1.

Introduction

Neuronal networks in vitro represent an intermediate model situated between single neurons and brain tissue for studying neural development, coding, pharmacology, pathology, and regeneration. Understanding network dynamics requires techniques capable of monitoring the activity of an elevated number of neurons at high temporal resolution. Over the last decade, two core approaches have been favored. One of them is based on optics, as a low invasive tool for imaging the activity of many cells with subcellular resolution over a field of view of a few hundred micrometers.¹ The other is the electrical extracellular recording of network activity by means of microelectrode array (MEA) devices,² which allow the multisite monitoring of several neurons with submillisecond resolution around a limited number of electrodes distributed over a few square millimeters. Several laboratories started to combine these two methods to compensate for their complementary shortcomings in resolving and extracting neural network properties.³

The properties of the culture substrate represent an important aspect of in vitro systems. The viscoelastic behavior of a neurite is assumed to result from the interaction of cytoskeletal components with the extracellular matrix (ECM).⁴ Indeed, not only chemical but also mechanical properties of the ECM can regulate cellular properties such as shape, polarity, motility, and morphological phenotypes. Alteration in the interactions between a cell and its surrounding ECM may result in apoptosis, malignant transformation, or loss of tissue architecture.⁵ The ability of a laser dissection system to selectively ablate subcellular compartments without damaging adjacent structures offers new possibilities for studying cell-ECM interactions^{6, 7} or axonal regeneration.^{8, 9}

In this work, we present the design and evaluate the performance of a system that combines two optical manipulation techniques, holographic optical tweezers (OTs), and a laser micro-dissector (LMD), to directly quantify the mechanical perturbation produced by laser ablation in a neurite, and to study in vitro regeneration of cellular processes after well-calibrated, laser-inflicted damage. Moreover, the system is configured to work in combination with fluorescence imaging and MEA electrophysiology to track and dissect neuronal interconnections for elucidating their local and network-wide role. For this purpose, we used low-density neural cultures to evaluate the architecture of the neural network, and to achieve higher spatial control of the neuronal connections we dissect and the structural changes we impose on the network itself.

The presented system will be applied to the study of processes involved in the early stages of neuronal differentiation, with particular focus on neurite regeneration and neural network dynamics.

2.

Material and Methods

2.1.

Optical Tweezers Setup

As depicted in Fig. 1, the OT setup (red and brown box) is based on an ytterbium continuous wave fiber laser operating at 1064 nm (IPG Laser GmbH). The laser beam is expanded with a telescope, composed of a 35 mm and 100 mm focal length doublet (L1 and L2, IR doublets – Thorlabs), and projected onto a spatial light modulator (SLM) (LCOS-SLM, model X10468–07, Hamamatsu: 800×600 pixel resolution, pixel dimension: 20×20 μm,² phase modulation: 0 to 4π, wavelength range: 680 to 1100 nm, 60 Hz refresh rate), before entering into a modified upright microscope (BX51, Olympus; green box). The computer-generated diffraction pattern at the SLM modulates the laser beam wavefront.

Fig. 1

Schematic optical layout of the instrument. Color box legend: Upright optical tweezers (red), a laser microdissector (violet), and a back-focal plane interferometer (brown) are operated on an Olympus BX51 microscope (green). AOM: acousto-optic modulator; BS_50/50: beam splitter 50/50%; CCD: charged coupled device camera; DM: dichroic mirror; F: filter; λ/2: half-wave plate; L: lens; MEA: microelectrode array amplifier; ND: neutral density filter; PBS: polarizing beam splitter; PD: photodiode; QPD: quadrant photodiode.

