Marco Dal Maschio

A hybrid bioorganic interface for neuronal photoactivation

A key issue in the realization of retinal prosthetic devices is reliable transduction of information carried by light into specific patterns of electrical activity in visual information processing networks. Soft organic materials can be used to couple artificial sensors with neuronal tissues. Here, we interface a network of primary neurons with an organic blend. We show that primary neurons can be successfully grown onto the polymer layer without affecting the optoelectronic properties of the active material or the biological functionality of neuronal network. Moreover, action potentials can be triggered in a temporally reliable and spatially selective manner with short pulses of visible light. Our results may lead to new neuronal communication and photo manipulation techniques, thus paving way to the development of artificial retinas and other neuroprosthetic interfaces based on organic photodetectors.

Layer-specific excitatory circuits differentially control recurrent network dynamics in the neocortex

In the absence of external stimuli, the mammalian neocortex shows intrinsic network oscillations. These dynamics are characterized by translaminar assemblies of neurons whose activity synchronizes rhythmically in space and time. How different cortical layers influence the formation of these spontaneous cellular assemblies is poorly understood. We found that excitatory neurons in supragranular and infragranular layers have distinct roles in the regulation of intrinsic low-frequency oscillations in mice in vivo. Optogenetic activation of infragranular neurons generated network activity that resembled spontaneous events, whereas photoinhibition of these same neurons substantially attenuated slow ongoing dynamics. In contrast, light activation and inhibition of supragranular cells had modest effects on spontaneous slow activity. This study represents, to the best of our knowledge, the first causal demonstration that excitatory circuits located in distinct cortical layers differentially control spontaneous low-frequency dynamics.

High-performance and site-directed in utero electroporation by a triple-electrode probe

In utero electroporation is a powerful tool to transfect and manipulate neural-precursor cells of the rodent parietal cortex and their progeny in vivo. Although this technique can potentially target numerous brain areas, reliability of transfection in some brain regions is low or physical access is limited. Here, we present a new in utero-electroporation configuration based on the use of three electrodes, the relative position and polarities of which can be adjusted. The technique allows easy access and exceedingly reliable monolateral or bilateral transfection at brain locations that could only be sporadically targeted before. By improvement in the efficiency of the electrical field distribution, demonstrated here by a mathematical simulation, the multi-electrode configuration also extends the developmental timeframe for reliable in utero electroporation, allowing for the first time specific transfection of Purkinje cells in the rat cerebellum.

Nanostructured superhydrophobic substrates trigger the development of 3D neuronal networks

The generation of 3D networks of primary neurons is a big challenge in neuroscience. Here, a novel method is presented for a 3D neuronal culture on superhydrophobic (SH) substrates. How nano-patterned SH devices stimulate neurons to build 3D networks is investigated. Scanning electron microscopy and confocal imaging show that soon after plating neurites adhere to the nanopatterned pillar sidewalls and they are subsequently pulled between pillars in a suspended position. These neurons display an enhanced survival rate compared to standard cultures and develop mature networks with physiological excitability. These findings underline the importance of using nanostructured SH surfaces for directing 3D neuronal growth, as well as for the design of biomaterials for neuronal regeneration.

Increased spontaneous activity of a network of hippocampal neurons in culture caused by suppression of inhibitory potentials mediated by anti-gad antibodies

Introduction: Anti-glutamic acid decarboxylase autoantibodies (GAD-Ab) are commonly considered the marker of autoimmune diabetes; they were first described in patients affected by stiff-person syndrome and recently, in ataxic or epileptic patients. The pathogenetic role of GAD-Ab remains unclear but inhibition of GABA synthesis or interference with GABA exocytosis are hypothesized. The aim of the study was to assess whether GAD-Ab interfere with neuronal transmission. Patients and methods: Serum from a GAD-Ab positive epileptic patient (by IHC and RIA), serum from a GAD-positive (only by RIA) diabetic case, sera from two epileptic GAD-Ab negative patients and a normal control were selected. Post-synaptic inhibitory potentials (IPSPs) were registered on hippocampal neurons in culture before and after the application of diluted sera in a patch clamp study. Results: A significant increase in the frequency of IPSPs was observed after application of GAD-positive epileptic serum, while no effect was noted using sera from negative controls. Conclusion: The inhibition in neuronal transmission only after application of GAD-positive epileptic serum, suggests an interference with GABA function and consequently with neuronal inhibition supporting a pathogenetic role of GAD-Ab in the development of epilepsy.

