Trophoblast glycoprotein is required for efficient synaptic vesicle exocytosis from retinal rod bipolar cells.

Introduction: Rod bipolar cells (RBCs) faithfully transmit light-driven signals from rod photoreceptors in the outer retina to third order neurons in the inner retina. Recently, significant work has focused on the role of leucine-rich repeat (LRR) proteins in synaptic development and signal transduction at RBC synapses. We previously identified trophoblast glycoprotein (TPBG) as a novel transmembrane LRR protein localized to the dendrites and axon terminals of RBCs. Methods: We examined the effects on RBC physiology and retinal processing of TPBG genetic knockout in mice using immunofluorescence and electron microscopy, electroretinogram recording, patch-clamp electrophysiology, and time-resolved membrane capacitance measurements. Results: The scotopic electroretinogram showed a modest increase in the b-wave and a marked attenuation in oscillatory potentials in the TPBG knockout. No effect of TPBG knockout was observed on the RBC dendritic morphology, TRPM1 currents, or RBC excitability. Because scotopic oscillatory potentials primarily reflect RBC-driven rhythmic activity of the inner retina, we investigated the contribution of TPBG to downstream transmission from RBCs to third-order neurons. Using electron microscopy, we found shorter synaptic ribbons in TPBG knockout axon terminals in RBCs. Time-resolved capacitance measurements indicated that TPBG knockout reduces synaptic vesicle exocytosis and subsequent GABAergic reciprocal feedback without altering voltage-gated Ca2+ currents. Discussion: TPBG is required for normal synaptic ribbon development and efficient neurotransmitter release from RBCs to downstream cells. Our results highlight a novel synaptic role for TPBG at RBC ribbon synapses and support further examination into the mechanisms by which TPBG regulates RBC physiology and circuit function.

Functional maturation of the rod bipolar to AII-amacrine cell ribbon synapse in the mouse retina.

Retinal ribbon synapses undergo functional changes after eye opening that remain uncharacterized. Using light-flash stimulation and paired patch-clamp recordings, we examined the maturation of the ribbon synapse between rod bipolar cells (RBCs) and AII-amacrine cells (AII-ACs) after eye opening (postnatal day 14) in the mouse retina at near physiological temperatures. We find that light-evoked excitatory postsynaptic currents (EPSCs) in AII-ACs exhibit a slow sustained component that increases in magnitude with advancing age, whereas a fast transient component remains unchanged. Similarly, paired recordings reveal a dual-component EPSC with a slower sustained component that increases during development, even though the miniature EPSC (mEPSC) amplitude and kinetics do not change significantly. We thus propose that the readily releasable pool of vesicles from RBCs increases after eye opening, and we estimate that a short light flash can evoke the release of ∼4,000 vesicles onto a single mature AII-AC.

Cell type specializations of the vocal-motor cortex in songbirds.

Identifying molecular specializations in cortical circuitry supporting complex behaviors, like learned vocalizations, requires understanding of the neuroanatomical context from which these circuits arise. In songbirds, the robust arcopallial nucleus (RA) provides descending cortical projections for fine vocal-motor control. Using single-nuclei transcriptomics and spatial gene expression mapping in zebra finches, we have defined cell types and molecular specializations that distinguish RA from adjacent regions involved in non-vocal motor and sensory processing. We describe an RA-specific projection neuron, differential inhibitory subtypes, and glia specializations and have probed predicted GABAergic interneuron subtypes electrophysiologically within RA. Several cell-specific markers arise developmentally in a sex-dependent manner. Our interactive apps integrate cellular data with developmental and spatial distribution data from the gene expression brain atlas ZEBrA. Users can explore molecular specializations of vocal-motor neurons and support cells that likely reflect adaptations key to the physiology and evolution of vocal control circuits and refined motor skills.

Motor cortex analogue neurons in songbirds utilize Kv3 channels to generate ultranarrow spikes.

Complex motor skills in vertebrates require specialized upper motor neurons with precise action potential (AP) firing. To examine how diverse populations of upper motor neurons subserve distinct functions and the specific repertoire of ion channels involved, we conducted a thorough study of the excitability of upper motor neurons controlling somatic motor function in the zebra finch. We found that robustus arcopallialis projection neurons (RAPNs), key command neurons for song production, exhibit ultranarrow spikes and higher firing rates compared to neurons controlling non-vocal somatic motor functions (dorsal intermediate arcopallium [AId] neurons). Pharmacological and molecular data indicate that this striking difference is associated with the higher expression in RAPNs of high threshold, fast-activating voltage-gated Kv3 channels, that likely contain Kv3.1 (KCNC1) subunits. The spike waveform and Kv3.1 expression in RAPNs mirror properties of Betz cells, specialized upper motor neurons involved in fine digit control in humans and other primates but absent in rodents. Our study thus provides evidence that songbirds and primates have convergently evolved the use of Kv3.1 to ensure precise, rapid AP firing in upper motor neurons controlling fast and complex motor skills.

