Yo Otsu

Differential regulation of synaptic and extra-synaptic NMDA receptors

A variety of processes limit NMDA (N-methyl-D-aspartate) receptor (NMDAR) activity in response to agonist exposure, including rundown—the decline of peak current with repeated, sustained agonist application. Here we report that calcium and tyrosine phosphorylation differentially regulate rundown of synaptic versus extrasynaptic NMDAR-mediated current in rat hippocampal pyramidal neurons.

Competition between Phasic and Asynchronous Release for Recovered Synaptic Vesicles at Developing Hippocampal Autaptic Synapses

Developing hippocampal neurons in microisland culture undergo rapid and extensive transmitter release-dependent depression of evoked (phasic) excitatory synaptic activity in response to 1 sec trains of 20 Hz stimulation. Although evoked phasic release was attenuated by repeated stimuli, asynchronous (miniature like) release continued at a high rate equivalent to ∼2.8 readily releasable pools (RRPs) of quanta/sec. Asynchronous release reflected the recovery and immediate release of quanta because it was resistant to sucrose-induced depletion of the RRP. Asynchronous and phasic release appeared to compete for a common limited supply of release-ready quanta because agents that block asynchronous release, such as EGTA-AM, led to enhanced steady-state phasic release, whereas prolongation of the asynchronous release time course by LiCl delayed recovery of phasic release from depression. Modeling suggested that the resistance of asynchronous release to depression was associated with its ability to out-compete phasic release for recovered quanta attributable to its relatively low release rate (up to 0.04/msec per vesicle) stimulated by bulk intracellular Ca2+ concentration ([Ca2+]i) that could function over prolonged intervals between successive stimuli. Although phasic release was associated with a considerably higher peak rate of release (0.4/msec per vesicle), the [Ca2+]i microdomains that trigger it are brief (1 msec), and with asynchronous release present, relatively few quanta can accumulate within the RRP to be available for phasic release. We conclude that despite depression of phasic release during train stimulation, transmission can be maintained at a near-maximal rate by switching to an asynchronous mode that takes advantage of a bulk presynaptic [Ca2+]i.

Optical monitoring of neuronal activity at high frame rate with a digital random-access multiphoton (RAMP) microscope

Two-photon microscopy offers the promise of monitoring brain activity at multiple locations within intact tissue. However, serial sampling of voxels has been difficult to reconcile with millisecond timescales characteristic of neuronal activity. This is due to the conflicting constraints of scanning speed and signal amplitude. The recent use of acousto-optic deflector scanning to implement random-access multiphoton microscopy (RAMP) potentially allows to preserve long illumination dwell times while sampling multiple points-of-interest at high rates. However, the real-life abilities of RAMP microscopy regarding sensitivity and phototoxicity issues, which have so far impeded prolonged optical recordings at high frame rates, have not been assessed. Here, we describe the design, implementation and characterisation of an optimised RAMP microscope. We demonstrate the application of the microscope by monitoring calcium transients in Purkinje cells and cortical pyramidal cell dendrites and spines. We quantify the illumination constraints imposed by phototoxicity and show that stable continuous high-rate recordings can be obtained. During these recordings the fluorescence signal is large enough to detect spikes with a temporal resolution limited only by the calcium dye dynamics, improving upon previous techniques by at least an order of magnitude.

T-Type and L-Type Ca2+ Conductances Define and Encode the Bimodal Firing Pattern of Vestibulocerebellar Unipolar Brush Cells

