Fast targeted gene transfection and optogenetic modification of single neurons using femtosecond laser irradiation

This work is supported by the UK Engineering Physical Sciences Research Council (EPSRC).A prevailing problem in neuroscience is the fast and targeted delivery of DNA into selected neurons. The development of an appropriate methodology would enable the transfection of multiple genes into the same cell or different genes into different neighboring cells as well as rapid cell selective functionalization of neurons. Here, we show that optimized femtosecond optical transfection fulfills these requirements. We also demonstrate successful optical transfection of channelrhodopsin-2 in single selected neurons. We extend the functionality of this technique for wider uptake by neuroscientists by using fast three-dimensional laser beam steering enabling an image-guided “point-and-transfect” user-friendly transfection of selected cells. A sub-second transfection timescale per cell makes this method more rapid by at least two orders of magnitude when compared to alternative single-cell transfection techniques. This novel technology provides the ability to carry out large-scale cell selective genetic studies on neuronal ensembles and perform rapid genetic programming of neural circuits.Publisher PDFPeer reviewe

An incubatable direct current stimulation system for in vitro studies of Mammalian cells.

The purpose of this study was to provide a simplified alternative technology and format for direct current stimulation of mammalian cells. An incubatable reusable stimulator was developed that effectively delivers a regulated current and does not require constant monitoring

The simulation of the activity dependent neural network growth

It is currently accepted that cortical maps are dynamic constructions that are altered in response to external input. Experience-dependent structural changes in cortical microcurcuts lead to changes of activity, i.e. to changes in information encoded. Specific patterns of external stimulation can lead to creation of new synaptic connections between neurons. The calcium influxes controlled by neuronal activity regulate the processes of neurotrophic factors released by neurons, growth cones movement and synapse differentiation in developing neural systems. We propose a model for description and investigation of the activity dependent development of neural networks. The dynamics of the network parameters (activity, diffusion of axon guidance chemicals, growth cone position) is described by a closed set of differential equations. The model presented here describes the development of neural networks under the assumption of activity dependent axon guidance molecules. Numerical simulation shows that morpholess neurons compromise the development of cortical connectivity.Comment: 10 pages, 2 figure

Molecular analysis of sensory axon branching unraveled a cGMP-dependent signaling cascade

Axonal branching is a key process in the establishment of circuit connectivity within the nervous system. Molecular-genetic studies have shown that a specific form of axonal branching - the bifurcation of sensory neurons at the transition zone between the peripheral and the central nervous System - is regulated by a cyclic guanosine monophosphate (cGMP)-dependent signaling cascade which is composed of C-type natriuretic peptide (CNP), the receptor guanylyl cyclase Npr2, and cGMP-dependent protein kinase Iα (cGKIα). In the absence of any one of these components, neurons in dorsal root ganglia (DRG) and cranial sensory ganglia no longer bifurcate, and instead turn in either an ascending or a descending direction. In contrast, collateral axonal branch formation which represents a second type of axonal branch formation is not affected by inactivation of CNP, Npr2, or cGKI. Whereas axon bifurcation was lost in mouse mutants deficient for components of CNP-induced cGMP formation; the absence of the cGMP-degrading enzyme phosphodiesterase 2A had no effect on axon bifurcation. Adult mice that lack sensory axon bifurcation due to the conditional inactivation of Npr2-mediated cGMP signaling in DRG neurons demonstrated an altered shape of sensory axon terminal fields in the spinal cord, indicating that elaborate compensatory mechanisms reorganize neuronal circuits in the absence of bifurcation. On a functional level, these mice showed impaired heat sensation and nociception induced by chemical irritants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are normal. These data point to a critical role of axon bifurcation for the processing of acute pain perception

Induction of Filopodia by Direct Local Elevation of Intracellular Calcium Ion Concentration

In neuronal growth cones, cycles of filopodial protrusion and retraction are important in growth cone translocation and steering. Alteration in intracellular calcium ion concentration has been shown by several indirect methods to be critically involved in the regulation of filopodial activity. Here, we investigate whether direct elevation of [Ca2+]i, which is restricted in time and space and is isolated from earlier steps in intracellular signaling pathways, can initiate filopodial protrusion. We raised [Ca2+]i level transiently in small areas of nascent axons near growth cones in situ by localized photolysis of caged Ca2+ compounds. After photolysis, [Ca2+]i increased from ∼60 nM to ∼1 μM within the illuminated zone, and then returned to resting level in ∼10–15 s. New filopodia arose in this area within 1–5 min, and persisted for ∼15 min. Elevation of calcium concentration within a single filopodium induced new branch filopodia. In neurons coinjected with rhodamine-phalloidin, F-actin was observed in dynamic cortical patches along nascent axons; after photolysis, new filopodia often emerged from these patches. These results indicate that local transient [Ca2+]i elevation is sufficient to induce new filopodia from nascent axons or from existing filopodia

The malleable brain: plasticity of neural circuits and behavior: A review from students to students

