Postsynaptic protein assembly in three and two dimensions studied by mesoscopic simulations.

Recently, cellular biomolecular condensates formed via phase separation have received considerable attention. While they can be formed either in cytosol (denoted as 3D) or beneath the membrane (2D), the underlying difference between the two has not been well clarified. To compare the phase behaviors in 3D and 2D, postsynaptic density (PSD) serves as a model system. PSD is a protein condensate located under the postsynaptic membrane that influences the localization of glutamate receptors and thus contributes to synaptic plasticity. Recent in vitro studies have revealed the formation of droplets of various soluble PSD proteins via liquid-liquid phase separation. However, it is unclear how these protein condensates are formed beneath the membrane and how they specifically affect the localization of glutamate receptors in the membrane. In this study, focusing on the mixture of a glutamate receptor complex, AMPAR-TARP, and a ubiquitous scaffolding protein, PSD-95, we constructed a mesoscopic model of protein-domain interactions in PSD and performed comparative molecular simulations. The results showed a sharp contrast in the phase behaviors of protein assemblies in 3D and those under the membrane (2D). A mixture of a soluble variant of the AMPAR-TARP complex and PSD-95 in the 3D system resulted in a phase-separated condensate, which was consistent with the experimental results. However, with identical domain interactions, AMPAR-TARP embedded in the membrane formed clusters with PSD-95, but did not form a stable separated phase. Thus, the cluster formation behaviors of PSD proteins in the 3D and 2D systems were distinct. The current study suggests that, more generally, stable phase separation can be more difficult to achieve in and beneath the membrane than in 3D systems.

Structural insights into the ligand binding domain of GluD1 and GluD2 receptors.

GluD1 and GluD2 subunits (also known as delta 1 and 2) are the members of the delta family of ionotropic glutamate receptors. They are particularly puzzling, since they are unable to bind glutamate, but rather bind glycine and d-serine via their classical ligand binding domain (LBD). While GluD2 has been the subject of intensive research over the past decades, it is only recently that GluD1 received similar interest and very few studies compare the properties of these two membrane proteins. In their research article included in this issue, Masternak et al. resolved the 3D structure of the GluD1 LBD, compared its d-serine sensitivity with that of GluD2 and identified critical residues involved in the dynamics of the LBD.

Mechanism of Calcium Permeation in a Glutamate Receptor Ion Channel.

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are neurotransmitter-activated cation channels ubiquitously expressed in vertebrate brains. The regulation of calcium flux through the channel pore by RNA-editing is linked to synaptic plasticity while excessive calcium influx poses a risk for neurodegeneration. Unfortunately, the molecular mechanisms underlying this key process are mostly unknown. Here, we investigated calcium conduction in calcium-permeable AMPAR using Molecular Dynamics (MD) simulations with recently introduced multisite force-field parameters for Ca2+. Our calculations are consistent with experiment and explain the distinct calcium permeability in different RNA-edited forms of GluA2. For one of the identified metal binding sites, multiscale Quantum Mechanics/Molecular Mechanics (QM/MM) simulations further validated the results from MD and revealed small but reproducible charge transfer between the metal ion and its first solvation shell. In addition, the ion occupancy derived from MD simulations independently reproduced the Ca2+ binding profile in an X-ray structure of an NaK channel mimicking the AMPAR selectivity filter. This integrated study comprising X-ray crystallography, multisite MD, and multiscale QM/MM simulations provides unprecedented insights into Ca2+ permeation mechanisms in AMPARs, and paves the way for studying other biological processes in which Ca2+ plays a pivotal role.

Stroke: Molecular mechanisms and therapies: Update on recent developments.