Cerebellar unipolar brush cells (UBCs) are glutamatergic interneurons that receive direct input from vestibular afferents in the form of a unique excitatory synapse on their dendritic brush. UBCs constitute independent relay lines for vestibular signals, and their inherent properties most likely determine how vestibular activity is encoded by the cerebellar cortex. We now demonstrate that UBCs are bimodal cells; they can either fire high-frequency bursts of action potentials when stimulated from hyperpolarized potentials or discharge tonically during sustained depolarizations. The two functional states can be triggered by physiological-like activity of the excitatory input and are encoded by distinct Ca2+-signaling systems. By combining complementary strategies, consisting of molecular and electrophysiological analysis and of ultrafast acousto-optical deflector-based two-photon imaging, we unraveled the identity and the subcellular localization of the Ca2+ conductances activating in each mode. Fast inactivating T-type Ca2+ channels produce low-threshold spikes, which trigger the high-frequency bursts and generate powerful Ca2+ transients in the brush and, to a much lesser extent, in the soma. The tonic firing mode is encoded by a signalization system principally composed of L-type channels. Ca2+ influx during tonic firing produces a linear representation of the spike rate of the cell in the form of a widespread and sustained Ca2+ concentration increase and regulates cellular excitability via BK potassium channels. The bimodal firing pattern of UBCs may underlie different coding strategies of the vestibular input by the cerebellum, thus likely increasing the computational power of this structure.

Miniature Transmitter Release: Accident of Nature or Careful Design?

Miniature transmitter release results from the constitutive low-level release of individual vesicles of neurotransmitter. Since the 1950s, this form of synaptic transmission has largely been thought to reflect a leaky evoked-release mechanism, and it was not clear whether it had a function of its own. Recent data challenge this view and suggest that miniature release can affect both the local chemistry of synapses and the network properties of neurons.

Activity-Dependent Gating of Calcium Spikes by A-type K+ Channels Controls Climbing Fiber Signaling in Purkinje Cell Dendrites

Summary In cerebellar Purkinje cell dendrites, heterosynaptic calcium signaling induced by the proximal climbing fiber (CF) input controls plasticity at distal parallel fiber (PF) synapses. The substrate and regulation of this long-range dendritic calcium signaling are poorly understood. Using high-speed calcium imaging, we examine the role of active dendritic conductances. Under basal conditions, CF stimulation evokes T-type calcium signaling displaying sharp proximodistal decrement. Combined mGluR1 receptor activation and depolarization, two activity-dependent signals, unlock P/Q calcium spikes initiation and propagation, mediating efficient CF signaling at distal sites. These spikes are initiated in proximal smooth dendrites, independently from somatic sodium action potentials, and evoke high-frequency bursts of all-or-none fast-rising calcium transients in PF spines. Gradual calcium spike burst unlocking arises from increasing inactivation of mGluR1-modulated low-threshold A-type potassium channels located in distal dendrites. Evidence for graded activity-dependent CF calcium signaling at PF synapses refines current views on cerebellar supervised learning rules.

Optical Postsynaptic Measurement of Vesicle Release Rates for Hippocampal Synapses Undergoing Asynchronous Release during Train Stimulation

Developing hippocampal neurons in microisland culture were found to undergo rapid depression of excitatory synaptic activity caused by consumption of their readily releasable pool (RRP) of vesicles in response to 20 Hz trains of stimulation. Associated with depression was a switch to an asynchronous release mode that maintained transmission at a high steady-state rate equivalent to ∼2.1 RRPs per second. We have applied postsynaptic Ca2+ imaging to directly monitor these asynchronous release events to estimate both the steady rate of transmitter release and the number of quanta within the RRP at individual hippocampal synapses. Based on the frequency of asynchronous release measured at individual synapses postsynaptically using Ca2+ imaging (5-17 sec after train stimulation) and with knowledge of the time course by which asynchronous release rates decay, we estimate that individual hippocampal synapses exhibit (in response to train stimulation) peak release rates of up to 21 quanta per second from an RRP that contains, on average, 10 quanta. Use-dependent block of evoked synaptic activity by MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine maleate] confirmed that synapses undergoing asynchronous release are not significantly different from the general population with regard to their composition of NMDA receptor and/or release probability. Given that high-frequency trains deplete the synapse of readily releasable quanta (and that these release rates can only be maintained for a few seconds), these high rates of asynchronous release likely reflect refilling of vesicles from a reserve pool and not necessarily the continuous action of a relatively slow clathrin- and endosome-dependent process.

