Tickling neurons with light - a new method to study epilepsy

Introduction

The field of optogenetics deals with activation and deactivation of neurons by means of neural light excitation phenomena. Our aim is to establish such an optogenetic interface in order to “communicate” with cortical neurons. For this purpose we shall design a cortical optical stimulation unit including electrical acquisition of the neural response. In order to achieve this, our biological-technical interface will utilize micro-machined sensors equipped with pre-processing and telemetric units. Our overall aim is to establish such innovative approaches in the field of epilepsy or similar diseases which allow novel insights in the behaviour of complex neural networks.

The foundations of optogenetics are based on the discovery of light-dependent ion channels of rhodopsin, known as channelrhodopsins in 2002/2003 by Ernst Bamberg Director of the Max-Planck-Institute for Biophysics in Frankfurt, Georg Nagel at the University of Würzburg and Peter Hegemann at the Humboldt University in Berlin (see for example [1,2]). These scientists established the presence of channelrhodopsins in mono-cellular algae. In this case the channels play a pivotal role for the phototaxis; the movement of algae in the direction of the source of light. Channelrhodopsins can also be successfully implanted in other cell types such as neurons where they allow a localized stimulation with high precision [1, 2, 3, 4, 5, 6]. This photonic stimulation is not only valid for in vitro experiments but also for in vivo interactions on living animals. Accordingly, using optical stimulation the modified neural tissue can be stimulated at a wavelength of around 480 nm (blue light) to open the ion channels and neural signalling. Yellow light emission at a wavelength of approximately 570 nm causes the ion channel to close, stopping the neural excitement. In this case a different protein, the so-called halorhodopsin, is activated which serves as an optical inhibitor of neural activity. In contrast to common electrical stimulation, not only single neurons are addressable by optogenetic methods but also entire specific cortical networks without the disadvantage of electrical methods.

The most common symptom of epilepsy is the presence of frequent seizures, which can cause damage to the neural networks leading to neurological deficiencies. An epileptic seizure at the cellular level is the result of a spontaneous synchronically neural signalling of the cortical network. One objective of research in the field of epilepsy is to improve understanding of this mechanism of neural synchronisation in order to develop new strategies for controlling such seizures. However, the complexity of neural networks in the brain allows only slow progress in the understanding of synchronisation [7, 8, 9, 10]. The currently used method of electronically based stimulation and acquisition techniques are only of limited used in the understanding of the complexity of epileptical seizures. Such techniques only allow stimulation and acquisition in small cortical areas. In contrast to the common electrical methods, optogenetic interaction with the cortex enables highly selective stimulation of large network complexes.

In cooperation with Prof. Dr. H. Beck from the Department of Epileptology at the University Hospital of Bonn we aim to develop a neural "tickling" with light in order to gain understanding of epileptic seizures at the level of neural networks. For this purpose, transgenic mouse brains are stimulated by optogenetic methods and the electrical response of neurons is monitored in-vivo. In contrast to “sliced mouse brain”, we hope to get new insights of epileptic seizures by means of this novel method on living mice.

This leads us to the following question: Is it possible to treat malfunctions in the human cortex by means of optogenetic methods? This could lead to remarkable improvements in the treatment of epilepsy or even the Parkinson disease by controlling the neural origin of seizures using fibre optics. Such diseases could be treated by controlling and suppressing seizures as needed.

Whether this future vision will be achieved is not yet known. In this project we shall demonstrate the significance and the pivotal role of optical stimulation of neurons in living animal models including cortical acquisition using wireless communication, which in itself is an immense progress not only from a medical medical standpoint, but more so from the technological side.

The biological-technical interface – our concept

Within the scope of a BMBF project (sponsored by FZ Jülich) we were able to build a successful hardware platform "Mausgehirnstecker". Our concept was based on a system which would enable and disable a neural stimulus while attached to the cranial bone of mice. In this so-called “topper” there are additional pre-amplifiers and adjustment units for the shaft including motor and integrated transmission elements (see figure 1).

Figure 1: The principle concept of the biological-technical interface. The interface consists of three components: the "topper" with the optical stimulation and electric acquisition units attached to the skull of the mouse, the "backpack" responsible for micro controlling, power supply and data transmission and finally the external hardware (workplace) connected wireless to the "backpack".

