Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

Podsumowanie

We have developed an in vitro unfolded hippocampus which preserves CA1-CA3 array of neurons. Combined with the penetrating micro-electrode array, neural activity can be monitored in both the longitudinal and transverse orientations. This method provides advantages over hippocampal slice preparations as the propagation in the entire hippocampus can be recorded simultaneously.

Streszczenie

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.

Wprowadzenie

Understanding the neural conduction or propagation of neural signals is crucial for determination of the mechanism of neural communication in both the normal function and pathological conditions in the brain ^1-3. The hippocampus is one of the most extensively studied structures in the brain since it plays fundamental role in several brain functions such as memory, and spatial tracking and is involved in several pathological changes that dramatically impact behavior as well ^1,6 . Although, the hippocampus exhibits a complex organization, the different elements of its structure can be readily identified and accessed in the slice preparation^4-6. In the transverse direction of the hippocampus, neural activity is known to propagate through the tri-synaptic pathway that comprise the Dentate Gyrus (DG), CA3, CA1 andsubiculum ^4,5. It is believed that synaptic transmission and axonal conduction play a major role for communication in this transverse circuit ^4,6. However, propagation of neural signal takes place in both transverse and longitudinal directions ^4,6. This implies that the hippocampus cannot be fully investigated by using slice preparations which limit the observation to a particular direction of propagation ⁴. The longitudinal slice was developed to investigate the axonal pathways along the longitudinal axis ⁵. Researchers have observed behavior-specific gamma and theta oscillations predominantly along the transverse and longitudinal axes respectively ⁶. These behaviors have been studied separately, yet simultaneous access to both directions is crucial to understand these behaviors. Even with the development of the intact hippocampus preparation, it is difficult to monitor the propagation throughout the entire tissue due to the folded-structure of the hippocampus ⁴. The unfolded hippocampus provides access to the packed neurons in a form of a flat two-dimensional cell layer ^7,8.

By unfolding the dentate gyrus (DG) (Figure 1), the hippocampus adopts a flattened shape with a rectangular configuration in which both transverse and longitudinal connections remain intact with the pyramidal cell layer arranged in a two-dimensional sheet containing both CA3 and CA1, leaving a flat piece of neural tissue that can be used to investigate neural propagation (Figure 2) ⁸. Neural activity can then be monitored with individual glass pipettes, microelectrode arrays, stimulating electrodes, as well as voltage sensitive dyes (VSD) ^3,7,8. In addition, genetically encoded voltage indicator from transgenic mice can be used to track the propagation pattern ⁹.

The flat configuration of the unfolded hippocampal network is well suited for optical method recording but also for a microelectrode array. Most of the commercially available arrays are fabricated with flat or low profile electrodes and can record neural activity in both tissue slices and cultured neurons ^10-12. However, the signal-to-noise ratio (SNR) decreases when the signals are obtained from an intact tissue since the soma of the neurons are located deeper into the tissue. Microelectrode electrode arrays with high aspect ratios are required to improve the SNR.

To this effect, a penetrating microelectrode array (PMEA) has been developed in our laboratory, and provides the ability to directly probe into the tissue by inserting 64 spikes with a diameter of 20 µm and height of 200 µm into the unfolded hippocampus ^7,13. This microelectrode array has higher SNR compared to the voltage sensitive dye imaging and the SNR remains stable during an experiment ^7,13. The combination of the unfolded hippocampal preparation and the PMEA provides a new way to investigate the neural propagation over a two-dimensional plane. Experiments using this technique have already yielded significant results about the mechanisms of neural signal propagation in the hippocampus whereby neural activity can propagate independently of synaptic or electric synapses ⁷.

Protokół

NOTE: Animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at the university. CD1 mice of either sex at the age of P10 to P20 are used in this study.

1. Solutions for Surgery and Experimental Recording

1. Prepare normal artificial cerebrospinal fluid (aCSF) buffer containing (mM): NaCl 124, KCl 3.75, KH₂PO4 1.25, MgSO₄ 2, NaHCO₃ 26, Dextrose 10, and CaCl₂ 2. Use this normal aCSF for tissue recovery after dissection, as well as for washing system at the beginning of the experiment.
2. Prepare sucrose aCSF that is used during the hippocampus dissection and contains (mM): Sucrose 220, KCl 2.95, NaH₂PO4 1.3, MgSO₄ 2, NaHCO₃ 26, Dextrose 10, and CaCl₂ 2. In order to induce epileptiform activity in the intact hippocampal tissue preparation, add 4-Aminopyridine (4-AP) to normal aCSF at the concentration of 100 µM.

