Simon Alford

Within neuronal circuits, ordered neurotransmission is contingent upon balance between excitatory glutamatergic and inhibitory GABAergic signaling. To study circuit-level processes, the paradigm of 4D cellular physiology has been developed, where, single cells and subcellular structures are studied as individual units in three-dimensional space over a continuous interval rather than as a single moment in time, or as a population-level average. Neurons are excitable cells expressing voltage-gated Ca ²⁺ channels and Ca ²⁺ fluxes subsequent to action potential firing are widely used as markers of neuronal activity. While the imaging of Ca ²⁺ dynamics at the soma is often performed, the imaging of Ca ²⁺ fluxes at presynaptic terminals has often proven to be an experimental challenge: existing imaging modalities suffer from inadequate acquisition speeds, insufficient penetration depths, insufficient spatial resolution to identify axonal structures, or spectral crosstalk issues. To visualize presynaptic Ca ²⁺ dynamics in both excitatory and inhibitory neurons, here we combine advanced lattice light-sheet microscopy with viral delivery of two genetically encoded calcium indicators (GECIs)- jRGECO1a and jGCaMP8f, to perform sequential imaging of Ca ²⁺ dynamics within acute ex vivo slice preparations. Our methodology, Biosensor Lattice light-sheet Imaging of Multidimensional Presynaptic Structure (BLIMPS), includes acute brain slice preparation, mounting on a temperature-controlled flow chamber within a LLSM, and imaging of electrically evoked Ca ²⁺ signals, with high adaptability to a range of genetic and pharmacological disease models. Our technique offers high spectral separation between evoked signals from each of the two GECIs and fast acquisition speeds of 0.1-0.3 KHz. Included within the BLIMPS technique is a robust, open-source data analysis pipeline to track highly responsive neuronal structures such as presynaptic terminals and quantify both the amplitudes and decay rates of evoked fluxes.

Negative regulation of exocytosis from secretory cells is accomplished through inhibitory signals from G_i/o G-protein-coupled-receptors by Gβγ subunit inhibition of two mechanisms: decreased calcium entry and direct interaction of Gβγ with Soluble N-ethylmaleimide-sensitive factor Attachment Protein (SNAP) Receptor (SNARE) plasma membrane fusion machinery. Previously, we disabled the second mechanism with a SNAP25 truncation (SNAP25Δ3) decreasing Gβγ affinity for the SNARE complex, leaving exocytotic fusion and modulation of calcium entry intact, and removing GPCR-Gβγ inhibition of SNARE-mediated exocytosis. Here, we report substantial metabolic benefit in mice carrying this mutation. SNAP25^Δ3/Δ3 mice exhibit enhanced insulin sensitivity and beiging of white fat. Metabolic protection was amplified in SNAP25^Δ3/Δ3 mice challenged with high fat diet. Glucose homeostasis, whole body insulin action, and insulin-mediated glucose uptake into white adipose tissue were improved along with resistance to diet-induced obesity. Metabolic protection in SNAP25^Δ3/Δ3 mice occurred without compromising the physiological response to fasting or cold. All metabolic phenotypes were reversed at thermoneutrality, suggesting basal autonomic activity is required. Direct electrode stimulation of sympathetic neuron exocytosis from SNAP25^Δ3/Δ3 inguinal adipose depots resulted in enhanced and prolonged norepinephrine release. Thus, the Gβγ-SNARE interaction represents a cellular mechanism that deserves further exploration as an additional avenue for combatting metabolic disease.

Progressive hippocampal degeneration is a key component of Alzheimer’s disease (AD) progression. Therefore, identifying how hippocampal neuronal function is modulated early in AD is an important approach to eventually prevent degeneration. AD-risk factors and signaling molecules likely modulate neuronal function, including APOE genotype and angiotensin II. Compared to APOE3, APOE4 increases AD risk up to 12-fold, and high levels of angiotensin II are hypothesized to disrupt neuronal function in AD. However, the extent that APOE and angiotensin II modulates the hippocampal neuronal phenotype in AD-relevant models is unknown. To address this issue, we used electrophysiological techniques to assess the impact of APOE genotype and angiotensin II on basal synaptic transmission, presynaptic, and post-synaptic activity in mice that express human APOE3 (E3FAD) or APOE4 (E4FAD) and overproduce Aβ. We found that compared to E3FAD mice, E4FAD mice have lower synaptic activity, but higher levels of paired-pulse facilitation (PPF) and long-term potentiation (LTP) in the Schaffer Collateral Commissural Pathway (SCCP) of the hippocampus. We also found that exogenous angiotensin II has a profound inhibitory effect on hippocampal LTP in both E3FAD and E4FAD mice. Collectively, our data suggests that APOE4 and Aβ are associated with a hippocampal phenotype comprised of lower basal activity and higher responses to high-frequency stimulation, the latter of which is suppressed by angiotensin II. These novel data suggest a potential mechanistic link between hippocampal activity, APOE4 genotype, and angiotensin II in AD.