Stroke, a neurological disease, is one of the leading causes of death worldwide, resulting in long-term disability in most survivors. Annual stroke costs in the United States alone were estimated at $46 billion recently. Stroke pathophysiology is complex, involving multiple causal factors, among which atherosclerosis, thrombus, and embolus are prevalent. The molecular mechanisms involved in the pathophysiology are essential to understanding targeted drug development. Some common mechanisms are excitotoxicity and calcium overload, oxidative stress, and neuroinflammation. In addition, various modifiable and non-modifiable risk factors increase the chances of stroke manifolds. Once a patient encounters a stroke, complete restoration of motor ability and cognitive skills is often rare. Therefore, shaping therapeutic strategies is paramount for finding a viable therapeutic agent. Apart from tPA, an FDA-approved therapy that is applied in most stroke cases, many other therapeutic strategies have been met with limited success. Stroke therapies often involve a combination of multiple strategies to restore the patient's normal function. Certain drugs like Gamma-aminobutyric receptor agonists (GABA), Glutamate Receptor inhibitors, Sodium, and Calcium channel blockers, and fibrinogen-depleting agents have shown promise in stroke treatment. Recently, a drug, DM199, a recombinant (synthetic) form of a naturally occurring protein called human tissue kallikrein-1 (KLK1), has shown great potential in treating stroke with fewer side effects. Furthermore, DM199 has been found to overcome the limitations presented when using tPA and/or mechanical thrombectomy. Cell-based therapies like Neural Stem Cells, Hematopoietic stem cells (HSCs), and Human umbilical cord blood-derived mesenchymal stem cells (HUCB-MSCs) are also being explored as a treatment of choice for stroke. These therapeutic agents come with merits and demerits, but continuous research and efforts are being made to develop the best therapeutic strategies to minimize the damage post-stroke and restore complete neurological function in stroke patients.

Enhanced AMPAR-dependent synaptic transmission by S-nitrosylation in the vmPFC contributes to chronic inflammatory pain-induced persistent anxiety in mice.

Chronic pain patients often have anxiety disorders, and some of them suffer from anxiety even after analgesic administration. In this study, we investigated the role of AMPAR-mediated synaptic transmission in the ventromedial prefrontal cortex (vmPFC) in chronic pain-induced persistent anxiety in mice and explored potential drug targets. Chronic inflammatory pain was induced in mice by bilateral injection of complete Freund's adjuvant (CFA) into the planta of the hind paws; anxiety-like behaviours were assessed with behavioural tests; S-nitrosylation and AMPAR-mediated synaptic transmission were examined using biochemical assays and electrophysiological recordings, respectively. We found that CFA induced persistent upregulation of AMPAR membrane expression and function in the vmPFC of anxious mice but not in the vmPFC of non-anxious mice. The anxious mice exhibited higher S-nitrosylation of stargazin (an AMPAR-interacting protein) in the vmPFC. Inhibition of S-nitrosylation by bilaterally infusing an exogenous stargazin (C302S) mutant into the vmPFC rescued the surface expression of GluA1 and AMPAR-mediated synaptic transmission as well as the anxiety-like behaviours in CFA-injected mice, even after ibuprofen treatment. Moreover, administration of ZL006, a small molecular inhibitor disrupting the interaction of nNOS and PSD-95 (20 mg·kg-1·d-1, for 5 days, i.p.), significantly reduced nitric oxide production and S-nitrosylation of AMPAR-interacting proteins in the vmPFC, resulting in anxiolytic-like effects in anxious mice after ibuprofen treatment. We conclude that S-nitrosylation is necessary for AMPAR trafficking and function in the vmPFC under chronic inflammatory pain-induced persistent anxiety conditions, and nNOS-PSD-95 inhibitors could be potential anxiolytics specific for chronic inflammatory pain-induced persistent anxiety after analgesic treatment.

Differences between the GluD1 and GluD2 receptors revealed by GluD1 X-ray crystallography, binding studies and molecular dynamics.

Ionotropic glutamate receptors are ligand-gated ion channels essential for fast excitatory neurotransmission in the brain. In contrast to most other members of the iGluR family, the subfamily of delta receptors, GluD1 and GluD2, does not bind glutamate but glycine/D-serine. GluD1 is widely expressed in the brain and the inner ear, where it is required for high-frequency hearing. Furthermore, it has been associated with schizophrenia, autism and depression. X-ray structures of the ligand-binding domain (LBD) of GluD2 have been published; however, no high-resolution structure is available for the ligand-binding domain of GluD1 (GluD1-LBD). Here, we report the X-ray crystal structure of the GluD1-LBD in its apo form at 2.57 Å resolution. Using isothermal titration calorimetry, we show that D-serine binds to the GluD1-LBD in an exothermic manner with a Kd of 160 µm, which is approximately five-fold greater than at GluD2. Furthermore, we identify Glu822 as a critical determinant of receptor activation in GluD1 A654T. In contrast to studies on the GluD2 lurcher mutant A654T, we did not observe any effect of 1 mm D-serine on the spontaneous currents at mouse GluD1 A654T by electrophysiological recordings of Xenopus laevis oocytes as previously also reported by others. These results point towards differences in the structure and dynamics between GluD1 and GluD2. Molecular dynamics simulations were employed to address this observation, suggesting that the apo structure of GluD1 is less flexible than the apo structure of GluD2 and that Pro725 in GluD1 may affect the interlobe closure of the ligand-binding domain of GluD1.

