Engineered fluidic device to achieve multiplexed monitoring of cell cultures with digital holographic microscopy.

We present a low-cost, 3D-printed, and biocompatible fluidic device, engineered to produce laminar and homogeneous flow over a large field-of-view. Such a fluidic device allows us to perform multiplexed temporal monitoring of cell cultures compatible with the use of various pharmacological protocols. Therefore, specific properties of each of the observed cell cultures can be discriminated simultaneously during the same experiment. This was illustrated by monitoring the agonists-mediated cellular responses, with digital holographic microscopy, of four different cell culture models of cystic fibrosis. Quantitatively speaking, this multiplexed approach provides a time saving factor of around four to reveal specific cellular features.

Image-Based Marker-Free Screening of GABAA Agonists, Antagonists, and Modulators.

The ionotropic GABAA receptors represent the main target for different groups of widely used drugs having hypnotic and anxiolytic effects. So far, most approaches used to assess GABA activity involve invasive low -throughput electrophysiological techniques or rely on fluorescent dyes, preventing the ability to conduct noninvasive and thus nonperturbing screens. To address this limitation, we have developed an automated marker-free cell imaging method, based on digital holographic microscopy (DHM). This technology allows the automatically screening of compounds in multiple plates without having to label the cells or use special plates. This methodological approach was first validated by screening the GABAA receptor expressed in HEK cells using a selection of active compounds in agonist, antagonist, and modulator modes. Then, in a second blind screen of a library of 3041 compounds (mostly composed of natural products), 5 compounds having a specific agonist action on the GABAA receptor were identified. The hits validated from this unbiased screen were the natural products muscimol, neurosteroid alphaxalone, and three compounds belonging to the avermectin family, all known for having an agonistic effect on the GABAA receptor. The results obtained were exempt from false negatives (structurally similar unassigned hits), and false-positive hits were detected and discarded without the need for performing electrophysiological measurements. The outcome of the screen demonstrates the applicability of our screening by imaging method for the discovery of new chemical structures, particularly regarding chemicals interacting with the ionotropic GABAA receptor and more generally with any ligand-gated ion channels and transporters.

Mapping of real-time morphological changes in the neuronal cytoskeleton with label-free wide-field second-harmonic imaging: a case study of nocodazole.

We demonstrate the use of wide-field high-throughput second-harmonic (SH) microscopy for investigating cytoskeletal morphological changes on the single-cell level. The method allows for real-time, in vitro, label-free measurements of cytoskeletal changes that can, under certain conditions, be quantified in terms of orientational distribution or in terms of changes in the number of microtubules. As SH generation is intrinsically sensitive to noncentrosymmetrically structured microtubules, but not to isotropic or centrosymmetric materials, we use it to probe the microtubule structure in the cytoskeleton when it undergoes dynamic changes induced by the application of nocodazole, a well-known microtubule-destabilizing drug that reversibly depolymerizes microtubules. In addition, the orientational directionality of microtubules in neurites and cell bodies is determined label-free using SH polarimetry measurements. Finally, we use spatiotemporal SH imaging to show label-free, real-time nocodazole-induced morphological changes in neurons of different age and in a single axon.

Lactate promotes plasticity gene expression by potentiating NMDA signaling in neurons.

L-lactate is a product of aerobic glycolysis that can be used by neurons as an energy substrate. Here we report that in neurons L-lactate stimulates the expression of synaptic plasticity-related genes such as Arc, c-Fos, and Zif268 through a mechanism involving NMDA receptor activity and its downstream signaling cascade Erk1/2. L-lactate potentiates NMDA receptor-mediated currents and the ensuing increase in intracellular calcium. In parallel to this, L-lactate increases intracellular levels of NADH, thereby modulating the redox state of neurons. NADH mimics all of the effects of L-lactate on NMDA signaling, pointing to NADH increase as a primary mediator of L-lactate effects. The induction of plasticity genes is observed both in mouse primary neurons in culture and in vivo in the mouse sensory-motor cortex. These results provide insights for the understanding of the molecular mechanisms underlying the critical role of astrocyte-derived L-lactate in long-term memory and long-term potentiation in vivo. This set of data reveals a previously unidentified action of L-lactate as a signaling molecule for neuronal plasticity.