This system for optical stimulus combined with electronic data is called an optrode. An optrode has therefore two functions: firstly the neural stimulus by means of light emission and secondly the acquisition of evoked electrical responses. In our case such an optrode is based on micro structured silicon (Si) with an assembled optical fibre including a light source (LED) (see figure 2a) and 16 electrodes are placed at the tip of the optrode (see figure 2b).

Figure 2: Schematic setup of the optrode. a. General view. The whole system has a length of 9.6 mm, a width of 3.5 mm and a maximum thickness of 525 µm at the rear end. The light is generated by a LED and guided along the shaft to the end of the tip by an optical fibre. The LED (green) and the optical fibre (yellow) are glued within a well on the main board. b. Schematic zoom of the tip of the shaft with light cone. 16 electrodes made of gold are arranged on two levels separated by an insulation of SiO2. The electrodes have a side lenght of 12 × 12 µm2 and a thickness of 0.5 µm.

In figure 3, a fully processed optrode is shown. Clearly visible is the main board with its LED, contact pads and the shaft. The optical fibre is assembled on the shaft, which includes also front-end electrodes.

Figure 3: Photo of fabricated optrode. a. Top view. b. Side view. The inset visualizes the optical fibre on the shaft.

In figure 4a, the magnified shaft is shown. In figure 4 b, the cone of light emitted by the optical fibre is shown.

Figure 4: Proof of optical activity. a. The light, generated on the main board by LED, is guided through the optical fibre. b. Spatial distribution of the cone of the light from the top of the shaft (above in gel and in future in neurons).

In preliminary experiments we tested our optrode electronically. First measurements proved the high precision of the signal taken in an electrolyte. All experiments showed an excellent signal to noise ration and almost no delay in verifying the electronic suitability.

Figure 5: Proof of electrical activity. a. Experimental setup. In a physiological NaCl solution signals were generated by an electrode and received at a distance of 6 mm by the optrode. b. Comparison of signals. Upper signal is the standard signal of an impulse generater and the lower signal as acquired by the optrode. There is no variation to be detected.

Data transmission and telemetric approach

The electrical signals of the neurons are acquired by the front-end electrodesof the optrode and submitted via an AD-converter to the microcontroller placed in the “Backpack”. This “Backpack” is transmitting all signals wireless to the external hardware. For better understanding of the signal processing and transmission the data flow is shown on figure 6.


Figure 6:
Schematic concept of signal processing and data transmission between the workplace the freely moving mouse. All components in the “topper” are controlled by the “backpack”. The “backpack” is additionally responding wireless to the external hardware.

The heart of the external hardware (workplace) consists of a personal computer (PC). The PC, equipped with a wireless communication system is connected to the “backpack” attached to the mouse. Furthermore, this mobile “backpack” is wired to the optrode, which is attached to the mouse head. Shaft-adjustment unit (micro-motor), pre-amplifier and LED in the “topper” are operated by a microcontroller in the “backpack”. Apart from the microcontroller, the power supply, the telemetric unit, and a µSD-chip are also integrated. One of the main aspects in the design of all mobile elements is weight and size in order to takeinto account the free movement and size of the mouse. Additionally a high signal quality and resolution of neural responses acquired from the 16 electrodes submitted after pre-processing at 10 kHz using a 12 Bit rate must be maintained. In case of data overflow or high amount of data, a µSD-chip incorperated in the "backpack" shall ensure the high signal quality and resolution.
The electronic components controlling the micro-motor are shown on figure 7. First tests were successful and impressively showed a stable and reliable behaviour.

Figure 7: Electronic components implemented in the “backpack” and “topper”. a. Preliminary setup for the “topper”. b. Electronic components including the battery are integrated in the “backpack”. The size of the “backpack” is limited to the commercially available 3V button battery.

The "topper"

The “topper” components include the micro-motor and electronics are placed on a printed circuit board on the shaft along with the optical fibre and the electrodes. Over the shaft the optrode is slowly inserted into the cortex up to 3.6 mm by means of a micro stepper motor with transmission and spindle. A main emphasis is the gentle and controlled insertion of the shaft into the cortex in order to avoid neural trauma or other injuries. Therefore speeds of 20 µm per day are chosen towards the end of the insertion procedure. A schema of the setup is shown on figure 8 in details.

