Cell signaling experiments driven by optical manipulation.

Cell signaling involves complex transduction mechanisms in which information released by nearby cells or extracellular cues are transmitted to the cell, regulating fundamental cellular activities. Understanding such mechanisms requires cell stimulation with precise control of low numbers of active molecules at high spatial and temporal resolution under physiological conditions. Optical manipulation techniques, such as optical tweezing, mechanical stress probing or nano-ablation, allow handling of probes and sub-cellular elements with nanometric and millisecond resolution. PicoNewton forces, such as those involved in cell motility or intracellular activity, can be measured with femtoNewton sensitivity while controlling the biochemical environment. Recent technical achievements in optical manipulation have new potentials, such as exploring the actions of individual molecules within living cells. Here, we review the progress in optical manipulation techniques for single-cell experiments, with a focus on force probing, cell mechanical stimulation and the local delivery of active molecules using optically manipulated micro-vectors and laser dissection.

Calcium control of gene regulation in rat hippocampal neuronal cultures.

Blockage of GABA-A receptors in hippocampal neuronal cultures triggers synchronous bursts of spikes initiating neuronal plasticity, partly mediated by changes of gene expression. By using specific pharmacological blockers, we have investigated which sources of Ca2+ entry primarily control changes of gene expression induced by 20 microM gabazine applied for 30 min (GabT). Intracellular Ca2+ transients were monitored with Ca2+ imaging while recording electrical activity with patch clamp microelectrodes. Concomitant transcription profiles were obtained using Affymetrix oligonucleotide microarrays and confirmed with quantitative RT-PCR. Blockage of NMDA receptors with 2-amino-5-phosphonovaleric acid (APV) did not reduce significantly somatic Ca2+ transients, which, on the contrary, were reduced by selective blockage of L, N, and P/Q types voltage gated calcium channels (VGCCs). Therefore, we investigated changes of gene expression in the presence of blockers of NMDA receptors and L, N, and P/Q VGCCs. Our results show that: (i) among genes upregulated by GabT, there are genes selectively dependent on NMDA activation, genes selectively dependent on L-type VGCCs and genes dependent on the activation of both channels; (ii) the majority of genes requires the concomitant activation of NMDA receptors and Ca2+ entry through VGCCs; (iii) blockage of N and P/Q VGCCs has an effect similar but not identical to blockage of L-type VGCCs.

Electroolfactogram responses from organotypic cultures of the olfactory epithelium from postnatal mice.

Organotypic cultures of the mouse olfactory epithelium connected to the olfactory bulb were obtained with the roller tube technique from postnatal mice aged between 13 and 66 days. To test the functionality of the cultures, we measured electroolfactograms (EOGs) at different days in vitro (DIV), up to 7 DIV, and we compared them with EOGs from identical acute preparations (0 DIV). Average amplitudes of EOG responses to 2 mixtures of various odorants at concentrations of 1 mM or 100 microM decreased in cultures between 2 and 5 DIV compared with 0 DIV. The percentage of responsive cultures was 57%. We also used the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) to trigger the olfactory transduction cascade bypassing odorant receptor activation. Average amplitudes of EOG responses to 500 microM IBMX were not significantly different in cultures up to 6 DIV or 0 DIV, and the average percentage of responsive cultures between 2 and 5 DIV was 72%. The dose-response curve to IBMX measured in cultures up to 7 DIV was similar to that at 0 DIV. Moreover, the percentage of EOG response to IBMX blocked by niflumic acid, a blocker of Ca-activated Cl channels, was not significantly different in cultured or acute preparations.

Improved neuron culture using scaffolds made of three-dimensional PDMS micro-lattices.