The human CFTR protein expressed in CHO cells activates aquaporin-3 in a cAMP-dependent pathway: study by digital holographic microscopy.

The transmembrane water movements during cellular processes and their relationship to ionic channel activity remain largely unknown. As an example, in epithelial cells it was proposed that the movement of water could be directly linked to cystic fibrosis transmembrane conductance regulator (CFTR) protein activity through a cAMP-stimulated aqueous pore, or be dependent on aquaporin. Here, we used digital holographic microscopy (DHM) an interferometric technique to quantify in situ the transmembrane water fluxes during the activity of the epithelial chloride channel, CFTR, measured by patch-clamp and iodide efflux techniques. We showed that the water transport measured by DHM is fully inhibited by the selective CFTR blocker CFTRinh172 and is absent in cells lacking CFTR. Of note, in cells expressing the mutated version of CFTR (F508del-CFTR), which mimics the most common genetic alteration encountered in cystic fibrosis, we also show that the water movement is profoundly altered but restored by pharmacological manipulation of F508del-CFTR-defective trafficking. Importantly, whereas activation of this endogenous water channel required a cAMP-dependent stimulation of CFTR, activation of CFTR or F508del-CFTR by two cAMP-independent CFTR activators, genistein and MPB91, failed to trigger water movements. Finally, using a specific small-interfering RNA against the endogenous aquaporin AQP3, the water transport accompanying CFTR activity decreased. We conclude that water fluxes accompanying CFTR activity are linked to AQP3 but not to a cAMP-stimulated aqueous pore in the CFTR protein.

High throughput second harmonic imaging for label-free biological applications.

Second harmonic generation (SHG) is inherently sensitive to the absence of spatial centrosymmetry, which can render it intrinsically sensitive to interfacial processes, chemical changes and electrochemical responses. Here, we seek to improve the imaging throughput of SHG microscopy by using a wide-field imaging scheme in combination with a medium-range repetition rate amplified near infrared femtosecond laser source and gated detection. The imaging throughput of this configuration is tested by measuring the optical image contrast for different image acquisition times of BaTiO3 nanoparticles in two different wide-field setups and one commercial point-scanning configuration. We find that the second harmonic imaging throughput is improved by 2-3 orders of magnitude compared to point-scan imaging. Capitalizing on this result, we perform low fluence imaging of (parts of) living mammalian neurons in culture.

Measurement of absolute cell volume, osmotic membrane water permeability, and refractive index of transmembrane water and solute flux by digital holographic microscopy.

A dual-wavelength digital holographic microscope to measure absolute volume of living cells is proposed. The optical setup allows us to reconstruct two quantitative phase contrast images at two different wavelengths from a single hologram acquisition. When adding the absorbing dye fast green FCF as a dispersive agent to the extracellular medium, cellular thickness can be univocally determined in the full field of view. In addition to the absolute cell volume, the method can be applied to derive important biophysical parameters of living cells including osmotic membrane water permeability coefficient and the integral intracellular refractive index (RI). Further, the RI of transmembrane flux can be determined giving an indication about the nature of transported solutes. The proposed method is applied to cultured human embryonic kidney cells, Chinese hamster ovary cells, human red blood cells, mouse cortical astrocytes, and neurons.

Simultaneous optical recording in multiple cells by digital holographic microscopy of chloride current associated to activation of the ligand-gated chloride channel GABA(A) receptor.