Three-dimensional in vivo scanning microscopy with inertia-free focus control

The acquisition of high-resolution images in three dimensions is of utmost importance for the morphological and functional investigation of biological tissues. Here, we present a laser scanning two-photon microscope with remote and motionless control of the focus position. The movement of the excitation spot along the propagation direction is achieved by shaping the laser wavefront with a spatial light modulator. Depending on the optical properties of the objective in use, this approach allows z movements in a range of tens to hundreds of micrometers with small changes of the point spread function. We applied this technique for the three-dimensional (3D) imaging of fluorescent cells in the mouse neocortex in vivo. The presented system bypasses the limitations of microscopes based on moving objectives, enabling high-resolution inertia-free 3D imaging.

Combined optical tweezers and laser dissector for controlled ablation of functional connections in neural networks

Regeneration of functional connectivity within a neural network after different degrees of lesion is of utmost clinical importance. To test pharmacological approaches aimed at recovering from a total or partial damage of neuronal connections within a circuit, it is necessary to develop a precise method for controlled ablation of neuronal processes. We combined a UV laser microdissector to ablate neural processes in vitro at single neuron and neural network level with infrared holographic optical tweezers to carry out force spectroscopy measurements. Simultaneous force spectroscopy, down to the sub-pico-Newton range, was performed during laser dissection to quantify the tension release in a partially ablated neurite. Therefore, we could control and measure the damage inflicted to an individual neuronal process. To characterize the effect of the inflicted injury on network level, changes in activity of neural subpopulations were monitored with subcellular resolution and overall network activity with high temporal resolution by concurrent calcium imaging and microelectrode array recording. Neuronal connections have been sequentially ablated and the correlated changes in network activity traced and mapped. With this unique combination of electrophysiological and optical tools, neural activity can be studied and quantified in response to controlled injury at the subcellular, cellular, and network level.

Simultaneous high-speed imaging and optogenetic inhibition in the intact mouse brain

Genetically encoded calcium indicators and optogenetic actuators can report and manipulate the activity of specific neuronal populations. However, applying imaging and optogenetics simultaneously has been difficult to establish in the mammalian brain, even though combining the techniques would provide a powerful approach to reveal the functional organization of neural circuits. Here, we developed a technique based on patterned two-photon illumination to allow fast scanless imaging of GCaMP6 signals in the intact mouse brain at the same time as single-photon optogenetic inhibition with Archaerhodopsin. Using combined imaging and electrophysiological recording, we demonstrate that single and short bursts of action potentials in pyramidal neurons can be detected in the scanless modality at acquisition frequencies up to 1 kHz. Moreover, we demonstrate that our system strongly reduces the artifacts in the fluorescence detection that are induced by single-photon optogenetic illumination. Finally, we validated our technique investigating the role of parvalbumin-positive (PV) interneurons in the control of spontaneous cortical dynamics. Monitoring the activity of cellular populations on a precise spatiotemporal scale while manipulating neuronal activity with optogenetics provides a powerful tool to causally elucidate the cellular mechanisms underlying circuit function in the intact mammalian brain.

Integrated brain circuits: neuron-astrocyte interaction in sleep-related rhythmogenesis.

Although astrocytes are increasingly recognized as important modulators of neuronal excitability and information transfer at the synapse, whether these cells regulate neuronal network activity has only recently started to be investigated. In this article, we highlight the role of astrocytes in the modulation of circuit function with particular focus on sleep-related rhythmogenesis. We discuss recent data showing that these glial cells regulate slow oscillations, a specific thalamocortical activity that characterizes non-REM sleep, and sleep-associated behaviors. Based on these findings, we predict that our understanding of the genesis and tuning of thalamocortical rhythms will necessarily go through an integrated view of brain circuits in which non-neuronal cells can play important neuromodulatory roles.

Targeted in vivo genetic manipulation of the mouse or rat brain by in utero electroporation with a triple-electrode probe

This protocol is an extension to:Nat. Protoc. 1, 1552–1558 (2006); doi:10.1038/nprot.2006.276; published online 9 November 2006This article describes how to reliably electroporate with DNA plasmids rodent neuronal progenitors of the hippocampus; the motor, prefrontal and visual cortices; and the cerebellum in utero. As a Protocol Extension article, this article describes an adaptation of an existing Protocol and offers additional applications. The earlier protocol describes how to electroporate mouse embryos using two standard forceps-type electrodes. In the present protocol, additional electroporation configurations are possible because of the addition of a third electrode alongside the two standard forceps-type electrodes. By adjusting the position and polarity of the three electrodes, the electric field can be directed with great accuracy to different neurogenic areas. Bilateral transfection of brain hemispheres can be achieved after a single electroporation episode. Approximately 75% of electroporated embryos survive to postnatal ages, and depending on the target area, 50–90% express the electroporated vector. The electroporation procedure takes 1 h 35 min. The protocol is suitable for the preparation of animals for various applications, including histochemistry, behavioral studies, electrophysiology and in vivo imaging.