Modulation of Neural Network Activity through Single Cell Ablation: An in Vitro Model of Minimally Invasive Neurosurgery.

Alessandro Soloperto,Marta Bisio,Paolo Bonifazi,Michela Chiappalone,Francesco Difato,Gemma Palazzolo

doi:10.3390/molecules21081018

Abstract

The technological advancement of optical approaches, and the growth of their applications in neuroscience, has allowed investigations of the physio-pathology of neural networks at a single cell level. Therefore, better understanding the role of single neurons in the onset and progression of neurodegenerative conditions has resulted in a strong demand for surgical tools operating with single cell resolution. Optical systems already provide subcellular resolution to monitor and manipulate living tissues, and thus allow understanding the potentiality of surgery actuated at single cell level. In the present work, we report an in vitro experimental model of minimally invasive surgery applied on neuronal cultures expressing a genetically encoded calcium sensor. The experimental protocol entails the continuous monitoring of the network activity before and after the ablation of a single neuron, to provide a robust evaluation of the induced changes in the network activity. We report that in subpopulations of about 1000 neurons, even the ablation of a single unit produces a reduction of the overall network activity. The reported protocol represents a simple and cost effective model to study the efficacy of single-cell surgery, and it could represent a test-bed to study surgical procedures circumventing the abrupt and complete tissue removal in pathological conditions.

Highlights

The incomparable precision of laser ablation [1], and the high resolution imaging of optical systems allows precise and repeatable targeting of single cell units in living samples [2]
The experimental model used to evaluate the effect of single cell ablation in a neuronal network was based on primary cortical neuronal cultures infected at 10 days in vitro (DIVs) with a viral vector expressing the genetically encoded calcium indicator GCaMP6s
Through a low magnification microscope objective (4ˆ Olympus air objective) we achieved a big field of view on the neural culture, and detect the activity up to about 1000 neurons with a frame rate of 65 Hz (Figure 1A; red marks the contours of the recorded cells)

Summary

Introduction

The incomparable precision of laser ablation [1], and the high resolution imaging of optical systems allows precise and repeatable targeting of single cell units in living samples [2]. The first application of laser ablation on neuronal cultures was implemented in the Gross laboratory using a CO2 laser, in order to characterize the inflicted structural changes on neuronal processes, and validating the possibility to use laser dissection to develop an in vitro model of brain injury. In this work they described three distinct mechanisms of laser ablation: direct vaporization, pressure shock waves, and non-thermal photo-ablation [5]. Compact pulsed laser sources [6] can be integrated in optical systems at reasonable cost, with respect to ultrafast laser sources which require complex and expensive maintenance

Methods

Results

Conclusion