Chloride channels represent a group of targets for major clinical indications. However, molecular screening for chloride channel modulators has proven to be difficult and time-consuming as approaches essentially rely on the use of fluorescent dyes or invasive patch-clamp techniques which do not lend themselves to the screening of large sets of compounds. To address this problem, we have developed a non-invasive optical method, based on digital holographic microcopy (DHM), allowing monitoring of ion channel activity without using any electrode or fluorescent dye. To illustrate this approach, GABA(A) mediated chloride currents have been monitored with DHM. Practically, we show that DHM can non-invasively provide the quantitative determination of transmembrane chloride fluxes mediated by the activation of chloride channels associated with GABA(A) receptors. Indeed through an original algorithm, chloride currents elicited by application of appropriate agonists of the GABA(A) receptor can be derived from the quantitative phase signal recorded with DHM. Finally, chloride currents can be determined and pharmacologically characterized non-invasively simultaneously on a large cellular sampling by DHM.

Early cell death detection with digital holographic microscopy.

BACKGROUND: Digital holography provides a non-invasive measurement of the quantitative phase shifts induced by cells in culture, which can be related to cell volume changes. It has been shown previously that regulation of cell volume, in particular as it relates to ionic homeostasis, is crucially involved in the activation/inactivation of the cell death processes. We thus present here an application of digital holographic microscopy (DHM) dedicated to early and label-free detection of cell death. METHODS AND FINDINGS: We provide quantitative measurements of phase signal obtained on mouse cortical neurons, and caused by early neuronal cell volume regulation triggered by excitotoxic concentrations of L-glutamate. We show that the efficiency of this early regulation of cell volume detected by DHM, is correlated with the occurrence of subsequent neuronal death assessed with the widely accepted trypan blue method for detection of cell viability. CONCLUSIONS: The determination of the phase signal by DHM provides a simple and rapid optical method for the early detection of cell death.

Determination of transmembrane water fluxes in neurons elicited by glutamate ionotropic receptors and by the cotransporters KCC2 and NKCC1: a digital holographic microscopy study.

Digital holographic microscopy (DHM) is a noninvasive optical imaging technique that provides quantitative phase images of living cells. In a recent study, we showed that the quantitative monitoring of the phase signal by DHM was a simple label-free method to study the effects of glutamate on neuronal optical responses (Pavillon et al., 2010). Here, we refine these observations and show that glutamate produces the following three distinct optical responses in mouse primary cortical neurons in culture, predominantly mediated by NMDA receptors: biphasic, reversible decrease (RD) and irreversible decrease (ID) responses. The shape and amplitude of the optical signal were not associated with a particular cellular phenotype but reflected the physiopathological status of neurons linked to the degree of NMDA activity. Thus, the biphasic, RD, and ID responses indicated, respectively, a low-level, a high-level, and an "excitotoxic" level of NMDA activation. Moreover, furosemide and bumetanide, two inhibitors of sodium-coupled and/or potassium-coupled chloride movement strongly modified the phase shift, suggesting an involvement of two neuronal cotransporters, NKCC1 (Na-K-Cl) and KCC2 (K-Cl) in the genesis of the optical signal. This observation is of particular interest since it shows that DHM is the first imaging technique able to monitor dynamically and in situ the activity of these cotransporters during physiological and/or pathological neuronal conditions.

Cell morphology and intracellular ionic homeostasis explored with a multimodal approach combining epifluorescence and digital holographic microscopy.

The authors have developed a live-cell multimodality microscope combining epifluorescence with digital holographic microscopy; it has been implemented with a decoupling procedure allowing to separately measure from the quantitative phase important cell parameters including absolute volume, shape and integral intracellular refractive index. In combination with the numerous different specific fluorescent cellular probes, this multimodality microscopy can address important issues in cell biology. This is demonstrated by the study of intracellular calcium homeostasis associated with the change in cell volume, which play a critical role in the excitotoxicity-induced neuronal death.

Glutamate exocytosis from astrocytes controls synaptic strength.

