Biology subjects

Hoppa, M. B.

Publications and source records attributed to Hoppa, M. B..

9 recordsLinked to original sources

bioRxiv2026

Kv2-VAP interactions enhance presynaptic ER and mitochondrial calcium influx and the mobilization of vesicles from the reserve pool

In neurons, the endoplasmic reticulum (ER) forms an extensive network that establishes membrane contact sites (MCSs) with various organelles including the plasma membrane (PM). While MCSs are known to regulate lipid exchange and Ca2+ signaling, their specific roles in synaptic transmission remain poorly understood. Here, we demonstrate that the ER resident proteins VAPA and VAPB are essential for organizing presynaptic Ca2+ exchange and mobilizing synaptic vesicles. We show that the loss of VAP impairs Ca2+ loading into both the ER and mitochondria during electrical activity. This regulation occurs primarily through VAP interactions with voltage-gated potassium channels (Kv2) at the PM. Our data suggest that the Kv2-VAP complex organizes presynaptic Ca2+ signaling outside of the active zone. Without this scaffold, synaptic vesicles become trapped in the reserve pool and fail to participate in exocytosis. These findings reveal a novel role for Kv2-VAP MCSs in coordinating organelle Ca2+ signaling and the synaptic vesicle cycle.

neuroscience↗

bioRxiv2025

Subthreshold electric fields bidirectionally modulate neurotransmitter release through axon polarization

1.Subthreshold electric fields modulate brain activity and demonstrate potential in several therapeutic applications. Electric fields are known to generate heterogenous membrane polarization within neurons due to their complex morphologies. While the effects of somatic and dendritic polarization in postsynaptic neurons have been characterized, the functional consequences of axonal polarization on neurotransmitter release from the presynapse are unknown. Here, we combined noninvasive optogenetic indicators of voltage, calcium and neurotransmitter release to study the subcellular response within single neurons to subthreshold electric fields. We first captured the detailed spatiotemporal polarization profile produced by uniform electric fields within individual neurons. Small polarization of presynaptic boutons produces rapid and powerful modulation of neurotransmitter release, with the direction - facilitation or inhibition - depending on the direction of polarization. We determined that subthreshold electric fields drive this effect by rapidly altering the number of synaptic vesicles participating in neurotransmission, producing effects which resemble short-term plasticity akin to presynaptic homeostatic plasticity. These results provide key insights into the mechanisms of subthreshold electric fields at the cellular level. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/639625v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@cf9016org.highwire.dtl.DTLVardef@fcc601org.highwire.dtl.DTLVardef@12b9a52org.highwire.dtl.DTLVardef@1a08232_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

A ratiometric ER calcium sensor for quantitative comparisons across cell types and subcellular regions

The endoplasmic reticulum (ER) is an important regulator of Ca2+ in cells and dysregulation of ER calcium homeostasis can lead to numerous pathologies. Understanding how various pharmacological and genetic perturbations of ER Ca2+ homeostasis impacts cellular physiology would likely be facilitated by more quantitative measurements of ER Ca2+ levels that allow easier comparisons across conditions. Here, we developed a ratiometric version of our original ER-GCaMP probe that allows for more quantitative comparisons of the concentration of Ca2+ in the ER across cell types and sub-cellular compartments. Using this approach we show that the resting concentration of ER Ca2+ in primary dissociated neurons is substantially lower than that in measured in embryonic fibroblasts.

cell biology↗

bioRxiv2023

Neuronal FOS reports synchronized activity of presynaptic neurons

The immediate early gene FOS is frequently used as a marker for highly active neurons. Implicit in this use is the assumption that there is a correlation between neuronal spiking and FOS expression. Here we use optogenetic stimulation of hippocampal neurons to investigate the relation between spike frequency and FOS expression, and report several surprising observations. First, FOS expression is cell-type specific, spiking CA2 pyramidal neurons rarely express FOS. Second, FOS has a U-shaped dependence on frequency: Spiking at 0.1 Hz is more effective than high frequency spiking (50 Hz) while intermediate frequencies do not induce FOS. Third, the pathway from spiking to FOS is not cell-autonomous. Instead, transmitter release and metabotropic glutamate receptor (mGluR) activation are required and, at 0.1 Hz, FOS is induced independently of CREB/calcineurin/MEK pathways. We propose that FOS does not primarily encode a neurons own spike frequency but indicates repeated participation in highly synchronized activity, e.g. sharp wave ripples.

