by increasing intraterminal l‐glutamate from 1 mm to 100 mm, even at room temperature, indicating that multi‐quantal glutamate saturated postsynaptic AMPARs. This saturation could be relieved by blocking AMPAR desensitization using cyclothiazide (100 μm). The concentration of ambient glutamate in the slice, estimated from NMDA receptor current fluctuations, was 55 nm; this was far below the concentration required for AMPAR desensitization. We conclude that rapid AMPAR desensitization, caused by glutamate released from multiple vesicles during synaptic transmission, underlies postsynaptic AMPAR saturation at this immature calyceal synapse before the onset of hearing.

Target-specific membrane potential dynamics of neocortical projection neurons during goal-directed behavior.

Goal-directed behavior involves distributed neuronal circuits in the mammalian brain, including diverse regions of neocortex. However, the cellular basis of long-range cortico-cortical signaling during goal-directed behavior is poorly understood. Here, we recorded membrane potential of excitatory layer 2/3 pyramidal neurons in primary somatosensory barrel cortex (S1) projecting to either primary motor cortex (M1) or secondary somatosensory cortex (S2) during a whisker detection task, in which thirsty mice learn to lick for water reward in response to a whisker deflection. Whisker stimulation in ‘Good performer’ mice, but not ‘Naive’ mice, evoked long-lasting biphasic depolarization correlated with task performance in S2-projecting (S2-p) neurons, but not M1-projecting (M1-p) neurons. Furthermore, S2-p neurons, but not M1-p neurons, became excited during spontaneous unrewarded licking in ‘Good performer’ mice, but not in ‘Naive’ mice. Thus, a learning-induced, projection-specific signal from S1 to S2 may contribute to goal-directed sensorimotor transformation of whisker sensation into licking motor output. DOI: http://dx.doi.org/10.7554/eLife.15798.001

A glycine receptor antagonist, strychnine, blocked NMDA receptor activation in the neonatal mouse neocortex

The NMDA receptor (NMDAR) is a Ca2+-permeable cation channel that plays a critical role in neural network formation during brain development. Since it is blocked in a voltage-dependent manner by extracellular Mg2+, in order for the NMDA to be activated, the membrane must be strongly depolarized. Immature neurons in the developing neocortex can be depolarized by ligand-gated Cl− channels, such as the glycine receptor (GlyR) or GABAA receptor (GABAAR). We here assess the contribution of GlyRs to Ca2+ influx via NMDARs in neonatal mouse cortical neurons. The GlyR antagonist, strychnine, was more effective in suppressing postsynaptic Ca2+ influx than the GABAAR antagonist, picrotoxin, suggesting greater potentiation of NMDARs by GlyRs than by GABAARs. The GlyR, known to be endogenously activated at this stage, may play a critical role in neocortical development.

Diverse Long-Range Axonal Projections of Excitatory Layer 2/3 Neurons in Mouse Barrel Cortex

Excitatory projection neurons of the neocortex are thought to play important roles in perceptual and cognitive functions of the brain by directly connecting diverse cortical and subcortical areas. However, many aspects of the anatomical organization of these inter-areal connections are unknown. Here, we studied long-range axonal projections of excitatory layer 2/3 neurons with cell bodies located in mouse primary somatosensory barrel cortex (wS1). As a population, these neurons densely projected to secondary whisker somatosensory cortex (wS2) and primary/secondary whisker motor cortex (wM1/2), with additional axon in the dysgranular zone surrounding the barrel field, perirhinal temporal association cortex and striatum. In three-dimensional reconstructions of 6 individual wS2-projecting neurons and 9 individual wM1/2-projecting neurons, we found that both classes of neurons had extensive local axon in layers 2/3 and 5 of wS1. Neurons projecting to wS2 did not send axon to wM1/2, whereas a small subset of wM1/2-projecting neurons had relatively weak projections to wS2. A small fraction of projection neurons solely targeted wS2 or wM1/2. However, axon collaterals from wS2-projecting and wM1/2-projecting neurons were typically also found in subsets of various additional areas, including the dysgranular zone, perirhinal temporal association cortex and striatum. Our data suggest extensive diversity in the axonal targets selected by individual nearby cortical long-range projection neurons with somata located in layer 2/3 of wS1.