Figure 8: Three-dimensional design of the “topper”. a. Whole unit including housing (Ti) and attachments. The unit is planned to be 22 mm in height and 8 g in weight. b. Base for attachment at the skull (dental cement), transmission and printed circuit board (orange). The main board is attached by mean of bayonet fitting. c. Main board including pre-processing (black), LED (green) and shaft.

Figure 9: Photos of fabricated “topper”. a. Complete integrated unit. b. Base plate, main board, motor and transmission elements. c. Main board with pre-processing unit and shaft.

Outlook


In the European Union approximately 8 million people suffer from epilepsy. Our research and development in this regard seeks to clarify and understand the complexity of the cortical neural networks. We hope to gain new insights, which may lead in a medium term to a more specific and successful treatment of patients with epileptic seizures. Over a longer term periode – as a vision – we hope to create a “brain pacemaker”. Towards this goal, our project and the results derived shall answers to many technological challenges. Further research along with partners from Spain, France and Israel is planned within the scope of an EU project (“EpiNET” at Era-Net NEURON; Project sponsorship DLR, Bonn).

References:

[1] Nagel, G., Ollig, D., Fuhrmann, M., Kateriya, S., Musti, A.-M., Bamberg, E., and Hegemann, P. (2002) “Channelrhodopsin-1, A Light-Gated Proton Channel in Green Algae”Science 296, 2395-2398
[2] Nagel, G., Szellas, T., Huhn, W., Kateriya, S., Adeishvili, N., Berthold, P., Ollig, D., Hegemann, P., and Bamberg, E. (2003) “Channelrhodopsin-2, a directly light-gated cation-selective membrane channel” Proc. Natl. Acad. Sci. 100, 13940-13945
[3] Li, X., Gutierrez, D.V., Hanson, M.G., Han, J., Mark, M.D., Chiel, H., Hegemann, P., Landmesser, L.T., and Herlitze, S. (2005) “Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin” Proc. Natl. Acad. Sci. 102, 17816-17821
[4] Boyden, E.S., F. Zhang, F., Bamberg, E., Nagel, G., and Deisseroth, K. (2005) “Millisecond-timescale, genetically targeted optical control of neural activity” Nature Neuroscience 8, 1263-1268
[5] Zhang, F., Wang, L., Brauner, M., Liewald, J. F., Kay, K., Watzke, N., Wood, P. G., Bamberg, E., Nagel, G., Gottschalk, A., and Deisseroth, K. (2007) “Multimodal fast optical interrogation of neural circuitry” Nature 446, 633-639
[6] Nagel, G., Brauner, M., Liewald, J.F., Adeishvili, N., Bamberg, E., and Gottschalk, A. (2005) “Lightactivation of Channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses” Current Biology 15, 2279-2284
[7] Remy, S., Csicsvari, J., and Beck, H. (2009) “Activity-dependent control of neuronal output by local and global dendritic spike attenuation” Neuron 61, 906-916
[8] Otte, D.M., Bilkei-Gorzó, A., Filiou, M.D., Turck, C.W., Yilmaz O., Holst, M.I., Schilling, K., Abou-Jamra, R., Schumacher, J., Benzel, I., Kunz, W.S., Beck, H., and Zimmer, A. (2009) “Behavioral changes in G72/G30 transgenic mice” Euro. Neuropsychopharmacol. 19, 339-348
[9] Wiese, C., Rolletschek, A., Kania, G., Navarrete-Santos, A., Anisimov, S.V., Steinfarz, B., Tarasov, K.V., Brugh, S.A., Zahanich, I., Rüschenschmidt, C., Beck, H., Blyszczuk, P., Czyz, J., Heubach, J.F. Ravens, U., Horstmann, O., St-Onge, L., Braun, T., Brüstle, O., Boheler, K.R., and Wobus, A.M. (2006) “Signals from embryonic fibroblasts induce adult intestinal epithelial cells to form nestin positive cells with proliferation and multilineage differentiation capacity in vitro” Stem Cells 24, 2085-2097
[10] Remy, C., Remy, S., Beck, H., Swandulla, D., and Hans, M. (2004) “Modulation of voltagedependent sodium channels by the delta-agonist SNC80 in acutely isolated rat hippocampal neurons” Neuropharmacology 47, 1102-1112