Mind-altering miniature neurotransmitter release?

It is well established that calcium is the trigger for fast action potential-evoked synaptic transmission (1). After elevation of intracellular calcium ([Ca2+]i) by action potential-mediated opening of voltage-dependent calcium channels (VDCCs), a low resting rate of neurotransmitter release of 0.01–0.03 vesicles per sec is elevated significantly to ≈20 per sec (2–4). Transmitter release occurring independently of action potential-mediated changes in [Ca2+]i is termed “miniature release” and involves the stochastic release of individual vesicles (quanta). The quantal nature of miniature activity has been used to elucidate basic functional parameters of central nervous system (CNS) and neuromuscular synapses (5). Although miniature transmission can occur at basal [Ca2+]i levels (≈80 nM), its frequency is greatly stimulated by even modest [Ca2+]i elevation (<1 μM) (6). Miniature release has been proposed recently to have a role in maintaining the function of developing synapses during periods without action potential-evoked synaptic activity (refs. 7 and 8, but also see ref. 9) and is regulated in parallel to evoked release (10). In addition to being the trigger for fast chemical synaptic transmission, calcium is also required for coupling nerve-induced excitation to cardiac and smooth muscle contraction (11). As a treatment for hypertension and angina agents that interfere with calcium entry such as dihydropyridine (DHP), VDCC blockers are commonly used. Drugs with core 1,4-DHP structures potently block the L-type VDCC, which is required for muscle contraction. In the article by Hirasawa and Pittman (12) in this issue of PNAS, a paradoxical effect of the DHP nifedipine was found on miniature excitatory postsynaptic currents (mEPSCs) recorded from magnocellular neurons of the supraoptic nucleus of the hypothalamus. Specifically, nifedipine but not close chemical cousins such as nimodipine or nitrendipine potentially induces up to a 15-fold increase …

Functional Principles of Posterior Septal Inputs to the Medial Habenula

Summary The medial habenula (MHb) is an epithalamic hub contributing to expression and extinction of aversive states by bridging forebrain areas and midbrain monoaminergic centers. Although contradictory information exists regarding their synaptic properties, the physiology of the excitatory inputs to the MHb from the posterior septum remains elusive. Here, combining optogenetics-based mapping with ex vivo and in vivo physiology, we examine the synaptic properties of posterior septal afferents to the MHb and how they influence behavior. We demonstrate that MHb cells receive sparse inputs producing purely glutamatergic responses via calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), heterotrimeric GluN2A-GluN2B-GluN1 N-methyl-D-aspartate (NMDA) receptors, and inhibitory group II metabotropic glutamate receptors. We describe the complex integration dynamics of these components by MHb cells. Finally, we combine ex vivo data with realistic afferent firing patterns recorded in vivo to demonstrate that efficient optogenetic septal stimulation in the MHb induces anxiolysis and promotes locomotion, contributing long-awaited evidence in favor of the importance of this septo-habenular pathway.

A novel two-site enzyme immunoassay reveals regional distributions and developmental changes of GluR1 and NMDAR1 proteins in the rat brain

Glutamate receptors including AMPA and NMDA receptors play important roles in neural development and synaptic plasticity in the brain. But it has been difficult to correlate individual biochemical phenomena with their quantitative and qualitative changes of the receptors occurring in specific neurons. In present study, we established a two-site enzyme immunoassay for AMPA and NMDA receptors whose detection limits were 30 pg for GluRl and 15 pg for NMDARl conteining C2 exon. Regional and developmental variations of the protein levels were much more remarkable than those of mRNA reported: Absolute protein content of GluRl was outstandingly enriched in the rat hippocampus, while that of NMDARI was high in all the forebrain regions examined. GluRl protein increased only in the postnatal second-third weeks while NMDARl gradually did in postnatal life. The difference observed between the distributions of the receptor proteins and mRNAs suggest that post-translational processes may play crucial roles in the expression of the glutamate receptors regulated during development. Supported in part by JSPS-RFTF-L00203.