A melanopsin ganglion cell subtype forms a dorsal retinal mosaic projecting to the supraoptic nucleus.

Visual input to the hypothalamus from intrinsically photosensitive retinal ganglion cells (ipRGCs) influences several functions including circadian entrainment, body temperature, and sleep. ipRGCs also project to nuclei such as the supraoptic nucleus (SON), which is involved in systemic fluid homeostasis, maternal behavior, social behaviors, and appetite. However, little is known about the SON-projecting ipRGCs or their relationship to well-characterized ipRGC subtypes. Using a GlyT2Cre mouse line, we show a subtype of ipRGCs restricted to the dorsal retina that selectively projects to the SON. These ipRGCs tile a dorsal region of the retina, forming a substrate for encoding ground luminance. Optogenetic activation of their axons demonstrates they release the neurotransmitter glutamate in multiple regions, including the suprachiasmatic nucleus (SCN) and SON. Our results challenge the idea that ipRGC dendrites overlap to optimize photon capture and suggests non-image forming vision operates to sample local regions of the visual field to influence diverse behaviors.

Resurgent Na+ currents promote ultrafast spiking in projection neurons that drive fine motor control.

The underlying mechanisms that promote precise spiking in upper motor neurons controlling fine motor skills are not well understood. Here we report that projection neurons in the adult zebra finch song nucleus RA display robust high-frequency firing, ultra-narrow spike waveforms, superfast Na+ current inactivation kinetics, and large resurgent Na+ currents (INaR). These properties of songbird pallial motor neurons closely resemble those of specialized large pyramidal neurons in mammalian primary motor cortex. They emerge during the early phases of song development in males, but not females, coinciding with a complete switch of Na+ channel subunit expression from Navß3 to Navß4. Dynamic clamping and dialysis of Navß4's C-terminal peptide into juvenile RA neurons provide evidence that Navß4, and its associated INaR, promote neuronal excitability. We thus propose that INaR modulates the excitability of upper motor neurons that are required for the execution of fine motor skills.

Glycine Release Is Potentiated by cAMP via EPAC2 and Ca2+ Stores in a Retinal Interneuron.

Neuromodulation via the intracellular second messenger cAMP is ubiquitous at presynaptic nerve terminals. This modulation of synaptic transmission allows exocytosis to adapt to stimulus levels and reliably encode information. The AII amacrine cell (AII-AC) is a central hub for signal processing in the mammalian retina. The main apical dendrite of the AII-AC is connected to several lobular appendages that release glycine onto OFF cone bipolar cells and ganglion cells. However, the influence of cAMP on glycine release is not well understood. Using membrane capacitance measurements from mouse AII-ACs to directly measure exocytosis, we observe that intracellular dialysis of 1 mm cAMP enhances exocytosis without affecting the L-type Ca2+ current. Responses to depolarizing pulses of various durations show that the size of the readily releasable pool of vesicles nearly doubles with cAMP, while paired-pulse depression experiments suggest that release probability does not change. Specific agonists and antagonists for exchange protein activated by cAMP 2 (EPAC2) revealed that the cAMP-induced enhancement of exocytosis requires EPAC2 activation. Furthermore, intact Ca2+ stores were also necessary for the cAMP potentiation of exocytosis. Postsynaptic recordings from OFF cone bipolar cells showed that increasing cAMP with forskolin potentiated the frequency of glycinergic spontaneous IPSCs. We propose that cAMP elevations in the AII-AC lead to a robust enhancement of glycine release through an EPAC2 and Ca2+ store signaling pathway. Our results thus contribute to a better understanding of how AII-AC crossover inhibitory circuits adapt to changes in ambient luminance.SIGNIFICANCE STATEMENT The mammalian retina operates over a wide dynamic range of light intensities and contrast levels. To optimize the signal-to-noise ratio of processed visual information, both excitatory and inhibitory synapses within the retina must modulate their gain in synaptic transmission to adapt to different levels of ambient light. Here we show that increases of cAMP concentration within AII amacrine cells produce enhanced exocytosis from these glycinergic interneurons. Therefore, we propose that light-sensitive neuromodulators may change the output of glycine release from AII amacrine cells. This novel mechanism may fine-tune the amount of tonic and phasic synaptic inhibition received by bipolar cell terminals and, consequently, the spiking patterns that ganglion cells send to the upstream visual areas of the brain.