Tissue engineering strives to create functional components of organs with different cell types in vitro. One of the challenges is to fabricate scaffolds for three-dimensional (3D) cell culture under physiological conditions. Of particular interest is the investigation of the morphology and function of the central nervous system cultured using such scaffolds. Here, we used an elastomer-polydimethylsiloxane (PDMS)-to produce lattice-type scaffolds from a photolithography-defined template. The photomask with antidot arrays was spin-coated by a thick layer of resist, and was downward mounted on a rotating stage at an angle of 45°. After the exposure was repeated three or more times, maintaining the same exposure plan but rotated by the same angle, a photoresist was developed to produce a 3D porous template. Afterwards, a pre-polymer mixture of PDMS was poured in and cured, followed by a resist etch, resulting in lattice-type PDMS features. Before cell culture, the PDMS lattices were surface functionalized. A culture test was conducted using NIH-3T3 cells and primary hippocampal cells from rats, showing homogenous cell infiltration and 3D attachment. As expected, a much higher cell number was found in the 3D PDMS lattices compared to the 2D culture. We also found a higher neuron-to-astrocyte ratio and a higher degree of cell ramification in the 3D culture compared to the 2D culture due to the change of scaffold topography and the elastic properties of the PDMS micro-lattices. Our results demonstrate that the 3D PDMS micro-lattices improve the survival and growth of cells, as well as the network formation of neurons. We believe that such an enabling technology is useful for research and clinical applications, including disease modeling, regenerative medicine, and drug discovery/drug cytotoxicity studies.

Highly variable spike trains underlie reproducible sensorimotor responses in the medicinal leech.

The nervous system of the leech is a particularly suitable model to investigate neural coding of sensorimotor responses because it allows both observation of behavior and the simultaneous measurement of a large fraction of its underlying neuronal activity. In this study, we used a combination of multielectrode recordings, videomicroscopy, and computation of the optical flow to investigate the reproducibility of the motor response caused by local mechanical stimulation of the leech skin. We analyzed variability at different levels of processing: mechanosensory neurons, motoneurons, muscle activation, and behavior. Spike trains in mechanosensory neurons were very reproducible, unlike those in motoneurons. The motor response, however, was reproducible because of two distinct biophysical mechanisms. First, leech muscles contract slowly and therefore are poorly sensitive to the jitter of motoneuron spikes. Second, the motor response results from the coactivation of a population of motoneurons firing in a statistically independent way, which reduces the variability of the population firing. These data show that reproducible spike trains are not required to sustain reproducible behaviors and illustrate how the nervous system can cope with unreliable components to produce reliable action.

Less than 5 Netrin-1 molecules initiate attraction but 200 Sema3A molecules are necessary for repulsion.

Guidance molecules, such as Sema3A or Netrin-1, induce growth cone (GC) repulsion or attraction. In order to determine the speed of action and efficiency of these guidance cues we developed an experimental procedure to deliver controlled amounts of these molecules. Lipid vesicles encapsulating 10-10(4) molecules of Sema3A or Netrin-1 were manipulated with high spatial and temporal resolution by optical tweezers and their photolysis triggered by laser pulses. Guidance molecules released from the vesicles diffused and reached the GC membrane in a few seconds. Following their arrival, GCs retracted or grew in 20-120 s. By determining the number of guidance molecules trapped inside vesicles and estimating the fraction of guidance molecules reaching the GC, we show that the arrival of less than 5 Netrin-1 molecules on the GC membrane is sufficient to induce growth. In contrast, the arrival of about 200 Sema3A molecules is necessary to induce filopodia repulsion.

Optical delivery of liposome encapsulated chemical stimuli to neuronal cells.

Spatially confined and precise time delivery of neuroactive molecules is an important issue in neurophysiology. In this work we developed a technique for delivering chemical stimuli to cultured neurons consisting in encapsulating the molecules of interest in liposomes. These vectors were then loaded in reservoirs consisting of glass capillaries. The reservoirs were placed in the recording chamber and single liposomes were trapped and transported out by optical tweezers to the site of stimulation on cultured neurons. Finally, the release of liposome content was induced by application of UV-pulses, breaking the liposome membrane. The efficiency of encapsulation and release were first evaluated by loading the liposomes with fluorescein. In order to test the effect of the UV-induced release, liposomes with diameter ranging from 1 to 10 µm (fL to pL volumes), were filled with KCl and tested on neuronal cells. Neuronal cultures, loaded with Ca(2+) dye, were monitored by imaging intracellular Ca(2+). An efficient release from the liposomes was demonstrated by detectable calcium signals, indicating stimulated depolarization of the neuronal cells by KCl. The present technique represents an alternative method for focal chemical stimulation of cultured cells that circumvents some of the limitations of microejection and photorelease of caged compounds.

