ABSTRACT
In this study, we demonstrate capabilities to selectively manipulate dissociated co-cultures of neurons plated in dual-compartment devices. Synaptic receptor antagonists and tetrodotoxin solutions were used to selectively control and study the network-wide burst propagation and cell firing in cortical-cortical and cortical-thalamic co-culture systems. The results show that in cortical-thalamic dissociated co-cultures, burst events initiate in the cortical region and propagate to the thalamic region and the burst events in thalamic region can be controlled by blocking the synaptic receptors in the cortical region. Whereas, in cortical-cortical co-culture system, one of the region acts as a site of burst initiation and facilitate propagation of bursts in the entire network. Tetrodotoxin, a sodium channel blocker, when applied to either of the regions blocks the firing of neurons in that particular region with significant influence on the firing of neurons in the other region. The results demonstrate selective pharmacological manipulation capabilities of co-cultures in a dual compartment device and helps understand the effects of neuroactive compounds on networks derived from specific CNS tissues and the dynamic interaction between them.
Subject(s)
Cerebral Cortex/cytology , Coculture Techniques/instrumentation , Microfluidic Analytical Techniques , Neurons/drug effects , Thalamus/cytology , Action Potentials/drug effects , Animals , Cell Communication/drug effects , Cerebral Cortex/embryology , Dimethylpolysiloxanes , Equipment Design , Excitatory Amino Acid Antagonists/pharmacology , GABA Antagonists/pharmacology , Genes, Reporter , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Microelectrodes , Nerve Net/physiology , Neurites/ultrastructure , Neurons/cytology , Neurotoxins/pharmacology , Nylons , Primary Cell Culture/methods , Rats , Rats, Wistar , Receptors, Neurotransmitter/drug effects , Receptors, Neurotransmitter/physiology , Sodium Channel Blockers/pharmacology , Tetrodotoxin/pharmacology , Thalamus/embryologyABSTRACT
Co-cultures containing dissociated cortical and thalamic cells may provide a unique model for understanding the pathophysiology in the respective neuronal sub-circuitry. In addition, developing an in vitro dissociated co-culture model offers the possibility of studying the system without influence from other neuronal sub-populations. Here we demonstrate a dual compartment system coupled to microelectrode arrays (MEAs) for co-culturing and recording spontaneous activities from neuronal sub-populations. Propagation of electrical activities between cortical and thalamic regions and their interdependence in connectivity is verified by means of a cross-correlation algorithm. We found that burst events originate in the cortical region and drive the entire cortical-thalamic network bursting behavior while mutually weak thalamic connections play a relevant role in sustaining longer burst events in cortical cells. To support these experimental findings, a neuronal network model was developed and used to investigate the interplay between network dynamics and connectivity in the cortical-thalamic system.
Subject(s)
Cerebral Cortex/physiology , Nerve Net/physiology , Neural Pathways/physiology , Thalamus/physiology , Algorithms , Animals , Cells, Cultured , Cerebral Cortex/cytology , Coculture Techniques , Computer Simulation , Data Interpretation, Statistical , Electrophysiological Phenomena , Female , Microelectrodes , Models, Neurological , Neurons/physiology , Pregnancy , Rats , Rats, Wistar , Thalamus/cytologyABSTRACT
We developed a dual-compartment neurofluidic system with inter-connecting microchannels to connect neurons from their respective compartments, placed on a planar microelectrode arrays. The design and development of the compartmented microfluidic device for neuronal cell culture, protocol for sustaining long-term cultures, and neurite growth through microchannels in such a closed compartment device are presented. Using electrophysiological measurements of spontaneous network activity in the compartments and selective pharmacological manipulation of cells in one compartment, the biological origin of network activity and the fluidic isolation between the compartments are demonstrated. The connectivity between neuronal populations via the microchannels and the crossing-over of neurites are verified using transfection experiments and immunofluorescence staining. In addition to the neurite cross-over to the adjacent compartment, functional connectivity between cells in both the compartments is verified using cross-correlation (CC) based techniques. Bidirectional signal propagation between the compartments is demonstrated using functional connectivity maps. CC analysis and connectivity maps demonstrate that the two neuronal populations are not only functionally connected within each compartment but also with each other and a well connected functional network was formed between the compartments despite the physical barrier introduced by the microchannels.
ABSTRACT
In this paper, we propose an experimental approach to develop an in vitro dissociated cortical-thalamic co-culture model using a dual compartment neurofluidic device. The device has two compartments separated by 10 µm wide and 3 µm high microchannels. The microchannels provide a physical isolation of neurons allowing only neurites to grow between the compartments. Long-term viable co-culture was maintained in the compartmented device, neurite growth through the microchannels was verified using immunofluorescence staining, and electrophysiological recordings from the co-culture system was investigated. Preliminary analysis of spontaneous activities from the co-culture shows a distinctively different firing pattern associated with cultures of individual cell types and further analysis is proposed for a deeper understanding of the dynamics involved in the network connectivity in such a co-culture system.
Subject(s)
Cerebral Cortex/cytology , Cerebral Cortex/physiology , Coculture Techniques/instrumentation , Microfluidic Analytical Techniques/instrumentation , Thalamus/cytology , Thalamus/physiology , Animals , Cell Proliferation , Cell Survival , Cells, Cultured , Equipment Design , Equipment Failure Analysis , Neural Pathways/physiology , Rats , Rats, WistarABSTRACT
This work investigates an approach to record electrophysiological measurements of neuronal cell cultures in a dual compartment neurofluidic system. The two compartments are separated by 10-microm-wide and 3-microm-high microchannels and this provides a physical isolation of neurons allowing only neurites to grow between the compartments. We present long-term cell viability in closed compartment devices, neurite growth across the microchannels and a recording setup for the long-term recording of the network activity over 21 Days-in-Vitro (DIV). Structural and fluidic isolation between the compartments are demonstrated using transfection experiments and neurotoxin exposure, respectively.