The SLM plane and the back focal plane of the microscope objectives are optically conjugated by a second telescope, made of two lenses in 4-f configuration both with focal lengths of 200 mm, to cover the distance between the SLM plane and the pupil plane (L3 and L4; IR doublets, Thorlabs). The structured laser light enters the microscope through an auxiliary port (adapted U-DP Olympus microscope component), which was originally designed for the lateral attachment of a CCD camera. The beam is reflected perpendicularly onto the optical axis of the microscope by a low pass dichroic mirror (DM2; 765dcspxr, Chroma). It then passes through a second dichroic mirror used for fluorescence imaging (DM3 selectable dichroic mirror: FF510-DI01, FF593-Di02, FF395/495/610-Di01, Semrock) before reaching the objective. DM3 is part of a standard filter cube situated in the filter cube wheel of the microscope. Emission filters have been relocated to external multiposition filter carriers (F3 and F4) in front of the two CCD cameras (CCD1 and CCD2), and excitation filters have been removed. In combination with the microscope tube lens (L10) and with an additional low pass emission filter (F2; ET750SP-2p8, Chroma) above DM2, this layout ensures that only the visible portion of the light returning from the sample can reach the CCD cameras. Stray, residual, or back-reflected infrared (IR) laser light is rejected. A dichroic mirror (DM7) in front of CCD1 and CCD2 separates and distributes the light coming from the sample onto the two cameras (CCD1: iXon V887ECSUVB EMCCD, Andor: 512×512 pixels, back illuminated sensor; CCD2: BASLER-Pilot, 04-PIA1000-48GM CCD, 1004×1004 pixels, 48 Hz full frame rate). Thus, with an appropriate choice of DM7, F3, and F4 (FF562-DI02, FF01-520/35, Semrock), bright field and fluorescence calcium imaging can be performed simultaneously.

A laser diode (TECBL-30G-488, World Star Tech. Inc.) that can be modulated by TTL pulses is the light source for fluorescence calcium imaging. A halogen lamp serves as the bright field illumination source (KL2500LCD COLD LIGHT 230VKL, Olympus). The latter is equipped with a filter wheel to select a color range which does not interfere with the emission spectra of the fluorochromes used to label the sample (BLP01-561R, Semrock).

Infrared light interference fringes in the back focal plane of the objective condenser are directed by the low pass dichroic broadband mirror DM4 onto a four-quadrant photodiode (QPD, S5980 with C5460SPL 6041 board, Hamamatsu) and a photodiode PD2 (PDA100A-EC, Thorlabs) for force-spectroscopy measurements, while visible light from the halogen lamp can pass through. The objective condenser, together with DM4, L5, L6, BS50/50 (700 to 1100 nm broadband nonpolarizing beam splitter cube, BS014, Thorlabs), F1 (LL01-1064-12.5, Semrock), and PD2 are mounted onto a metal framework (brown box). It can be aligned manually with respect to the microscope stage and to the QPD by three manual translation stages (PT3/M, Thorlabs). The QPD is mounted on a separate x, y, z stage and quantifies the lateral displacement of a probe in the optical trap. PD2 with a pinhole mask in front of it provides a signal proportional to the axial position of the trapped probe. Electrical signals coming from the four quadrants of the QPD and PD2 are amplified 100-fold (2 amplifier boards: MULTIBOARD, Sg-lux) and then digitized independently by an analog-to-digital (A/D) data acquisition converter (PCI-4462, 24 bit, 4 channels, 204.8 k Samples/second, National Instruments).

The stage of the microscope is composed of a 3-axis linear dc motor micro-positioning system (M-126.CG1, Physics-Instruments) carrying a separate 3-axis piezoelectric nanopositioning stage (P-733.3DD, Physics-Instruments) to combine coarse movement of the sample with the sub-nanometer resolution of the piezo stage.

3.