Light sheet microscopy in live cells requires minimal excitation intensity and resolves three-dimensional (3D) information rapidly. Lattice light sheet microscopy (LLSM) works similarly but uses a lattice configuration of Bessel beams to generate a flatter, diffraction-limited z-axis sheet suitable for investigating subcellular compartments, with better tissue penetration. We developed a LLSM method for investigating cellular properties of tissue in situ. Neural structures provide an important target. Neurons are complex 3D structures, and signaling between cells and subcellular structures requires high resolution imaging. We developed an LLSM configuration based on the Janelia Research Campus design or in situ recording that allows simultaneous electrophysiological recording. We give examples of using LLSM to assess synaptic function in situ. In presynapses, evoked Ca²⁺ entry causes vesicle fusion and neurotransmitter release. We demonstrate the use of LLSM to measure stimulus-evoked localized presynaptic Ca²⁺ entry and track synaptic vesicle recycling. We also demonstrate the resolution of postsynaptic Ca²⁺ signaling in single synapses. A challenge in 3D imaging is the need to move the emission objective to maintain focus. We have developed an incoherent holographic lattice light-sheet (IHLLS) technique to replace the LLS tube lens with a dual diffractive lens to obtain 3D images of spatially incoherent light diffracted from an object as incoherent holograms. The 3D structure is reproduced within the scanned volume without moving the emission objective. This eliminates mechanical artifacts and improves temporal resolution. We focus on LLS and IHLLS applications and data obtained in neuroscience and emphasize increases in temporal and spatial resolution using these approaches.

We previously developed an incoherent holography technique for use in lattice light sheet (LLS) microscopes that represents a specialized adaptation of light sheet microscopy. Light sheet instruments resolve 3D information by illuminating the sample at 90° to the imaging plane with a sheet of laser light that excites fluorophores in the sample only in a narrow plane. Imaging this plane and then moving it in the imaging z-axis allows construction of the sample volume. Among these types of instruments, LLS microscopy gives higher z-axis resolution and tissue depth penetration. It has a similar working principle to light sheet fluorescence microscopy but uses a lattice configuration of Bessel beams instead of Gaussian beams. Our incoherent light detection technique replaces the glass tube lens of the original LLS with a dual diffractive lens system to retrieve the axial depth of the sample. Here, we show that the system is applicable to all light sheet instruments. To make a direct comparison in the same emission light path, we can imitate the nature of non-Bessel light sheet systems by altering the mask annuli used to create Bessel beams in the LLS system. We change the diffractive mask annuli from a higher NA anulus to a smaller NA anulus. This generates a Gaussian excitation beam similar to conventional light sheet systems. Using this approach, we propose an incoherent light detection system for light sheet 3D imaging by choosing a variable NA and moving only the light sheet while keeping the sample stage and detection microscope objective stationary.

While the ability of G protein βγ subunits (Gβγ) to bind to and functionally inhibit the neuronal SNARE proteins Stx1A, SNAP25, and synaptobrevin in the presence of the calcium sensor synaptotagmin I is well documented, these three SNARE proteins, which form the core SNARE complex for synchronous evoked release in neurons, are but a subset of the larger family of SNARE proteins, which participate in many other exocytic processes within the cell and in other populations of secretory cells throughout the body, from which the release of neurotransmitters, hormones, and other factors is regulated by G _i/o -coupled GPCRs. The ability of Gβγ to regulate these processes is unknown. To investigate the feasibility of this mechanism to inhibit SNARE function more broadly, we utilized a series of biochemical assays of binding and function with four Qa-SNAREs (Stx1A, Stx2, Stx3, and Stx4) and four Qb,c-SNAREs (SNAP25, SNAP23, SNAP29, and SNAP47) in tandem with the R-SNARE synaptobrevin, synaptotagmin I, and Gβγ. Gβγ was found to bind to multiple Qa-SNARE isoforms as well as SNAP23, and inhibit the lipid mixing function of these SNAREs, as well as SNAP29. Together, this data suggests a more broad role for the Gβγ-SNARE pathway in the regulation of exocytosis beyond cells that express Stx1A or SNAP25.

