Neural activity propagation in an unfolded hippocampal preparation with a penetrating micro-electrode array.

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in the hippocampus. The transverse hippocampal slice preparation is the most common tissue preparation to study hippocampus electrophysiology. A longitudinal hippocampal slice was also developed in order to investigate longitudinal connections in the hippocampus. The intact mouse hippocampus can also be maintained in vitro because its thickness allows adequate oxygen diffusion. However, these three preparations do not provide direct access to neural propagation since some of the tissue is either missing or folded. The unfolded intact hippocampus provides both transverse and longitudinal connections in a flat configuration for direct access to the tissue to analyze the full extent of signal propagation in the hippocampus in vitro. In order to effectively monitor the neural activity from the cell layer, a custom made penetrating micro-electrode array (PMEA) was fabricated and applied to the unfolded hippocampus. The PMEA with 64 electrodes of 200 µm in height could record neural activity deep inside the mouse hippocampus. The unique combination of an unfolded hippocampal preparation and the PMEA provides a new in-vitro tool to study the speed and direction of propagation of neural activity in the two-dimensional CA1-CA3 regions of the hippocampus with a high signal to noise ratio.

A high aspect ratio microelectrode array for mapping neural activity in vitro.

A novel high-aspect-ratio penetrating microelectrode array was designed and fabricated for the purpose of recording neural activity. The array allows two dimensional recording of 64 sites in vitro with high aspect ratio penetrating electrodes. Traditional surface electrode arrays, although easy to fabricate, do not penetrate to the viable tissue such as central hippocampus slices and thus have a lower signal/noise ratio and lower selectivity than a penetrating array. In the unfolded hippocampus preparation, the CA1-CA3 pyramidal cell layer in the whole unfolded rodent hippocampus preparation is encased by the alveus on one side and the Schaffer tract on the other and requires penetrating electrodes for high signal to noise ratio recording. An array of 64 electrode spikes, each with a target height of 200µm and diameter of 20µm, was fabricated in silicon on a transparent glass substrate. The impedance of the individual electrodes was measured to be approximately 1.5MΩ±497kΩ. The signal to noise ratio was measured and found to be 19.4±3dB compared to 3.9±0.8dB S/N for signals obtained with voltage sensitive dye RH414. A mouse unfolded hippocampus preparation was bathed in solution containing 50 micro-molar 4-amino pyridine and a complex two dimensional wave of activity was recorded using the array. These results indicate that this novel penetrating electrode array is able to obtain data superior to that of voltage sensitive dye techniques for broad field two-dimensional neuronal activity recording. When used with the unfolded hippocampus preparation, the combination forms a uniquely capable tool for imaging hippocampal network activity in the entire hippocampus.

Orthogonal wave propagation of epileptiform activity in the planar mouse hippocampus in vitro.

PURPOSE: In vitro brain preparations have been used extensively to study the generation and propagation of epileptiform activity. Transverse and longitudinal slices of the rodent hippocampus have revealed various patterns of propagation. Yet intact connections between the transverse and longitudinal pathways should generate orthogonal (both transverse and longitudinal) propagation of seizures involving the entire hippocampus. This study utilizes the planar unfolded mouse hippocampus preparation to reveal simultaneous orthogonal epileptiform propagation and to test a method of arresting propagation. METHODS: This study utilized an unfolded mouse hippocampus preparation. It was chosen due to its preservation of longitudinal neuronal processes, which are thought to play an important role in epileptiform hyperexcitability. 4-Aminopyridine (4-AP), microelectrodes, and voltage-sensitive dye imaging were employed to investigate tissue excitability. KEY FINDINGS: In 50-µm 4-AP, stimulation of the stratum radiatum induced transverse activation of CA3 cells but also induced a longitudinal wave of activity propagating along the CA3 region at a speed of 0.09 m/s. Without stimulation, a wave originated at the temporal CA3 and propagated in a temporal-septal direction could be suppressed with glutamatergic receptor antagonists. Orthogonal propagation traveled longitudinally along the CA3 pathway, secondarily invading the CA1 region at a velocity of 0.22 ± 0.024 m/s. Moreover, a local lesion restricted to the CA3 region could arrest wave propagation. SIGNIFICANCE: These results reveal a complex two-dimensional epileptiform wave propagation pattern in the hippocampus that is generated by a combination of synaptic transmission and axonal propagation in the CA3 recurrent network. Epileptiform propagation block via a transverse selective CA3 lesion suggests a potential surgical technique for the treatment of temporal lobe epilepsy.