neuroscience↗

bioRxiv2022

Activity-driven synaptic translocation of LGI1 controls excitatory neurotransmission

The fine control of synaptic function requires robust trans-synaptic molecular interactions. However, it remains poorly understood how the landscape of trans-synaptic bridges dynamically remodels to reflect functional states of the synapse. Here we developed novel optical tools to visualize in firing synapses the molecular behavior of a particular secreted trans-synaptic protein, LGI1, and its presynaptic receptor, ADAM23, and discovered that neuronal activity acutely rearranges the abundance of these proteins at the synaptic cleft. Surprisingly, LGI1 in synapses was not secreted, as described elsewhere, but exo- and endocytosed through its interaction with ADAM23. Activity-driven translocation of LGI1 facilitated the formation of trans-synaptic connections proportionally to the history of activity of the synapse, adjusting excitatory transmission to synaptic firing rates. Thus, our findings reveal that LGI1 abundance at the synaptic cleft can be acutely remodeled and serves as a critical point of activity-dependent control of synaptic function. Highlights- Neuronal activity translocates LGI1 and ADAM23 to the presynaptic surface - LGI1 and ADAM23 are not located in synaptic vesicles - Stable cleft localization of LGI1 depends on the history of synaptic activity - LGI1 abundance at the synaptic cleft controls glutamate release

neuroscience↗

bioRxiv2021

Activity-dependent endoplasmic reticulum Ca2+ uptake depends on Kv2.1-mediated endoplasmic reticulum/plasma membrane junctions to promote synaptic transmission

The endoplasmic reticulum (ER) forms a continuous and dynamic network throughout a neuron, extending from dendrites to axon terminals, and axonal ER dysfunction is implicated in several neurological disorders. In addition, tight junctions between the ER and plasma membrane (PM) are formed by several molecules including Kv2 channels, but the cellular functions of many ER-PM junctions remain unknown. Dynamic Ca2+ uptake into the ER during electrical activity plays an essential role in synaptic transmission as failure to allow rapid ER Ca2+ filling during stimulation activates stromal interaction molecule 1 (STIM1) and decreases both presynaptic Ca2+ influx and synaptic vesicle exocytosis. Our experiments demonstrate that Kv2.1 channels are necessary for enabling ER Ca2+ uptake during electrical activity as genetic depletion of Kv2.1 rendered both the somatic and axonal ER unable to accumulate Ca2+ during electrical stimulation. Moreover, our experiments show that the loss of Kv2.1 in the axon impairs synaptic vesicle fusion during stimulation via a mechanism unrelated to modulation of membrane voltage. Thus, our data demonstrate that the non-conducting role of Kv2.1 in forming stable junctions between the ER and PM via ER VAMP-associated protein (VAP) binding couples ER Ca2+ uptake with electrical activity. Our results further suggest that Kv2.1 has a critical function in neuronal cell biology for Ca2+-handling independent of voltage and reveals a novel and critical pathway for maintaining ER lumen Ca2+ levels and efficient neurotransmitter release. Taken together these findings reveal an essential non-classical role for both Kv2.1 and the ER-PM junctions in synaptic transmission. SignificanceThe endoplasmic reticulum (ER) extends throughout the neuron as a continuous organelle, and its dysfunction is associated with several neurological disorders. During electrical activity, the ER takes up Ca2+ from the cytosol which has been shown to support synaptic transmission. This close choreography of ER Ca2+ uptake with electrical activity suggests functional coupling of the ER to sources of voltage-gated Ca2+ entry through an unknown mechanism. Here we report a non-conducting role for Kv2.1 through its ER binding domain that is necessary for ER Ca2+ uptake during neuronal activity. Loss of Kv2.1 profoundly disables neurotransmitter release without altering presynaptic voltage suggesting that Kv2.1-mediated signaling hubs play an important neurobiological role in Ca2+ handling and synaptic transmission independent of ion conduction.

neuroscience↗

bioRxiv2020

The Photoconvertible Fluorescent Probe, CaMPARI, Labels Active Neurons in Freely-Moving Intact Adult Fruit Flies