Molecular mechanism of prestin electromotive signal amplification.

Hearing involves two fundamental processes: mechano-electrical transduction and signal amplification. Despite decades of studies, the molecular bases for both remain elusive. Here, we show how prestin, the electromotive molecule of outer hair cells (OHCs) that senses both voltage and membrane tension, mediates signal amplification by coupling conformational changes to alterations in membrane surface area. Cryoelectron microscopy (cryo-EM) structures of human prestin bound with chloride or salicylate at a common "anion site" adopt contracted or expanded states, respectively. Prestin is ensconced within a perimeter of well-ordered lipids, through which it induces dramatic deformation in the membrane and couples protein conformational changes to the bulk membrane. Together with computational studies, we illustrate how the anion site is allosterically coupled to changes in the transmembrane domain cross-sectional area and the surrounding membrane. These studies provide insight into OHC electromotility by providing a structure-based mechanism of the membrane motor prestin.

Encoding sound in the cochlea: from receptor potential to afferent discharge.

Ribbon-class synapses in the ear achieve analog to digital transformation of a continuously graded membrane potential to all-or-none spikes. In mammals, several auditory nerve fibres (ANFs) carry information from each inner hair cell (IHC) to the brain in parallel. Heterogeneity of transmission among synapses contributes to the diversity of ANF sound-response properties. In addition to the place code for sound frequency and the rate code for sound level, there is also a temporal code. In series with cochlear amplification and frequency tuning, neural representation of temporal cues over a broad range of sound levels enables auditory comprehension in noisy multi-speaker settings. The IHC membrane time constant introduces a low-pass filter that attenuates fluctuations of the receptor potential above 1-2 kHz. The ANF spike generator adds a high-pass filter via its depolarization-rate threshold that rejects slow changes in the postsynaptic potential and its phasic response property that ensures one spike per depolarization. Synaptic transmission involves several stochastic subcellular processes between IHC depolarization and ANF spike generation, introducing delay and jitter that limits the speed and precision of spike timing. ANFs spike at a preferred phase of periodic sounds in a process called phase-locking that is limited to frequencies below a few kilohertz by both the IHC receptor potential and the jitter in synaptic transmission. During phase-locking to periodic sounds of increasing intensity, faster and facilitated activation of synaptic transmission and spike generation may be offset by presynaptic depletion of synaptic vesicles, resulting in relatively small changes in response phase. Here we review encoding of spike-timing at cochlear ribbon synapses.

How to Build a Fast and Highly Sensitive Sound Detector That Remains Robust to Temperature Shifts.

Frogs must have sharp hearing abilities during the warm summer months to successfully find mating partners. This study aims to understand how frog hair cell ribbon-type synapses preserve both sensitivity and temporal precision during temperature changes. Under room (∼24°C) and high (∼32°C) temperature, we performed in vitro patch-clamp recordings of hair cells and their afferent fibers in amphibian papillae of either male or female bullfrogs. Afferent fibers exhibited a wide heterogeneity in membrane input resistance (Rin) from 100 mΩ to 1000 mΩ, which may contribute to variations in spike threshold and firing frequency. At higher temperatures, most fibers increased their frequency of spike firing due to an increase in spontaneous EPSC frequencies. Hair cell resting membrane potential (Vrest) remained surprisingly stable during temperature increases, because Ca2+ influx and K+ outflux increased simultaneously. This increase in Ca2+ current likely enhanced spontaneous EPSC frequencies. These larger "leak currents" at Vrest also lowered Rin and produced higher electrical resonant frequencies. Lowering Rin will reduce the hair cells receptor potential and presumably moderate the systems sensitivity. Using membrane capacitance measurements, we suggest that hair cells can partially compensate for this reduced sensitivity by increasing exocytosis efficiency and the size of the readily releasable pool of synaptic vesicles. Furthermore, paired recordings of hair cells and their afferent fibers showed that synaptic delays shortened and multivesicular release becomes more synchronous at higher temperatures, which should improve temporal precision. Together, our results explain many previous in vivo observations on the temperature dependence of spikes in auditory nerves.SIGNIFICANCE STATEMENT The vertebrate inner ear detects and transmits auditory information over a broad dynamic range of sound frequency and intensity. It achieves remarkable sensitivity to soft sounds and precise frequency selectivity. How does the ear of cold-blooded vertebrates maintain its performance level as temperature changes? More specifically, how does the hair cell to afferent fiber synapse in bullfrog amphibian papilla adjust to a wide range of physiological temperatures without losing its sensitivity and temporal fidelity to sound signals? This study uses in vitro experiments to reveal the biophysical mechanisms that explain many observations made from in vivo auditory nerve fiber recordings. We find that higher temperature facilitates vesicle exocytosis and electrical tuning to higher sound frequencies, which benefits sensitivity and selectivity.