Results

During differentiation, neurons change their shape to explore the extracellular environment and extrude processes to establish connections with other cells. Once the connections are formed, the components of the cytoskeleton rearrange to produce tension on the connection site and stabilize the architecture of the network.¹¹ Here, we demonstrated how the release of tension in a neural connection could be directly quantified through back focal plane interferometric (BFPI) tracking of a membrane-bound bead kept in an optical trap,^{12, 13} while a laser dissector was progressively damaging a neurite and perturbing the mechanical equilibrium of its cytoskeleton. Rat hippocampal neurons (7 DIV) were loaded in vitro with the calcium indicator Fluo-4 AM to monitor calcium entry upon dissection of their processes. During laser ablation of a neurite, simultaneous time-lapse bright field, and fluorescence imaging documented the thinning of the neurite until its complete disruption [(Fig. 2a] and the resulting calcium influx into the cell (see supplementary Videos 1 and 2) as quantified by (F-F₀)/F₀ in the region of interest [(Fig. 2b, red ROI in the last frame].

Video 1

Bright field time lapse imaging during neurite laser dissection. Time lapse bright field imaging of rat hippocampal neuron (7 DIV) to monitor the thinning of the neurite upon dissection until its complete disruption. Numbers indicate seconds. Bar is 10 mm. Frame rate is 7 Hz (see Fig. 2). (QuickTime, 3.7 MB)1

Video 2

Fluorescence calcium time lapse imaging during neurite laser dissection. Time lapse fluorescence imaging of rat hippocampal neuron (7 DIV) loaded with the calcium indicator Fluo-4 AM to monitor calcium entry upon dissection of its process. Numbers indicate seconds. Bar is 12 mm. Frame rate is 5 Hz (see Fig. 2). (QuickTime, 3.7 MB)2

Fibronectin-coated beads were optically trapped and moved into close proximity of the neurite to be dissected.¹⁴ The position of the trapped probe with respect to the neurite and to the focal spot of the UV laser dissection, was controlled by displaying a dynamically generated hologram on the SLM. The trapped probe was positioned 2 μm above the coverslip and about 20 μm away from the UV laser spot. The trap stiffness, K _x,y,z (k _x, k _y, k _z), and the detector sensitivity were calibrated using the power spectrum method, when the bead had no contact with any structure.¹⁵ The UV laser pulses had no influence on the power spectrum of the trapped bead.¹⁶ Adhesion events between the fibronectin-coated bead and the integrins in the plasma membrane of a neurite were monitored by changes in the variance of the Brownian motion of the bead. When the bead was far from the neurite, the total variance ( [TeX:] $\sigma _t ^2 = \sigma _x ^2 + \sigma _y ^2 + \sigma _z ^2$ ${\sigma }_t^2={\sigma }_x^2+{\sigma }_y^2+{\sigma }_z^2$ ) was 568 nm²; it decreased to 265 nm² when the bead adhered to the membrane.¹⁷

BFPI therefore allowed the simultaneous quantification of the released tension in the damaged neurite with nanometer and submillisecond resolution [Fig. 2d]. Upon complete retraction of the neurite, the bead remained in the laser trap. The total variance in its Brownian motion came back to the value measured before it adhered to the cell membrane. This approach neither required any assumptions on the organization of the cytoskeleton components in the neurite nor its modeling as a homogeneous viscoelastic solid. Figure 2c depicts the cumulative energy release by the UV laser during the experiment. The damage to the neurite could be controlled from partial to complete ablation by varying either the number of delivered pulses or the energy per pulse.

During the first 25 s, 2000 pulses with energy of 20 nJ per pulse were delivered. Local calcium influx at the stimulated site and its subsequent spread toward the soma was visible shortly after the first pulses had been delivered at 3.6 s [Fig. 2b]. Administering a high number of low-energy pulses led to partial damage only, even at 70 s [Fig. 2a]. For the period of subsequent five exposures to UV light pulses [Fig. 2c], we monitored the force on the adhered probe. However, if the energy per pulse was increased while the number of pulses was decreased, a complete ablation of the process required only 200 pulses at energies of 65 nJ per pulse [Figs. 2a and 2c after 70 s). Once the UV laser damage inflicted to a neuron was calibrated and quantified, UV surgery could be performed with high spatio-temporal control. Figures 3, 4, 5 show the importance of this approach for neural regeneration and functional network interconnectivity studies.