Distinct molecular pathways govern presynaptic homeostatic plasticity.

Presynaptic homeostatic plasticity (PHP) stabilizes synaptic transmission by counteracting impaired neurotransmitter receptor function through neurotransmitter release potentiation. PHP is thought to be triggered by impaired receptor function and to involve a stereotypic signaling pathway. However, here we demonstrate that different receptor perturbations that similarly reduce synaptic transmission result in different responses at the Drosophila neuromuscular junction. While receptor inhibition by the glutamate receptor (GluR) antagonist γ-D-glutamylglycine (γDGG) is not compensated by PHP, the GluR inhibitors Philanthotoxin-433 (PhTx) and Gyki-53655 (Gyki) induce compensatory PHP. Intriguingly, PHP triggered by PhTx and Gyki involve separable signaling pathways, including inhibition of distinct GluR subtypes, differential modulation of the active-zone scaffold Bruchpilot, and short-term plasticity. Moreover, while PHP upon Gyki treatment does not require genes promoting PhTx-induced PHP, it involves presynaptic protein kinase D. Thus, synapses not only respond differentially to similar activity impairments, but achieve homeostatic compensation via distinct mechanisms, highlighting the diversity of homeostatic signaling.

Sodium-calcium exchanger mediates sensory-evoked glial calcium transients in the developing retinotectal system.

Various types of sensory stimuli have been shown to induce Ca2+ elevations in glia. However, a mechanistic understanding of the signaling pathways mediating sensory-evoked activity in glia in intact animals is still emerging. During early development of the Xenopus laevis visual system, radial astrocytes in the optic tectum are highly responsive to sensory stimulation. Ca2+ transients occur spontaneously in radial astrocytes at rest and are abolished by silencing neuronal activity with tetrodotoxin. Visual stimulation drives temporally correlated increases in the activity patterns of neighboring radial astrocytes. Following blockade of all glutamate receptors (gluRs), visually evoked Ca2+ activity in radial astrocytes persists, while neuronal activity is suppressed. The additional blockade of either glu transporters or sodium-calcium exchangers (NCX) abolishes visually evoked responses in glia. Finally, we demonstrate that blockade of NCX alone is sufficient to prevent visually evoked responses in radial astrocytes, highlighting a pivotal role for NCX in glia during development.

ß-Amyloid disruption of LTP/LTD balance is mediated by AKAP150-anchored PKA and Calcineurin regulation of Ca2+-permeable AMPA receptors.

Regulated insertion and removal of postsynaptic AMPA glutamate receptors (AMPARs) mediates hippocampal long-term potentiation (LTP) and long-term depression (LTD) synaptic plasticity underlying learning and memory. In Alzheimer's disease ß-amyloid (Aß) oligomers may impair learning and memory by altering AMPAR trafficking and LTP/LTD balance. Importantly, Ca2+-permeable AMPARs (CP-AMPARs) assembled from GluA1 subunits are excluded from hippocampal synapses basally but can be recruited rapidly during LTP and LTD to modify synaptic strength and signaling. By employing mouse knockin mutations that disrupt anchoring of the kinase PKA or phosphatase Calcineurin (CaN) to the postsynaptic scaffold protein AKAP150, we find that local AKAP-PKA signaling is required for CP-AMPAR recruitment, which can facilitate LTP but also, paradoxically, prime synapses for Aß impairment of LTP mediated by local AKAP-CaN LTD signaling that promotes subsequent CP-AMPAR removal. These findings highlight the importance of PKA/CaN signaling balance and CP-AMPARs in normal plasticity and aberrant plasticity linked to disease.

Single molecule FRET methodology for investigating glutamate receptors.