Use of optical tweezers technology for long-term, focal stimulation of specific subcellular neuronal compartments.

Spatial regulation of secretory molecule release is a sophisticated mechanism used by the nervous system to control network development and finely tune the activity of each synapse. Great efforts have been made to develop techniques that mimic secretory molecule release with the aim of stimulating neurons as close as possible to physiological conditions. However, current techniques have poor spatial resolution or low flexibility. Here, we propose a novel approach to achieve focal and prolonged stimulation of neurons using optical tweezers and single microbeads functionalized with a secretory molecule, the neurotrophin brain-derived neurotrophic factor (BDNF). BDNF is a key regulator of neuronal development and plasticity. We show that single BDNF-coated microbeads can be extracted with optical tweezers from small reservoirs and positioned with submicrometric precision to specific sites on the dendrites of cultured hippocampal neurons. Localized contact of microbeads functionalized with BDNF, but not with bovine serum albumin (BSA), induced focal increase of calcium signaling in the stimulated dendrite, specific activation of the TrkB receptor pathway and influenced the development of growth cones. Remarkably, a single BDNF-coated bead localized on a dendrite was found to be enough for TrkB phosphorylation, an efficient and long-lasting activation of calcium signaling in the soma, and c-Fos signaling in the nucleus, comparable to bath stimulation conditions. These findings support the use of optical tweezer technology for long-term, localized stimulation of specific subcellular neuronal compartments.

Elevation of somatic Ca2+ upregulates genes Nr4a1 and Egr2, but not Bdnf and Arc.

Blockage of Ca2+ uptake with thapsigargin, a specific antagonist of sarco/endoplasmic reticulum Ca2+-ATPase pumps, causes an increase of somatic Ca2+, with negligible changes of Ca2+ levels in dendrites. Treatment with thapsigargin in the presence of blockers of NMDA (N-methyl-D-aspartic acid) receptors upregulates some activity-dependent genes (Egr2 and Nr4a1), leaving unaltered the expression level of other activity-dependent genes (Bdnf and Arc). These results show that the elevation of somatic Ca2+ can initiate transcription of specific genes, independently of activation of NMDA receptors, but that transcription of other genes is not initiated by a simple elevation of intracellular Ca2+.

Embryonic stem cell-derived neurons form functional networks in vitro.

Embryonic stem (ES) cells provide a flexible and unlimited source for a variety of neuronal types. Because mature neurons establish neuronal networks very easily, we tested whether ES-derived neurons are capable of generating functional networks and whether these networks, generated in vitro, are capable of processing information. Single-cell electrophysiology with pharmacological antagonists demonstrated the presence of both excitatory and inhibitory synaptic connections. Extracellular recording with planar multielectrode arrays showed that spontaneous bursts of electrical activity are present in ES-derived networks with properties remarkably similar to those of hippocampal neurons. When stimulated with extracellular electrodes, ES-derived neurons fired action potentials, and the evoked electrical activity spread throughout the culture. A statistical analysis indicated that ES-derived networks discriminated between stimuli of different intensity at a single trial level, a key feature for an efficient information processing. Thus, ES-derived neurons provide a novel in vitro strategy to create functional networks with defined computational properties.

Electrophysiological properties and modeling of murine vomeronasal sensory neurons in acute slice preparations.

The vomeronasal system is involved in the detection of pheromones in many mammals. Vomeronasal sensory neurons encode the behaviorally relevant information into action potentials that are directly transmitted to the accessory olfactory bulb. We developed a model of the electrical activity of mouse basal vomeronasal sensory neurons, which mimics both the voltage-gated current properties and the firing behavior of these neurons in their near-native state, using a minimal number of parameters. Data were obtained by recordings with the whole-cell voltage-clamp or current-clamp techniques from mouse basal vomeronasal sensory neurons in acute slice preparations. The resting potential ranged from -50 to -70 mV, and current injections of less than 2-10 pA induced tonic firing in most neurons. The experimentally determined firing frequency as a function of injected current was well described by a Michaelis-Menten equation and was exactly reproduced by the model, which could be used in combination with future models that will include details of the mouse vomeronasal transduction cascade.

Dendritic sodium spikelets and low-threshold calcium spikes in turtle olfactory bulb granule cells.