Efficient and reliable neurotransmission requires precise coupling between action potentials (APs), Ca²⁺entry and neurotransmitter release. However, Ca²⁺requirements for release, including the number of channels required, their subtypes, and their location with respect to primed vesicles, remains to be precisely defined for central synapses. Indeed, Ca²⁺entry may occur through small numbers or even single open Ca²⁺channels, but these questions remain largely unexplored in simple active zone (AZ) synapses common in the nervous system, and key to addressing Ca²⁺channel and synaptic dysfunction underlying numerous neurologic and neuropsychiatric disorders. Here, we present single channel analysis of evoked AZ Ca²⁺entry, using cell-attached patch clamp and lattice light-sheet microscopy (LLSM), resolving small channel numbers evoking Ca²⁺entry following depolarization, at single AZs in individual central lamprey reticulospinal presynaptic terminals from male and females. We show a small pool (mean of 23) of Ca²⁺channels at each terminal, comprising N-(CaV2.2), P/Q-(CaV2.1), and R-(CaV2.3) subtypes, available to gate neurotransmitter release. Significantly, of this pool only one to seven channels (mean of 4) open on depolarization. High temporal fidelity lattice light-sheet imaging reveals AP-evoked Ca²⁺transients exhibiting quantal amplitude variations of 0–6 event sizes between individual APs and stochastic variation of precise locations of Ca²⁺entry within the AZ. Further, total Ca²⁺channel numbers at each AZ correlate to the number of presynaptic primed synaptic vesicles. Dispersion of channel openings across the AZ and the similar number of primed vesicles and channels indicate that Ca²⁺entry via as few as one channel may trigger neurotransmitter release.SIGNIFICANCE STATEMENTPresynaptic Ca²⁺entry through voltage-gated calcium channels (VGCCs) causes neurotransmitter release. To understand neurotransmission, its modulation, and plasticity, we must quantify Ca²⁺entry and its relationship to vesicle fusion. This requires direct recordings from active zones (AZs), previously possible only at calyceal terminals containing many AZs, where few channels open following action potentials (APs; Sheng et al., 2012), and even single channel openings may trigger release (Stanley, 1991, 1993). However, recording from more conventional terminals with single AZs commonly found centrally has thus far been impossible. We addressed this by cell-attached recordings from acutely dissociated single lamprey giant axon AZs, and by lattice light sheet microscopy of presynaptic Ca²⁺entry. We demonstrate nanodomains of presynaptic VGCCs coupling with primed vesicles with 1:1 stoichiometry.

In presynaptic terminals, membrane-delimited G_i/o-mediated presynaptic inhibition is ubiquitous and acts via Gβγ to inhibit Ca²⁺entry, or directly at SNARE complexes to inhibit Ca²⁺-dependent synaptotagmin-SNARE complex interactions. At CA1-subicular presynaptic terminals, 5-HT_1Band GABA_Breceptors colocalize. GABA_Breceptors inhibit Ca²⁺entry, whereas 5-HT_1Breceptors target SNARE complexes. We demonstrate in male and female rats that GABA_Breceptors alter P_r, whereas 5-HT_1Breceptors reduce evoked cleft glutamate concentrations, allowing differential inhibition of AMPAR and NMDAR EPSCs. This reduction in cleft glutamate concentration was confirmed by imaging glutamate release using a genetic sensor (iGluSnFR). Simulations of glutamate release and postsynaptic glutamate receptor currents were made. We tested effects of changes in vesicle numbers undergoing fusion at single synapses, relative placement of fusing vesicles and postsynaptic receptors, and the rate of release of glutamate from a fusion pore. Experimental effects of P_rchanges, consistent with GABA_Breceptor effects, were straightforwardly represented by changes in numbers of synapses. The effects of 5-HT_1Breceptor-mediated inhibition are well fit by simulated modulation of the release rate of glutamate into the cleft. Colocalization of different actions of GPCRs provides synaptic integration within presynaptic terminals. Train-dependent presynaptic Ca²⁺accumulation forces frequency-dependent recovery of neurotransmission during 5-HT_1Breceptor activation. This is consistent with competition between Ca²⁺-synaptotagmin and Gβγ at SNARE complexes. Thus, stimulus trains in 5-HT_1Breceptor agonist unveil dynamic synaptic modulation and a sophisticated hippocampal output filter that itself is modulated by colocalized GABA_Breceptors, which alter presynaptic Ca²⁺. In combination, these pathways allow complex presynaptic integration.SIGNIFICANCE STATEMENTTwo G protein-coupled receptors colocalize at presynaptic sites, to mediate presynaptic modulation by Gβγ, but one (a GABA_Breceptor) inhibits Ca²⁺entry whereas another (a 5-HT_1Breceptor) competes with Ca²⁺-synaptotagmin binding to the synaptic vesicle machinery. We have investigated downstream effects of signaling and integrative properties of these receptors. Their effects are profoundly different. GABA_Breceptors alter P_rleaving synaptic properties unchanged, whereas 5-HT_1Breceptors fundamentally change properties of synaptic transmission, modifying AMPAR but sparing NMDAR responses. Coactivation of these receptors allows synaptic integration because of convergence of GABA_Breceptor alteration on Ca²⁺and the effect of this altered Ca²⁺signal on 5-HT_1Breceptor signaling. This presynaptic convergence provides a novel form of synaptic integration.

Fresnel incoherent correlation holography (FINCH) was a milestone in incoherent holography. In this roadmap, two pathways, namely the development of FINCH and applications of FINCH explored by many prominent research groups, are discussed. The current state-of-the-art FINCH technology, challenges, and future perspectives of FINCH technology as recognized by a diverse group of researchers contributing to different facets of research in FINCH have been presented.