The release of transmitters from glia influences synaptic functions. The modalities and physiological functions of glial release are poorly understood. Here we show that glutamate exocytosis from astrocytes of the rat hippocampal dentate molecular layer enhances synaptic strength at excitatory synapses between perforant path afferents and granule cells. The effect is mediated by ifenprodil-sensitive NMDA ionotropic glutamate receptors and involves an increase of transmitter release at the synapse. Correspondingly, we identify NMDA receptor 2B subunits on the extrasynaptic portion of excitatory nerve terminals. The receptor distribution is spatially related to glutamate-containing synaptic-like microvesicles in the apposed astrocytic processes. This glial regulatory pathway is endogenously activated by neuronal activity-dependent stimulation of purinergic P2Y1 receptors on the astrocytes. Thus, we provide the first combined functional and ultrastructural evidence for a physiological control of synaptic activity via exocytosis of glutamate from astrocytes.

The mental retardation protein PAK3 contributes to synapse formation and plasticity in hippocampus.

Mutations of the gene coding for PAK3 (p21-activated kinase 3) are associated with X-linked, nonsyndromic forms of mental retardation (MRX) in which the only distinctive clinical feature is the cognitive deficit. The mechanisms through which PAK3 mutation produces the mental handicap remain unclear, although an involvement in the mechanisms that regulate the formation or plasticity of synaptic networks has been proposed. Here we show, using a transient transfection approach, that antisense and small interfering RNA-mediated suppression of PAK3 or expression of a dominant-negative PAK3 carrying the human MRX30 mutation in rat hippocampal organotypic slice cultures results in the formation of abnormally elongated dendritic spines and filopodia-like protrusions and a decrease in mature spine synapses. Ultrastructural analysis of the changes induced by expression of PAK3 carrying the MRX30 mutation reveals that many elongated spines fail to express postsynaptic densities or contact presynaptic terminals. These defects are associated with a reduced spontaneous activity, altered expression of AMPA-type glutamate receptors, and defective long-term potentiation. Together, these data identify PAK3 as a key regulator of synapse formation and plasticity in the hippocampus and support interpretations that these defects might contribute to the cognitive deficits underlying this form of mental retardation.

Calcium/calmodulin-dependent protein kinase II contributes to activity-dependent filopodia growth and spine formation.

Remodeling of synaptic networks through an activity-dependent formation or elimination of synaptic connections is believed to contribute to information processing and long-term memory. Recent work showed that enhanced synaptic activation, including induction of long-term potentiation and sensory stimulation, promote a rapid growth of dendritic filopodia and the formation of new spines or new types of synapses. Here, we investigated whether calcium/calmodulin-dependent protein kinase II (CaMKII), an enzyme implicated in the control of synaptic efficacy, also participated in these mechanisms. We show that the intracellular application of autophosphorylated CaMKII reproduced these morphological changes and triggered filopodia growth and spine formation. In addition, we find that activation of endogenous kinase through the inhibition of phosphatases or the application of calmodulin in the cell produced similar effects. Conversely, blockade of CaMKII activity prevented the synaptic enhancement, the growth of filopodia and formation of new spines triggered by LTP induction, and a short anoxia/hypoglycemia. Together, these results support the interpretation that CaMKII contributes to the control of activity-dependent structural plasticity.

Presynaptic remodeling contributes to activity-dependent synaptogenesis.

Induction of long-term potentiation and application of short periods of anoxia/hypoglycemia result in the growth of dendritic filopodia and formation of new spines. Here we investigated whether these conditions also affected the morphology of presynaptic structures. Using confocal imaging of DiI-labeled axons, electron microscopy, and stereological analyses, we show that short anoxia/hypoglycemia and theta burst stimulation induced rapid, calcium-dependent growth of presynaptic filopodia-like protrusions and remodeling of presynaptic varicosities. Three-dimensional reconstruction of axonal outgrowths revealed that, within 30 min, they made contacts and triggered the formation of a postsynaptic density on the target cell. Interestingly, these axonal filopodia first established synapses with the dendritic shaft and later mostly with spines. They also contributed to the formation of multi-innervated spines. Because these presynaptic growth mechanisms depended on NMDA receptor activation, we investigated whether a diffusing messenger could be involved. We found that blockade of nitric oxide synthase prevented these changes, and conversely, a nitric oxide donor could reproduce them. A model is presented that proposes that activation of NMDA receptors and subsequent release of nitric oxide could trigger the growth of presynaptic filopodia, which, in turn, play an active role in synaptogenesis and spine formation.