Presynaptic Diversity Revealed by Ca2+-Permeable AMPA Receptors at the Calyx of Held Synapse.

GluA2-lacking Ca2+-permeable AMPARs (CP-AMPARs) play integral roles in synaptic plasticity and can mediate excitotoxic cellular signaling at glutamatergic synapses. However, the developmental profile of functional CP-AMPARs at the auditory brainstem remains poorly understood. Through a combination of electrophysiological and live-cell Ca2+ imaging from mice of either sex, we show that the synaptic release of glutamate from the calyx of Held nerve terminal activates CP-AMPARs in the principal cells of the medial nucleus of the trapezoid body in the brainstem. This leads to significant Ca2+ influx through these receptors before the onset of hearing at postnatal day 12 (P12). Using a selective open channel blocker of CP-AMPARs, IEM-1460, we estimate that ∼80% of the AMPAR population are permeable to Ca2+ at immature P4-P5 synapses. However, after the onset of hearing, Ca2+ influx through these receptors was greatly reduced. We estimate that CP-AMPARs comprise approximately 40% and 33% of the AMPAR population at P18-P22 and P30-P34, respectively. By quantifying the rate of EPSC block by IEM-1460, we found an increased heterogeneity in glutamate release probability for adult-like calyces (P30-P34). Using tetraethylammonium (TEA), a presynaptic potassium channel blocker, we show that the apparent reduction of CP-AMPARs in more mature synapses is not a consequence of presynaptic action potential (AP) speeding. Finally, through postsynaptic AP recordings, we show that inhibition of CP-AMPARs reduces spike fidelity in juvenile synapses, but not in more mature synapses. We conclude that the expression of functional CP-AMPARs declines over early postnatal development in the calyx of Held synapse.SIGNIFICANCE STATEMENT The calyx of Held synapse is pivotal to the circuitry that computes sound localization. Postsynaptic Ca2+ influx via AMPARs may be critical for signaling the maturation of this brainstem synapse. The GluA4 subunit may dominate the AMPAR complex at mature synapses because of its fast gating kinetics and large unitary conductance. The expectation is that AMPARs dominated by GluA4 subunits should be highly Ca2+ permeable. However, we find that Ca2+-permeable AMPAR expression declines during postnatal development. Using the rate of EPSC block by IEM-1460, an open channel blocker of Ca2+-permeable AMPARs, we propose a novel method to determine glutamate release probability and uncover an increased heterogeneity in release probability for more mature calyces of Held nerve terminals.

Clustered Ca2+ Channels Are Blocked by Synaptic Vesicle Proton Release at Mammalian Auditory Ribbon Synapses.

A Ca2+ current transient block (ICaTB) by protons occurs at some ribbon-type synapses after exocytosis, but this has not been observed at mammalian hair cells. Here we show that a robust ICaTB occurs at post-hearing mouse and gerbil inner hair cell (IHC) synapses, but not in immature IHC synapses, which contain non-compact active zones, where Ca2+ channels are loosely coupled to the release sites. Unlike ICaTB at other ribbon synapses, ICaTB in mammalian IHCs displays a surprising multi-peak structure that mirrors the EPSCs seen in paired recordings. Desynchronizing vesicular release with intracellular BAPTA or by deleting otoferlin, the Ca2+ sensor for exocytosis, greatly reduces ICaTB, whereas enhancing release synchronization by raising Ca2+ influx or temperature increases ICaTB. This suggests that ICaTB is produced by fast multivesicular proton-release events. We propose that ICaTB may function as a submillisecond feedback mechanism contributing to the auditory nerve's fast spike adaptation during sound stimulation.

Vision: Microcircuits Rage against the Dimming of the Light.

At sundown when light levels fall, rod photoreceptors take the night shift from the daylight-sensitive cones and a specialized mammalian microcircuit 'wires' the rods into the ancestral cone pathway. A recent study combines serial electron microscopy and simultaneous patch clamp recordings to shed light on this microcircuit in unprecedented detail.