Single molecule Förster Resonance Energy Transfer (smFRET) allows us to measure variation in distances between donor and acceptor fluorophores attached to a protein, providing the conformational landscape of the protein with respect to this specific distance. smFRET can be performed on freely diffusing molecules or on tethered molecules. Here, we describe the tethered method used to study ionotropic glutamate receptors, which allows us to track the changes in FRET as a function of time, thus providing information on the conformations sampled and kinetics of conformational changes in the millisecond to second time scale. Strategies for attaching fluorophores to the proteins, methods for acquiring and analyzing the smFRET trajectories, and limitations are discussed.

Structure of the Arabidopsis thaliana glutamate receptor-like channel GLR3.4.

Glutamate receptor-like channels (GLRs) play vital roles in various physiological processes in plants, such as wound response, stomatal aperture control, seed germination, root development, innate immune response, pollen tube growth, and morphogenesis. Despite the importance of GLRs, knowledge about their molecular organization is limited. Here we use X-ray crystallography and single-particle cryo-EM to solve structures of the Arabidopsis thaliana GLR3.4. Our structures reveal the tetrameric assembly of GLR3.4 subunits into a three-layer domain architecture, reminiscent of animal ionotropic glutamate receptors (iGluRs). However, the non-swapped arrangement between layers of GLR3.4 domains, binding of glutathione through S-glutathionylation of cysteine C205 inside the amino-terminal domain clamshell, unique symmetry, inter-domain interfaces, and ligand specificity distinguish GLR3.4 from representatives of the iGluR family and suggest distinct features of the GLR gating mechanism. Our work elaborates on the principles of GLR architecture and symmetry and provides a molecular template for deciphering GLR-dependent signaling mechanisms in plants.

Structure of the Arabidopsis Glutamate Receptor-like Channel GLR3.2 Ligand-Binding Domain.

Glutamate receptor-like channels (GLRs) play important roles in numerous plant physiological processes. GLRs are homologous to ionotropic glutamate receptors (iGluRs) that mediate neurotransmission in vertebrates. Here we determine crystal structures of Arabidopsis thaliana GLR3.2 ligand-binding domain (LBD) in complex with glycine and methionine to 1.58- and 1.75-Å resolution, respectively. Our structures show a fold similar to that of iGluRs, but with several secondary structure elements either missing or different. The closed clamshell conformation of GLR3.2 LBD suggests that both glycine and methionine act as agonists. The mutation R133A strongly increases the constitutive activity of the channel, suggesting that the LBD mutated at the residue critical for agonist binding produces a more stable closed clamshell conformation. Furthermore, our structures explain the promiscuity of GLR activation by different amino acids, confirm evolutionary conservation of structure between GLRs and iGluRs, and predict common molecular principles of their gating mechanisms driven by bilobed clamshell-like LBDs.

D-Serine Potently Drives Ligand-Binding Domain Closure in the Ionotropic Glutamate Receptor GluD2.

Despite their classification as ionotropic glutamate receptors, GluD receptors are not functional ligand-gated ion channels and do not bind glutamate. GluD2 receptors bind D-serine and coordinate transsynaptic complexes that regulate synaptic plasticity. Instead of opening the ion channel pore, mechanical tension produced from closure of GluD2 ligand-binding domains (LBDs) drives conformational rearrangements for non-ionotropic signaling. We report computed conformational free energy landscapes for the GluD2 LBD in apo and D-serine-bound states. Unexpectedly, the conformational free energy associated with GluD2 LBD closure upon D-serine binding is greater than that for AMPA, NMDA, and kainate receptor LBDs upon agonist binding. This excludes insufficient force generation as an explanation for lack of ion channel activity in GluD2 receptors and suggests that non-ionotropic conformational rearrangements do more work than pore opening. We also report free energy landscapes for GluD2 LBD harboring a neurodegenerative mutation and demonstrate selective stabilization of closed conformations in the apo state.

Distinct Cell Surface Expression Patterns of N-Glycosylation Site Mutants of AMPA-Type Glutamate Receptor under the Homo-Oligomeric Expression Conditions.