2. Surgical Procedure for the Intact Hippocampus Preparation

1. Drop isoflurane (1 ml) into the chamber at the bottom of a desiccator glass jar (No.1 in Specific Materials and Equipment) and use a regular paper towel to cover the surface of the bottom stage so the animal does not get in contact with the liquid. Then, place the CD1 mouse into the jar and close the lid. Keep the animal in the closed jar for about 1 min to 2 min. When breath frequency is about one per second, remove the mouse from the jar.
2. Place the mouse on the surgery stage and decapitate with a suitable scissors. Immediately after decapitation, place the head into the ice-cold (3-4 °C), oxygenated sucrose aCSF for about 30 sec.
3. Use fine scissors (No.5 in Specific Materials and Equipment) to remove the skin on top of the skull and to cut the skull along the middle line of the head as well as at two ends near the temporal lobe. Use forceps (No. 6 in Specific Materials and Equipment) to peel the cut skull towards each side of the head in order to expose the brain.
4. Insert a micro lab spatula (No. 8 in Specific Materials and Equipment) into the gap between the parietal lobes of the brain and skull to carefully peel the brain from the bottom of the skull and then drop it onto a prepared ice-cold (3-4 °C) surgical stage covered with wet filter paper (No.2 in Specific Materials and Equipment). Remove the cerebellum with an ice-chilled blade and separate the two hemispheres by cutting the midline of the brain. Then, place the two separated hemispheres into a beaker filled with the ice-cold sucrose aCSF bubbled with 95% O₂/ 5% CO₂.
5. Take one half of the hemisphere and place on the ice-cold filter paper stage. Place regular paper towels or filter paper around the brain to suck the extra solution. Use two fire-polished glass pipette tools (No. 10 in Materials and Equipment) to separate the cortex from the rest of the central part of the brain.
6. After the hippocampus is exposed from inside of the cortex and put on the ice-cold stage, place two or three drops of ice-cold sucrose aCSF on the tissue and then remove extra solution around the hippocampus. Cut the connections with the cortex at two ends of the hippocampus. Then, dissect the hippocampus out of the brain with the fire polished glass tools and remove the remaining part of the brain.
7. Separate the whole hippocampus with its alveus side facing up and hippocampal sulcus facing down. Quickly drop two or three drops of ice-cold sucrose aCSF on the tissue again and remove the extra solution around the tissue using a piece of paper towel. Use a fire polished glass tool to turn over the whole hippocampus to expose the sulcus (Figure 1B, C).
8. Under a normal optical microscope, insert a custom made glass needle (No. 11 in Specific Materials and Equipment) into one end of the sulcus and cut the fiber connections, from dentate gyrus (DG) to subiculum or the CA1 field, along the direction of sulcus (Figure 1C). Apply an ice-cold blade to trim the septal and temporal ends of the hippocampus if necessary. Insert a custom made metal wire loop (No. 12 in Specific Materials and Equipment) into the cut sulcus and pull over the DG away from the tissue while holding the subiculum/CA1 end of the hippocampus by a fire polished glass tool (Figure 1D).
9. Following the unfolding procedure above, add another two or three drops of the ice-cold sucrose aCSF onto the tissue and remove the extra solution around the tissue. Then, trim the unfolded hippocampus with the ice-cold blade at the edges (Figure 1E) and place the preparation by a spatula into the recovering chamber filled with normal aCSF and bubbled with 95% O₂/ 5% CO₂ at RT (about 25 °C). Leave the unfolded hippocampus tissue to recover about 1 hr before placing it into the recording chamber.
10. Take the other brain hemisphere and use it to go through steps from 2.5 to 2.9. Usually, take about 1 to 2 min to finish the whole procedure to unfold a single hippocampus and drop ice-cold sucrose aCSF continuously to keep the tissue oxygenated and hydrated.

3. Experimental System Setup

1. Glue a custom fabricated PMEA on a pin grid array (PGA) package and use micro wire bonding to connect each pad from a single microelectrode to the pad of a pin on the package (Figure 3B). Then, insert the package into the socket on a custom-made circuit board ¹³.
2. Individually connect each microelectrode to its filters with band pass cut-off frequency from 1 Hz to 4 KHz and amplifiers with gain of 100 on the custom made circuit board ¹³. Digitize the analog output from the circuit board by an A/D converting system, and acquire and store the data on a computer.
3. Glue a custom-made plastic recording chamber around the array with inlet and outlet tubing for the solution flow (Figure 4A). Use at least two bottles to keep different solutions in the system, and join and control the output tubing of the bottles by a tri-valve. Connect the output of the tri-valve to a tubing system with an IV drip chamber which is guiding the solution into the recording chamber.
4. Between the tri-valve and the inlet of the recording chamber, add an electrical heater to heat the solution to a controlled temperature (35 °C) before the solution is guided to the recording chamber. Attach the outlet of the recording chamber to a vacuum tube to collect the solution into a vacuum tube connected flask. Do not recycle any solution in any experiment in this study.