Linking neural circuitry to behavior by mapping active neurons in vivo is a challenge. Both genetically encoded calcium indicators (GECIs) and intermediate early genes (IEGs) have been used to pinpoint active neurons during a stimulus or a behavior but have drawbacks such as limiting the movement of the organism, requiring a priori knowledge of the active region or having poor temporal resolution. CaMPARI (calcium-modulated photoactivatable ratiometric integrator) was engineered to overcome these spatial-temporal challenges. CaMPARI is a photoconvertible protein that only converts from green to red fluorescence in the presence of high calcium concentration and violet (405 nm) light. This allows the experimenter to precisely mark active neurons within defined temporal windows. The photoconversion can then be quantified by taking the ratio of the red fluorescence to the green. CaMPARI promises the ability to trace active neurons during specific stimulus; however, CaMPARIs uses in adult Drosophila have been limited to photoconversion during fly immobilization. Here, we demonstrate a method that allows photoconversion of multiple freely-moving intact adult flies during a stimulus. Flies were placed in a dish with filter paper wet with acetic acid (pH=2) or neutralized acetic acid (pH=7) and exposed to photoconvertible light (60 mW) for 30 min (500 ms on, 200 ms off). Immediately following photoconversion, whole flies were fixed and imaged by confocal microscopy. The red:green ratio was quantified for the DC4 glomerulus, a bundle of neurons expressing Ir64a, an ionotropic receptor that senses acids in the Drosophila antennal lobe. Flies exposed to acetic acid showed 1.3-fold greater photoconversion than flies exposed to neutralized acetic acid. This finding was recapitulated using a more physiological stimulus of apple cider vinegar. These results indicate that CaMPARI can be used to label neurons in intact, freely-moving adult flies and will be useful for identifying the circuitry underlying complex behaviors.

neuroscience↗

bioRxiv2020

The Potassium Channel Subunit Kvβ1 is Required for Synaptic Facilitation

Analysis of the presynaptic action potentials (APsyn) role in synaptic facilitation in hippocampal pyramidal neurons has been difficult due to size limitations of axons. We overcame these size barriers by combining high resolution optical recordings of membrane potential, exocytosis and Ca2+ in cultured hippocampal neurons. These recordings revealed a critical and selective role for Kv1 channel inactivation in synaptic facilitation of excitatory hippocampal neurons. Presynaptic Kv1 channel inactivation was mediated by the Kv{beta}1 subunit, and had a surprisingly rapid onset that was readily apparent even in brief physiological stimulation paradigms including paired-pulse stimulation. Genetic depletion of Kv{beta}1 blocked all broadening of the APsyn during high frequency stimulation and eliminated synaptic facilitation without altering the initial probability of vesicle release. Thus using all quantitative optical measurements of presynaptic physiology, we reveal a critical role for presynaptic Kv channels in synaptic facilitation at small presynaptic terminals of the hippocampal neurons upstream of exocytic machinery. SignificanceNerve terminals generally engage in two opposite and essential forms of synaptic plasticity (facilitation or depression) during high frequency stimulation that play critical roles in learning and memory. Measurements of the electrical impulses (action potentials) underlying these two forms of plasticity has been difficult in small nerve terminals due to their size. In this study we deployed a combination of optical measurements of vesicle fusion and membrane voltage to overcome this previous size barrier. Here, we found a unique molecular composition of Kv1 channel {beta}-subunits that causes broadening of the presynaptic action essential to synaptic facilitation. Disruption of the Kv{beta}1 inactivation mechanism switches excitatory nerve terminals into a depressive state, without any disruption to initial probability of vesicle fusion.

neuroscience↗

bioRxiv2019

Freeze-frame imaging of synaptic activity using SynTagMA

Information within the brain travels from neuron to neuron across synapses. At any given moment, only a few synapses within billions will be active and are thought to transmit key information about the environment, a behavior being executed or memory being recalled. Here we present SynTagMA, which marks active synapses within a ~2 s time window. Upon violet illumination, the genetically expressed tag converts from green to red fluorescence if bound to calcium. Targeted to presynaptic terminals, preSynTagMA allows discrimination between active and silent axons. Targeted to excitatory postsynapses, postSynTagMA creates a snapshot of synapses active just before photoconversion. To analyze large datasets, we developed an analysis program that automatically identifies and tracks the fluorescence of thousands of individual synapses in tissue. Together, these tools provide a high throughput method for repeatedly mapping active synapses in vitro and in vivo.

neuroscience↗