Ca2+-Permeable AMPARs Mediate Glutamatergic Transmission and Excitotoxic Damage at the Hair Cell Ribbon Synapse.

We report functional and structural evidence for GluA2-lacking Ca2+-permeable AMPARs (CP-AMPARs) at the mature hair cell ribbon synapse. By using the methodological advantages of three species (of either sex), we demonstrate that CP-AMPARs are present at the hair cell synapse in an evolutionarily conserved manner. Via a combination of in vivo electrophysiological and Ca2+ imaging approaches in the larval zebrafish, we show that hair cell stimulation leads to robust Ca2+ influx into afferent terminals. Prolonged application of AMPA caused loss of afferent terminal responsiveness, whereas blocking CP-AMPARs protects terminals from excitotoxic swelling. Immunohistochemical analysis of AMPAR subunits in mature rat cochlea show regions within synapses lacking the GluA2 subunit. Paired recordings from adult bullfrog auditory synapses demonstrate that CP-AMPARs mediate a major component of glutamatergic transmission. Together, our results support the importance of CP-AMPARs in mediating transmission at the hair cell ribbon synapse. Further, excess Ca2+ entry via CP-AMPARs may underlie afferent terminal damage following excitotoxic challenge, suggesting that limiting Ca2+ levels in the afferent terminal may protect against cochlear synaptopathy associated with hearing loss.SIGNIFICANCE STATEMENT A single incidence of noise overexposure causes damage at the hair cell synapse that later leads to neurodegeneration and exacerbates age-related hearing loss. A first step toward understanding cochlear neurodegeneration is to identify the cause of initial excitotoxic damage to the postsynaptic neuron. Using a combination of immunohistochemical, electrophysiological, and Ca2+ imaging approaches in evolutionarily divergent species, we demonstrate that Ca2+-permeable AMPARs (CP-AMPARs) mediate glutamatergic transmission at the adult auditory hair cell synapse. Overexcitation of the terminal causes Ca2+ accumulation and swelling that can be prevented by blocking CP-AMPARs. We demonstrate that CP-AMPARs mediate transmission at this first-order sensory synapse and that limiting Ca2+ accumulation in the terminal may protect against hearing loss.

Distinct Actions of Voltage-Activated Ca2+ Channel Block on Spontaneous Release at Excitatory and Inhibitory Central Synapses.

At chemical synapses, voltage-activated calcium channels (VACCs) mediate Ca2+ influx to trigger action potential-evoked neurotransmitter release. However, the mechanisms by which Ca2+ regulates spontaneous transmission have not been fully determined. We have shown that VACCs are a major trigger of spontaneous release at neocortical inhibitory synapses but not at excitatory synapses, suggesting fundamental differences in spontaneous neurotransmission at GABAergic and glutamatergic synapses. Recently, VACC blockers were reported to reduce spontaneous release of glutamate and it was proposed that there was conservation of underlying mechanisms of neurotransmission at excitatory and inhibitory synapses. Furthermore, it was hypothesized that the different effects on excitatory and inhibitory synapses may have resulted from off-target actions of Cd2+, a nonselective VACC blocker, or other variations in experimental conditions. Here we report that in mouse neocortical neurons, selective and nonselective VACC blockers inhibit spontaneous release at inhibitory but not at excitatory terminals, and that this pattern is observed in culture and slice preparations as well as in synapses from acute slices of the auditory brainstem. The voltage dependence of Cd2+ block of VACCs accounts for the apparent lower potency of Cd2+ on spontaneous release of GABA than on VACC current amplitudes. Our findings indicate fundamental differences in the regulation of spontaneous release at inhibitory and excitatory synapses by stochastic VACC activity that extend beyond the cortex to the brainstem.SIGNIFICANCE STATEMENT Presynaptic Ca2+ entry via voltage-activated calcium channels (VACCs) is the major trigger of action potential-evoked synaptic release. However, the role of VACCs in the regulation of spontaneous neurotransmitter release (in the absence of a synchronizing action potential) remains controversial. We show that spontaneous release is affected differently by VACCs at excitatory and inhibitory synapses. At inhibitory synapses, stochastic openings of VACCs trigger the majority of spontaneous release, whereas they do not affect spontaneous release at excitatory synapses. We find this pattern to be wide ranging, holding for large and small synapses in the neocortex and brainstem. These findings indicate fundamental differences of the Ca2+ dependence of spontaneous release at excitatory and inhibitory synapses and heterogeneity of the mechanisms of release across the CNS.