LTP, memory and structural plasticity.

Our current understanding of the mechanisms of information processing and storage in the brain, based on the concept proposed more than fifty years ago by D. Hebb, is that a key role is played by changes in synaptic efficacy induced by coincident pre- and postsynaptic activity. Decades of studies of the properties of long-term potentiation (LTP) have shown that this form of plasticity adequately fulfills these requirements and is likely to contribute to several models of learning and memory. Recent analyses of the molecular events implicated in LTP are consistent with the view that modifications of receptor properties or insertion of new receptors account for the potentiation of synaptic transmission. These experiments, however, have also uncovered an unexpected structural plasticity of synapses. Dendritic spines appear to be dynamic structures that can be formed, modified in their shape or eliminated under the influence of activity. Furthermore, recent studies suggest that LTP, in addition to changes in synaptic function, is also associated with mechanisms of synaptogenesis. We review here the evidence pointing to this activity-dependent remodeling and discuss the possible role of this structural plasticity for synaptic potentiation, learning and memory.

Activity-induced changes of spine morphology.

Spine morphology has been shown in recent years to exhibit a high degree of plasticity. In developing tissue such as organotypic slice cultures, shape changes in spines as well as reorganization of the postsynaptic density (PSD) occur within minutes. Furthermore, several studies have shown that these and other changes can be induced by or are dependent on synaptic activation. Formation of filopodia, enlargement of spines, formation of spines with perforated PSDs, appearance of new spines, and formation of specific types of synapses such as multiple synapse boutons (MSBs), in which two spines contact the same terminal, have all been reported to be induced in an activity-dependent manner. The common denominator of most of these different processes is that they are calcium and NMDA receptor dependent. Their time course, however, may vary. Some appear quite rapidly after stimulation (e.g., filopodia, perforated synapses), while others are clearly more delayed (e.g., formation of spines, appearance of MSBs). How these different structural changes relate to each other, as well as their functional significance, have therefore become intriguing issues. The characteristics of these different types of morphological changes are reviewed, with a discussion of the possibility that structural plasticity contributes to changes in synaptic efficacy.

Remodeling of hippocampal synaptic networks by a brief anoxia-hypoglycemia.

Cerebral ischemia is a major cause of brain dysfunction. Using a model of delayed death induced by a brief, transient oxygen and glucose deprivation, we studied here how this affected the structural organization of hippocampal synaptic networks. We report that brief anoxic-hypoglycemic episodes rapidly modified the structure of synapses. This was characterized, at the electron microscopic level, by a transient increase in the proportion of perforated synapses, followed after 2 hr by an increase in images of multiple synapse boutons. These changes were considerable because 10-20% of all synapses were affected. This structural remodeling was correlated by three kinds of modifications observed using two-photon confocal microscopy: the growth of filopodia, occurring shortly (5-20 min) after anoxia-hypoglycemia, enlargements of existing spines, and formation of new spines, both seen mainly 20-60 min after the insult. All of these structural changes were calcium and NMDA receptor dependent and thus reproduced, to a larger scale, those associated with synaptic plasticity. Concomitantly and related to the severity of anoxia-hypoglycemia, we could also observe spine loss and images of spine, dendrite, or presynaptic terminal swellings that evolved up to membrane disruption. These changes were also calcium dependent and reduced by NMDA receptor antagonists. Thus, short anoxic-hypoglycemic episodes, through NMDA receptor activation and calcium influx, resulted in a profound structural remodeling of synaptic networks, through growth, formation, and elimination of spines and synapses.