Active dendritic membrane properties were investigated by whole cell recordings from adult turtle olfactory bulb granule cells. The laminar structure of the olfactory bulb allowed differential polarization of the distal apical dendrites versus the somatic part of the cells by an external electric field. Dendritic depolarization evoked small (approximately 10 mV) all-or-none depolarizing events of approximately 10-ms duration. These spikelets often occurred in bursts at high frequency (< or = 250 Hz); they were present despite the application of synaptic and gap junction antagonists, but were abolished by TTX and intracellularly applied QX314. The spikelets were interpreted as attenuated sodium spikes initiated in different branches of the granule cells dendrites. They occurred spontaneously, but could also be evoked by excitatory postsynaptic potentials (EPSPs) to the distal dendrites. Spikelets initiated by distal excitation could function as prepotentials for full sodium spikes, in part depending on the level of proximal depolarization. Somatic depolarization by the electric field evoked full sodium spikes as well as low-threshold calcium spikes (LTSs). Calcium imaging revealed that the electrophysiologically identified LTS evoked from the soma was associated with calcium transients in the proximal and the distal dendrites. Our data suggest that the LTS in the soma/proximal dendrites plays a major role in boosting excitability, thus contributing to the initiation of sodium spiking in this compartment. The results furthermore suggest that the LTS and the sodium spikes may act independently or cooperatively to regulate dendritic calcium influx.

Regulation of granule cell excitability by a low-threshold calcium spike in turtle olfactory bulb.

Granule cells excitability in the turtle olfactory bulb was analyzed using whole cell recordings in current- and voltage-clamp mode. Low-threshold spikes (LTSs) were evoked at potentials that are subthreshold for Na spikes in normal medium. The LTSs were evoked from rest, but hyperpolarization of the cell usually increased their amplitude so that they more easily boosted Na spike initiation. The LTS persisted in the presence of TTX but was antagonized by blockers of T-type calcium channels. The voltage dependence, kinetics, and inactivation properties of the LTS were characteristic of a low-threshold calcium spike. The threshold of the LTS was slightly above the resting potential but well below the Na spike threshold, and the LTS was often evoked in isolation in normal medium. Tetraethylammonium (TEA) and 4-aminopyridine (4-AP) had only minimal effects on the LTS but revealed the presence of a high-threshold Ca2+ spike (HTS), which was antagonized by Cd2+. The LTS displayed paired-pulse attenuation, with a timescale for recovery from inactivation of about 2 s at resting membrane potential. The LTS strongly boosted Na spike initiation; with repetitive stimulation, the long recovery of the LTS governed Na spike initiation. Thus the olfactory granule cells possess an LTS, with intrinsic kinetics that contribute to sub- and suprathreshold responses on a timescale of seconds. This adds a new mechanism to the early processing of olfactory input.

Dynamics and reproducibility of a moderately complex sensory-motor response in the medicinal leech.

Local bending, a motor response caused by mechanical stimulation of the leech skin, has been shown to be remarkably reproducible, in its initial phase, despite the highly variable firing of motoneurons sustaining it. In this work, the reproducibility of local bending was further analyzed by monitoring it over a longer period of time and by using more intact preparations, in which muscle activation in an entire body segment was studied. Our experiments showed that local bending is a moderately complex motor response, composed of a sequence of four different phases, which were consistently identified in all leeches. During each phase, longitudinal and circular muscles in specific areas of the body segment acted synergistically, being co-activated or co-inhibited depending on their position relative to the stimulation site. Onset and duration of the first phase were reproducible across different trials and different animals as a result of the massive co-activation of excitatory motoneurons sustaining it. The other phases were produced by the inhibition of excitatory and activation of inhibitory motoneurons, and also by the intrinsic relaxation dynamics of leech muscles. As a consequence, their duration and relative timing was variable across different preparations, whereas their order of appearance was conserved. These results suggest that, during local bending, the leech neuromuscular system 1) operates a reduction of its available degrees of freedom, by simultaneously recruiting groups of otherwise antagonistic muscles and large populations of motoneurons; and 2) ensures reliability and effectiveness of this escape reflex, by guaranteeing the reproducibility of its crucial initial phase.