The AMPA-type glutamate receptor (AMPAR) is a homotetrameric or heterotetrameric ion channel composed of various combinations of four subunits (GluA1-4), and its abundance in the synapse determines the strength of synaptic activity. The formation of oligomers in the endoplasmatic reticulum (ER) is crucial for AMPAR subunits' ER-exit and translocation to the cell membrane. Although N-glycosylation on different AMPAR subunits has been shown to regulate the ER-exit of hetero-oligomers, its role in the ER-exit of homo-oligomers remains unclear. In this study, we investigated the role of N-glycans at GluA1N63/N363 and GluA2N370 in ER-exit under the homo-oligomeric expression conditions, whose mutants are known to show low cell surface expressions. In contrast to the N-glycosylation site mutant GluA1N63Q, the cell surface expression levels of GluA1N363Q and GluA2N370Q increased in a time-dependent manner. Unlike wild-type (WT) GluA1, GluA2WT rescued surface GluA2N370Q expression. Additionally, the expression of GluA1N63Q reduced the cell surface expression level of GluA1WT. In conclusion, our findings suggest that these N-glycans have distinct roles in the ER-exit of GluA1 and GluA2 homo-oligomers; N-glycan at GluA1N63 is a prerequisite for GluA1 ER-exit, whereas N-glycans at GluA1N363 and GluA2N370 control the ER-exit rate.

Poly-arginine-18 (R18) Confers Neuroprotection through Glutamate Receptor Modulation, Intracellular Calcium Reduction, and Preservation of Mitochondrial Function.

Recent studies have highlighted that a novel class of neuroprotective peptide, known as cationic arginine-rich peptides (CARPs), have intrinsic neuroprotective properties and are particularly effective anti-excitotoxic agents. As such, the present study investigated the mechanisms underlying the anti-excitotoxic properties of CARPs, using poly-arginine-18 (R18; 18-mer of arginine) as a representative peptide. Cortical neuronal cultures subjected to glutamic acid excitotoxicity were used to assess the effects of R18 on ionotropic glutamate receptor (iGluR)-mediated intracellular calcium influx, and its ability to reduce neuronal injury from raised intracellular calcium levels after inhibition of endoplasmic reticulum calcium uptake by thapsigargin. The results indicate that R18 significantly reduces calcium influx by suppressing iGluR overactivation, and results in preservation of mitochondrial membrane potential (ΔΨm) and ATP production, and reduced ROS generation. R18 also protected cortical neurons against thapsigargin-induced neurotoxicity, which indicates that the peptide helps maintain neuronal survival when intracellular calcium levels are elevated. Taken together, these findings provide important insight into the mechanisms of action of R18, supporting its potential application as a neuroprotective therapeutic for acute and chronic neurological disorders.

Structures of metabotropic GABAB receptor.

Stimulation of the metabotropic GABAB receptor by γ-aminobutyric acid (GABA) results in prolonged inhibition of neurotransmission, which is central to brain physiology1. GABAB belongs to family C of the G-protein-coupled receptors, which operate as dimers to transform synaptic neurotransmitter signals into a cellular response through the binding and activation of heterotrimeric G proteins2,3. However, GABAB is unique in its function as an obligate heterodimer in which agonist binding and G-protein activation take place on distinct subunits4,5. Here we present cryo-electron microscopy structures of heterodimeric and homodimeric full-length GABAB receptors. Complemented by cellular signalling assays and atomistic simulations, these structures reveal that extracellular loop 2 (ECL2) of GABAB has an essential role in relaying structural transitions by ordering the linker that connects the extracellular ligand-binding domain to the transmembrane region. Furthermore, the ECL2 of each of the subunits of GABAB caps and interacts with the hydrophilic head of a phospholipid that occupies the extracellular half of the transmembrane domain, thereby providing a potentially crucial link between ligand binding and the receptor core that engages G proteins. These results provide a starting framework through which to decipher the mechanistic modes of signal transduction mediated by GABAB dimers, and have important implications for rational drug design that targets these receptors.

Mutational Analysis and Modeling of Negative Allosteric Modulator Binding Sites in AMPA Receptors.

