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Manipulating neuronal outgrowth using a novel pneumatic micro gene gun

Final Report Summary - MANIPULATING TILING (Manipulating neuronal outgrowth using a novel pneumatic micro gene gun)

The 'Manipulating Tiling' project was motivated by the question how a single neuron gains a specific structure for a specific function, a question of great importance for understanding the brain in health and disease. Our long-term research goal was to reveal factors that direct and govern neuronal growth in order to manipulate and control neuronal development, thus, to enable the engineering of neuronal trees. We aimed to develop the ability to study and manipulate single neurons at the single cell level, both in vitro and in the intact animal.
In this project we have studied the nervous system of the medicinal leech (Hirudo Medicinalis) which is ideal for in vivo and in vitro studies. The leech neurons are identified and their activity and morphology are well characterized. We focused on the mechanosensory neurons that are sensitive to light and pressure touch that show diverse, yet stereotyped, morphologies as tiling the leech body wall. We studied the role of the axon guidance cue netrin, as a candidate for directing mechanosensory tiling. To this end, we characterized the pattern of expression of netrin and its receptors UNC-5 and DCC. Prior to the current project Netrin and the receptor UNC-5 were examined (UNC-5 only partially resolved). DCC in the leech was still unknown.
In addition, we grew dissociated neurons in culture and followed their growth for several days and up to two weeks. The leech neurons can develop in culture in extremely low densities, thus, to allow detailed analysis of the dendritic tree along the regeneration process. We used this culture system in order to study mechanisms of directing neuronal growth including physical guidance.
In order to modulate the expression of molecular candidates locally we used a traditional microinjection set-up and a novel technology we have developed, a pneumatic capillary gene gun. With this technology we can deliver reagents such as DNA and RNA molecules to a well-defined, confined region in the skin and the nervous system of intact embryos and adults, in order to knock in and knock down genes respectively. A main goal of this proposal was to broaden the use of the gene gun, to improve its performance and to develop the ability to deliver functional proteins or drugs directly into internal layers of the target tissue. Such capability is essential for a therapeutic use.
During the first period of the project I have focused on the establishment of the lab including the integration of the pneumatic capillary gene gun; establishment of an adult leech colony and a breeding colony; developing neuronal cell culture model system; developing manipulation abilities at the single cell level; developing image processing tools and modeling tools for neuronal activity.
Based on the specific aims of the project, during the second period we have advanced the project as follows. We have developed additional delivery assays by improving the set-up and by adding more types of carriers to broaden the use of the gene gun set-up.
Using on the cell culture model we have performed neuronal cell culture experiments and were able to direct neuronal growth and align the neuronal processes to patterned substrates. Most importantly, we have characterized further the role of the Netrin receptor UNC-5 and for the first time we have identified and sequenced the Netrin receptor DCC and studied its pattern of expression in the leech embryo.

Achievements for the reporting period:
(i) Delivery set-up: the Pneumatic Capillary Gene Gun. We have established two models of the pneumatic capillary gene guns, including a high power set-up for a wide range of penetration depths (collaboration with Prof. Groisman, UCSD, USA). We have used the set-ups for the delivery of DNA molecules, RNA molecules and drugs. The set-ups enable highly localized targeting at controlled depths. The delivery parameters are affected by the type and size of the carriers. In order to broaden the use of the gun we have installed the new generation gene gun (courtesy of Prof. Groisman) with higher negative pressure that allows higher accelerating He pressure. The novel set-up enables to target deeper layers of cells and to deliver lighter carriers. The use of the new generation gene gun has facilitated the development of novel methodologies for gene manipulations and drug delivery.
 The work has been presented in the NanoIsrael Annual meeting, Tel-Aviv, 2012; Israeli Bio-medical engineering meeting, 2012; ICRS meeting for controlled drug delivery, 2012 (Zilony, Tzur-Balter, Segal and Shefi).
 The work has been published: Zilony N., Tzur-Balter A., Segal E., Shefi O., Scientific Reports, 3:2499, 2013.

(ii) Leech colony. We have established a productive leech breeding colony. We import between 800 to 1000 adult leeches per year and use them for neuronal cultures (dissociated from adult leeches) and for breeding. The colonies are separated and maintained by the lab manger and the students.

(iii) Manipulating neuronal growth culture model. We grew adult leech neurons in culture with controlled number of cells per culture dish. We have followed the regeneration process using time lapse technique and characterized the different stages of development between single isolated neurons to fully connected network. We grew the primary neurons in conditioned medium and tested the effect of Nerve Growth Factors on the neuronal morphology. For the topographic manipulations we used photolithography to fabricate substrates with repeatable line-pattern ridges of nano-scale heights. We plated the leech neurons atop the patterned-substrates and compared their growth pattern to neurons plated atop non-patterned substrates. These interactions of the neuronal process dominantly affected the neuronal growth direction and the response of the entire neuronal branching tree to the patterned substrates that is significantly affected.
(In collaboration with Dr. Amos Sharoni, Bar Ilan University, Israel).
The work has been presented in several meetings including the annual SFN meeting (Society Neuroscience Meeting), Washington DC, 2011, the ISFN (Israeli neuroscience meeting), Eilat, Israel, 2011 and the NanoIsrael annual meeting, Tel Aviv, 2012. I have presented the work in an oral presentation in the SFN meeting (Society Neuroscience Meeting), New Orleans, 2012.
The work has been published in:
 Baranes, Chejanovsky, Alon, Sharoni and Shefi, Topographic cues of nano-scale height direct neuronal growth pattern. Biotechnology and Bioengineering, 109(7):1791-1797, 2012.
 Baranes, Kollmar, Chejanovsky, Sharoni and Shefi, Interactions of neurons with topographic nano cues affect branching morphology mimicking neuron-neuron interactions. Journal of Molecular Histology, 43(4):437-447, 2012.
(iv) Analyzing Netrin receptors: Based on our preliminary results we have extracted mRNA of leech embryos and analyzed the cDNA to isolate the two netrin receptors UNC-5 and DCC. We have used PCR to combine the partial sequences we had of the UNC-5 to a single sequence of the receptor. In addition, we have performed the transcriptome of the adult CNS. We have used bioinformatics tools following the RNA sequencing and have fish transcript containing the DCC. Based on the DCC sequence we have successfully demonstrated the expression of DCC in the P cells that innervate the ventral territory, in agreement with our hypothesis (we have used in situ hybridization procedure). The rest of the sequencing results will be analyzed in parallel to partially sequenced genome of the medicinal leech (joint effort led by Prof. Macagno, UCSD).
 The recent results have been summarized for publication, currently under revision in Bioinformatics.
(v) Image processing and modeling: We developed an algorithm for the segmentation of the complex neuronal trees. The algorithm is based on the division of the images into patches denoted as superpixels that are connected to clusters using min-cut spectral graph approach. Currently, the results are summarized for publication.
In addition, we have developed the full Hodgkin Huxley cable model that takes into account the morphology of the neuronal tissue. This tool enables us to analyze neuronal activity for different dendritic patterns, including the activity along axons and branching points. A detailed analysis we have performed was summarized for publication.
 Currently under revision in the IEEE Transactions on Neural Networks and Learning Systems.

Personnel: currently in the lab there are 5 PhD students, 3 master students, a postdoc (50%) and a lab manager (50%). The background of the personnel is diverse including chemists, biologists computer scientists and engineers.