One of the most intriguing features of the brain is its ability to be malleable, allowing it to adapt continually to changes in the environment. Specific neuronal activity patterns drive long-lasting increases or decreases in the strength of synaptic connections, referred to as long-term potentiation (LTP) and long-term depression (LTD) respectively. Such phenomena have been described in a variety of model organisms, which are used to study molecular, structural, and functional aspects of synaptic plasticity. This review originated from the first International Society for Neurochemistry (ISN) and Journal of Neurochemistry (JNC) Flagship School held in Alpbach, Austria (Sep 2016), and will use its curriculum and discussions as a framework to review some of the current knowledge in the field of synaptic plasticity. First, we describe the role of plasticity during development and the persistent changes of neural circuitry occurring when sensory input is altered during critical developmental stages. We then outline the signaling cascades resulting in the synthesis of new plasticity-related proteins, which ultimately enable sustained changes in synaptic strength. Going beyond the traditional understanding of synaptic plasticity conceptualized by LTP and LTD, we discuss system-wide modifications and recently unveiled homeostatic mechanisms, such as synaptic scaling. Finally, we describe the neural circuits and synaptic plasticity mechanisms driving associative memory and motor learning. Evidence summarized in this review provides a current view of synaptic plasticity in its various forms, offers new insights into the underlying mechanisms and behavioral relevance, and provides directions for future research in the field of synaptic plasticity.Fil: Schaefer, Natascha. University of Wuerzburg; AlemaniaFil: Rotermund, Carola. University of Tuebingen; AlemaniaFil: Blumrich, Eva Maria. Universitat Bremen; AlemaniaFil: Lourenco, Mychael V.. Universidade Federal do Rio de Janeiro; BrasilFil: Joshi, Pooja. Robert Debre Hospital; FranciaFil: Hegemann, Regina U.. University of Otago; Nueva ZelandaFil: Jamwal, Sumit. ISF College of Pharmacy; IndiaFil: Ali, Nilufar. Augusta University; Estados UnidosFil: García Romero, Ezra Michelet. Universidad Veracruzana; MéxicoFil: Sharma, Sorabh. Birla Institute of Technology and Science; IndiaFil: Ghosh, Shampa. Indian Council of Medical Research; IndiaFil: Sinha, Jitendra K.. Indian Council of Medical Research; IndiaFil: Loke, Hannah. Hudson Institute of Medical Research; AustraliaFil: Jain, Vishal. Defence Institute of Physiology and Allied Sciences; IndiaFil: Lepeta, Katarzyna. Polish Academy of Sciences; ArgentinaFil: Salamian, Ahmad. Polish Academy of Sciences; ArgentinaFil: Sharma, Mahima. Polish Academy of Sciences; ArgentinaFil: Golpich, Mojtaba. University Kebangsaan Malaysia Medical Centre; MalasiaFil: Nawrotek, Katarzyna. University Of Lodz; ArgentinaFil: Paid, Ramesh K.. Indian Institute of Chemical Biology; IndiaFil: Shahidzadeh, Sheila M.. Syracuse University; Estados UnidosFil: Piermartiri, Tetsade. Universidade Federal de Santa Catarina; BrasilFil: Amini, Elham. University Kebangsaan Malaysia Medical Centre; MalasiaFil: Pastor, Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia ; ArgentinaFil: Wilson, Yvette. University of Melbourne; AustraliaFil: Adeniyi, Philip A.. Afe Babalola University; NigeriaFil: Datusalia, Ashok K.. National Brain Research Centre; IndiaFil: Vafadari, Benham. Polish Academy of Sciences; ArgentinaFil: Saini, Vedangana. University of Nebraska; Estados UnidosFil: Suárez Pozos, Edna. Instituto Politécnico Nacional; MéxicoFil: Kushwah, Neetu. Defence Institute of Physiology and Allied Sciences; IndiaFil: Fontanet, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia ; ArgentinaFil: Turner, Anthony J.. University of Leeds; Reino Unid

Two-way communication with neural networks in vivo using focused light

Neuronal networks process information in a distributed, spatially heterogeneous manner that transcends the layout of electrodes. In contrast, directed and steerable light offers the potential to engage specific cells on demand. We present a unified framework for adapting microscopes to use light for simultaneous in vivo stimulation and recording of cells at fine spatiotemporal resolutions. We use straightforward optics to lock onto networks in vivo, to steer light to activate circuit elements and to simultaneously record from other cells. We then actualize this 'free' augmentation on both an 'open' two-photon microscope and a leading commercial one. By following this protocol, setup of the system takes a few days, and the result is a noninvasive interface to brain dynamics based on directed light, at a network resolution that was not previously possible and which will further improve with the rapid advance in development of optical reporters and effectors. This protocol is for physiologists who are competent with computers and wish to extend hardware and software to interface more fluidly with neuronal networks.National Institutes of Health (U.S.) (Postdoctoral Fellowship)Simons Foundation (Postdoctoral Fellowship)National Institutes of Health (U.S.) (Predoctoral Fellowship)National Institutes of Health (U.S.)Simons Foundatio

A micro-LED array based platform for spatio-temporal optogenetic control of various cardiac models

Optogenetics relies on dynamic spatial and temporal control of light to address emerging fundamental and therapeutic questions in cardiac research. In this work, a compact micro-LED array, consisting of 16 × 16 pixels, is incorporated in a widefield fluorescence microscope for controlled light stimulation. We describe the optical design of the system that allows the micro-LED array to fully cover the field of view regardless of the imaging objective used. Various multicellular cardiac models are used in the experiments such as channelrhodopsin-2 expressing aggregates of cardiomyocytes, termed cardiac bodies, and bioartificial cardiac tissues derived from human induced pluripotent stem cells. The pacing efficiencies of the cardiac bodies and bioartificial cardiac tissues were characterized as a function of illumination time, number of switched-on pixels and frequency of stimulation. To demonstrate dynamic stimulation, steering of calcium waves in HL-1 cell monolayer expressing channelrhodopsin-2 was performed by applying different configurations of patterned light. This work shows that micro-LED arrays are powerful light sources for optogenetic control of contraction and calcium waves in cardiac monolayers, multicellular bodies as well as three-dimensional artificial cardiac tissues