Fig. 2

Laser dissection of a single neurite and measurements of the released tension. (a) Bright field images of a differentiating rat hippocampal neuron (7 DIV). The violet arrow indicates the dissection site on the neurite (Video 1). Frame rate was 7 Hz. The numbers indicate seconds. Bar: 10 μm. The pulse repetition of the UV laser was 100 Hz. A bead (Ø 4 μm) coated with fibronectin was trapped and positioned on the neurite to adhere to integrins in the plasma membrane. The power of the IR laser at the sample was 40 mW. Objective: 60×, NA 0.9 (Olympus). The trapping stiffness was calibrated by the power spectra method: k _{x ,y} = 80 pN/μm, k _z = 13 pN/μm. (b) Fluo-4 AM fluorescence of the neuron in (a). Average power at the sample of the 488 nm laser diode was 50 μW. Repetition rate of the laser diode was 5 Hz with a duty cycle of 4%. Frame acquisition rate was 5 Hz. Numbers indicate seconds. Bar: 12 μm (Video 2). The red ROI in the last frame indicates the region for quantifying the (F-F₀)/ F₀ fluctuations in color-coded fluorescence intensity (F₀ was the intensity value in the first frame of the video). (c) Time course of the energy delivered by the UV laser to the sample. (d) Displacement of and force on the trapped bead versus time. Green and blue traces are the x, y components; the red trace is the z component. Sampling rate was 10 kHz.

Fig. 3

Laser dissection of single neurons in neural networks. (a) Complete neurite dissection and regeneration of a hippocampal neuron (rat, 4 DIV). The white arrow indicates the dissection site. The numbers indicate minutes. The pulse repetition of the UV laser was 100 Hz, the average power at the sample about 7 μW. The black arrows in the last two frames illustrate the orientation of the exploratory motion of the regenerating growth cone after dissection. Bars: 12 μm (Video 3). (b) Partial dissection and regeneration of a hippocampal neuron (rat, 3 DIV). The white arrow indicates the dissection site. Numbers indicate minutes. The pulse repetition rate of the UV laser was 100 Hz, the average power at the sample 3 μW. As seen in the last frame, the neurite appears completely recovered after 20 min. Bar: 25 m (Video 4).

Fig. 4

Laser dissection of individual connections in a neural network on a MEA. (a) Electrode layout of the MEA chip: 6×10 matrix of electrodes with a diameter of 30 μm and an electrode spacing of 500 μm (= black bar). The strongly correlated activity on electrodes 41 and 27 was used for the analysis shown in Fig. 5. (b) Mosaic of two bright field images of the neural network around one electrode (#27) of such 60-electrodes MEA. White lines indicate the dissected connections and numbers the dissection sequence. (c) Mosaic of five fluorescence images of the network in (b) loaded with Fluo-4 AM. Circles and numbers indicate cells and approximate areas from which calcium fluctuations were quantified.

Fig. 5

Spontaneous activity of a neural network recorded simultaneously by MEA electrodes and calcium fluorescence imaging during subsequent ablation of neuronal connections. The recording of network activity was divided into eight experimental sessions (S1÷S8) each lasting 625 s; four of them are depicted. Color-coded calcium fluctuations from the seven cells shown in Fig. 4b were temporally aligned with extracellularly recorded electrical activity from electrodes 41 and 27. Calcium fluctuations were quantified as (F- F₀)/F₀, with F₀ equal to the minimum intensity measured in each video session, to obtain fluctuation values above zero. Moreover, intensities of individual cells were normalized to their maximum intensity during an experimental session to obtain the same color legend for all experimental sessions. Dissection events during the experimental sessions are marked by vertical red lines; numbers correspond to those in Fig. 4. The inset displays the change in the total number of recorded spikes from electrodes 27 and 41 during each session.