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) constitute a subclass of the ionotropic glutamate receptor superfamily, which functions as glutamate-gated cation channels to mediate the majority of excitatory neurotransmission in the central nervous system. AMPARs are therapeutic targets in a range of brain disorders associated with abnormal glutamate hyperactivity. Multiple classes of AMPAR inhibitors have been developed during the past decades, including competitive antagonists, ion channel blockers, and negative allosteric modulators (NAMs). At present, the NAM is the only class of AMPAR ligands that have been developed into safe and useful drugs in humans in the form of perampanel (Fycompa), which was recently approved for treatment of epilepsy. Compared with the detailed understanding of other AMPAR ligand classes, surprisingly little information has been available regarding the molecular mechanism of perampanel and other classes of NAMs at AMPARs; including the location and structure of NAM binding pockets in the receptor complex. However, structures of the AMPAR GluA2 in complex with NAMs were recently reported that unambiguously identified the NAM binding sites. In parallel with this work, our aim with the present study was to identify specific residues involved in the formation of the NAM binding site for three prototypical AMPAR NAMs. Hence, we have performed a mutational analysis of the AMPAR region that links the four extracellular ligand-binding domains to the central ion channel in the transmembrane domain region. Furthermore, we perform computational ligand docking of the NAMs into structural models of the homomeric GluA2 receptor and optimize side chain conformations around the NAMs to model how NAMs bind in this specific site. The new insights provide potentially valuable input for structure-based drug design of new NAMs. SIGNIFICANCE STATEMENT: The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are glutamate-gated ion channels that mediate the majority of excitatory neurotransmission in the brain. Negative allosteric modulators of AMPA receptors are considered to have significant therapeutic potential in diseases linked to glutamate hyperactivity. The present work employs mutational analysis and molecular modeling of the binding site for prototypical NAMs to provide new molecular insight into how NAMs interact with the AMPA receptor, which is of potential use for future design of new types of NAMs.

Structural biology of glutamate receptor ion channels: towards an understanding of mechanism.

Ionotropic glutamate receptors (iGluRs) are tetrameric ion channels that mediate signal transmission at neuronal synapses, where they contribute centrally to the postsynaptic plasticity that underlies learning and memory. Receptor activation by l-glutamate triggers complex allosteric cascades that are transmitted through the layered and highly flexible receptor assembly culminating in opening a cation-selective pore. This process is shaped by the arrangement of the four core subunits as well as the presence of various auxiliary subunits, and is subject to regulation by an array of small molecule modulators targeting a number of sites throughout the complex. Here, we discuss recent structures of iGluR homomers and heteromers illuminating the organization and subunit arrangement of the core tetramer, co-assembled with auxiliary subunits and in complex with allosteric modulators.

On-cell coordination chemistry: Chemogenetic activation of membrane-bound glutamate receptors in living cells.

Investigating functions of membrane-bound receptors provides invaluable information about cellular signaling and physiological events. Recently, chemical genetic methods to design chemical switches on the target proteins have intensely been developed for interrogation of the cellular signaling of individual receptor proteins. We recently reported coordination chemistry-based chemogenetics to allosterically activate two types of neurotransmitter receptors, ionotropic and metabotropic glutamate receptors, in living cells. Based on their well-studied structure-activity relationships, we semi-rationally incorporated two His mutations into glutamate receptors near ligand binding pockets as an allosteric site. The engineered glutamate receptors could be allosterically activated upon treatment of Pd(bpy) complex (bpy: 2,2'-bipyridine) through stabilization of the activated conformation in mammalian cells and cultured neurons. Here, we describe the detailed protocol of our approach including the receptor design and activation of the His-engineered receptors and the downstream of the signal transduction cascade in living cells.

Holographic two-photon activation for synthetic optogenetics.

Optogenetic tools provide users the ability to photocontrol the activity of cells. Commonly, activation is achieved by expression of proteins from photosynthetic organisms, for example, microbial opsins (e.g., ChR2). Alternatively, a sister approach, synthetic optogenetics, enables photocontrol over proteins of mammalian origin by use of photoswitches, visible light (typically), and genetic modification. Thus, synthetic optogenetics facilitates interrogation of native neuronal signaling mechanisms. However, the poor tissue penetration of visible wavelengths impedes the use of the technique in tissue, as two-photon excitation (2PE) is typically required to access the near-infrared window. Here, we describe an alternative technique that uses 2PE-compatible photoswitches (section 1) for photoactivation of genetically modified glutamate receptors (section 2). Furthermore, for fast, multi-region photoactivation, we describe the use of 2P-digital holography (2P-DH) (section 3). We detail how to combine 2P-DH and synthetic optogenetics with electrophysiology, or with red fluorescence Ca2+ recordings, for all-optical neural interrogation. The time required to complete the methods, aside from obtaining the necessary reagents and illumination equipment, is ~3 weeks.