Video 3

Complete neurite dissection and growth cone formation. Complete cut of a neurite by 150 pulses of the UV laser at energy of 67 nJ per pulse. After complete dissection of the neurite, a new growth cone is formed at the tip and starts to navigate to find the path to re-establish a connection. Numbers indicate seconds. Bar is 10 mm. Frame rate 0.3 Hz (see Fig. 3). (QuickTime, 3.1 MB)3

Video 4

Partial neurite dissection and regeneration. Partial damage inflicted to a neurite by 190 pulses of the UV laser at energy of 30 nJ per pulse. The video shows the thinning of the process at the dissection site, without loss of the connection with the distal part of the neurite, and its fast regeneration. Numbers indicate seconds. Bar is 10 mm. Frame rate 0.3 Hz (see Fig. 3). (QuickTime, 3.0 MB)4

Two types of localized surgery on a neural network are shown in Fig. 3. In Fig. 3a, 150 pulses at energy of 67 nJ per pulse were sufficient to completely cut a neurite at a distance of about 30 μm from the soma. In consequence of the cut, the distal part of the neurite degenerated while the proximal part of the neurite formed an exploring growth cone after 7 min. The last two frames show how the growth cone changes orientation to explore its surroundings, and to recreate the connection to the network (see Video 3).

Figure 3b shows the partial dissection of a neurite of a differentiating rat hippocampal neuron at 4 DIV upon delivery of 190 pulses at energy of 30 nJ per pulse. In this case, only a thinning of the process was observed at the dissection site without loss of the connection with the distal part of the neurite. With the cut performed at low energy per pulse, the distal part of the neurite did not degenerate, and the complex motility of the growth cone was preserved during the dissection and thereafter (see Video 4). Six minutes later, healing and fast regeneration of the neurite occurred. After 20 min, the process integrity appeared completely recovered.

These experiments not only demonstrate the high degree of control over the site and type of induced damage, but also the possibility of tracking and documenting the regeneration process in real time. Moreover, these results show that UV laser ablation experiments can be performed during the first days after cell plating, usually a critical differentiation period in in vitro experiments. In most cases, laser dissection experiments in vivo are carried out with lasers having longer wavelengths.⁹ However, for in vitro preparations, a pulsed UV laser has two advantages: it creates a smaller diffraction-limited spot and exposes the cell to a low average power, thus shortening the exposure time and minimizing undesirable thermal effects at the sample.^{8, 18} This gives high spatial control over the surgical process, narrowing the nonlethal ablation distance to the cell body to a few tens of micrometers and avoiding irreversible damage to the cell during differentiation.

The precision of the system opens the door to functional interconnectivity studies. Spontaneous activity of a low-density network of hippocampal neurons (rat, 18 DIV) on a MEA was recorded continuously at 25 kHz while sequentially dissecting individual connections to pinpoint correlated activity changes. Simultaneous calcium imaging of a subpopulation of neurons near the dissection site revealed local activity with single cell resolution. Although neurons plated at low-densities are more difficult to maintain, and less neurons are sufficiently close to the electrode to yield recordable signals, single units and connections in the network can be visualized more easily. Thus, their network architecture is more accessible to modifications. Figure 4a shows a mosaic of two bright field images acquired around 1 (electrode 27) out of 60 electrodes of the MEA chip. Numbered lines indicate the location and succession of cutting connections. Figure 4b displays the same region as a mosaic of five fields of view acquired by fluorescence calcium imaging. Numbered circles indicate the single units and integration areas for plotting fluorescence fluctuations over time (Fig. 5). Considering that the laser dissector spot was positioned on the neurite to be ablated by motion of the stage, the number of cells from which we recorded calcium fluctuations depended on the field of view of the acquired images.

By combining electrophysiological and fluorescence recording techniques, network-wide activity could be monitored electrically by the 60 electrodes while the contribution of individual neurons to the signal recorded from a specific electrode could be estimated from calcium fluctuations (Fig. 4). Dissecting individual connections without irreversibly damaging the neuron allows identifying its contribution to the overall network activity.

Figure 5 shows the changes in the network activity during the lesion of some of its processes [as depicted in Fig. 4b] in eight subsequent experimental sessions (S1÷S8). S1 shows basal calcium fluctuations in cell 1, which was the cell closest to electrode 27. The dissection of processes in its vicinity [white bars in Fig 4b] started after 480 s. Until S3, after the ablation of six different processes, cell 1 presented spontaneous activity. At the seventh cut, cell 1 showed a high calcium influx and became silent. In S5, average activity on electrode 27 decreased to 1/3. In contrast, activity on electrode 41, which was about 2.5 mm away from electrode 27, became less sparse and mainly organized in bursts. The trend is summarized in the inset graph as the total number of recorded spikes from these two electrodes in each experimental session.

In S5, the 11th cut produced a calcium wave in the neighboring cells. After a while (about 200 s), cells recovered their basal calcium levels with the exception of cell 3, which maintained an elevated calcium level until S8. This suggests that the damage inflicted to the neurite in cut 11 belonged to cell 3. Nonetheless, spontaneous activity stayed until synchronized with activity in other cells and those contributing to the spiking on electrodes 27 and 41.

4.

Discussion

The integration of optical and electrophysiological techniques is becoming an emerging method for studying neural circuits in vitro. In the present study, we demonstrated how precisely calibrated laser surgery on neural networks can serve as an experimental model for network computation and regeneration studies. In the first experimental configuration, we illustrated how the energy of the laser dissection system can be fine-tuned by just two parameters, the number of pulses per time and the energy per pulse. In particular, we showed how the modulation of the energy delivered to the sample differently affected the morphological, chemical, and mechanical damage inflicted to a cell. Changes in the morphology of the process were documented by bright field imaging, while calcium imaging gave an estimate of the extracellular solution influx into the cell before the resealing of its membrane.

Furthermore, we could directly quantify the released tension in a neurite upon disruption of the cytoskeletal components by interferometric force spectroscopy without need for any theoretical cell modeling.^{6, 7} Several papers reported on the importance of combining optical manipulation techniques as laser dissectors and optical tweezers.¹⁹ In this work, we present, for the first time to our knowledge, the possibility of performing laser dissection and force spectroscopy simultaneously, to calibrate and quantify the laser ablation process with sub-pico–Newton and millisecond resolution.

An increasing number of works point out the importance of a mechanical equilibrium between the tensile force of the cytoskeleton and the elastic resistance of the extracellular matrix,^{20, 21} and how the cell is exploring the mechanical properties of the substrate during differentiation.¹² The quantification of the released tension during laser surgery, besides giving an estimate of the damage inflicted to a cell,²² can be used to characterize and catalogue type age-specific tensile forces in different neurites and neurons. This knowledge will lead to a better understanding of how the intercellular organization of a network is steered toward mechanical equilibrium,^{23, 24} and how neuro-degenerative pathologies affecting cytoskeleton components may alter this equilibrium. Moreover, it offers the possibility to study how changes in the extracellular matrix²⁵ of cells in culture can modify the cell-substrate interactions.

The use of a pulsed UV-A laser with very short pulse duration (400 ps) permits laser surgery at extremely low average power (few μW) at the sample, thereby confining unwanted thermal effects. We exemplarily demonstrated this by dissecting neural processes close to their cell bodies during the first days of differentiation. In future studies, this feature may be exploited to better characterize neural polarization and regeneration after neurite abalation.²²

In the last experimental configuration, we presented the simultaneous local calcium imaging and electrophysiological MEA recording of network-wide activity.³ We reported on its implementation with a low invasive laser dissection system for getting new insights into network architectures and the functional contributions of individual neurons or their sub-cellular compartments.

Future improvements of the system are underway, such as the development of a multipoint force spectroscopy system which combines the holographic optical tweezers with fast optical imaging.²⁶ This will allow the tracing and quantification of the tension release and its propagation in space and time in order to characterize the mechanical organization of the network, quantify its damage, and study the effect on its activity. Furthermore, the holographic optical tweezers will be used for local cell stimulation with functionalized beads²⁷ to study and stimulate the regeneration process.