4. Placing the Unfolded Hippocampus onto the PMEA to Record the Neural Activity

1. To prepare the experimental setup, fill one bottle with normal aCSF and another bottle with 4-AP aCSF. In both bottles, bubble 95% O₂/ 5% CO₂ from the very beginning of each experiment. Use a tri-valve connector to control which solution will be selected during an experiment. Connect a vacuum tube at the outlet of the chamber to pump the solution into a dust container. Heat the pipeline before delivering it into the recording chamber and keep the solution at a controlled temperature level (35 °C).
2. When the inlet and outlet of recording chamber are closed, use a custom made glass pipette dropper to transfer and place the unfolded hippocampus into the recording chamber. Under the microscope, position the unfolded hippocampus using a regular small paint brush while the tissue is floating in the solution. Place the unfolded hippocampus with its alveus side facing down, CA3 area pointing away, and CA1 field pointing towards the researcher.
3. Carefully suck away the solution in the chamber using a vacuum pipette from the edge of the recording chamber to lower the solution level until the chamber is dried and the tissue is lowered onto the array. Then, carefully place a custom made tissue anchor (Figure 4) (No. 14 in Specific Materials and Equipment) on top of the tissue to hold the unfolded hippocampus onto the array. Put a few drops of solution into the recording chamber to refill it, and gradually open the inlet and outlet to adjust flow rate to about two drops per second in the IV drip chamber.
4. Incubate the tissue in the recording chamber with normal aCSF for about 1 min to recover, then switch the solution supply to 4-AP dissolved aCSF and adjust the flow rate properly. Incubate the tissue in 4-AP dissolved aCSF for about 5 to 10 min and then the researcher could start the software to record the signal when spontaneous activity appears.

5. Removing the Tissue from the PMEA After an Experiment

1. Control the tissue anchor by a micromanipulator and gradually lift the tissue from the recording chamber. Shut down both inlet and outlet to stop the flow in the recording chamber. The recording chamber should be full with solution or add a few drops of solution to fill the chamber if the recording chamber is not full.
2. Use a small paint brush to lift each corner of the tissue. If the tissue is not floating in the solution, then employ the vacuum tube to dry the chamber carefully with the tissue still sitting on the array. Then carefully open the inlet to gradually refill the chamber and shut down the inlet to stop the flow when the recording chamber is full. Apply the small paint brush to lift each corner of the tissue again.
3. Repeat step 5.2 until the tissue is detached from the array and floating in the solution. If the tissue is floating in the solution, then use the vacuum tube to suck the tissue away. Open the flow in the inlet and open the vacuum in the outlet. Wash the system with distilled water and dry it out.

Wyniki

The data shown in the figures here were recorded in the unfolded hippocampus preparation with 4-AP (100 µM) aCSF added during incubation of the tissue in the recording chamber at RT (25 °C). Normally activity starts within 5 min, but in some hippocampal tissues from the older animals it may take longer. The 4-AP-induced neuronal firing observed with the PMEA is the same as previously reported ^14,15. Since the electrodes have a height of 200 µm, the electrode tips are located just below the cell ...

Dyskusje

The development of the unfolded hippocampus preparation, where the longitudinal and transverse axes of the hippocampus are preserved in combination with a penetrating microelectrode array, provides a powerful tool to investigate the anatomy connections or neural propagation in the hippocampus ⁷. This unfolding procedure is also applicable for studying hippocampus in adult mice. Recent studies with this preparation showed that the 4-AP-induced epileptiform activity could propagate with a diagonal wave front acr...

Materiały

Name	Company	Catalog Number	Comments
desiccator jar	LABRECYCLERS Inc.	5410	Place regular paper towels at the bottome of the jar for animal anesthesia use.
A blade and Custome made surgical stage for unfolding hippocampus	N/A	N/A	A petri dish is place upside down (in the center) in the ice with a wet filter paper place on top of it.
Custom made tissue recovery chamber	N/A	N/A	Plastic tubes were glued with plastic mesh at the bottom and bubbled with 95% O2/ 5% CO2 in the aCSF.
Straight Operating Scissors	Fisher Scientific	S17336B                                            Medco Instruments No.:81995	This scissors is used to   decapitate the mice.
Integra Miltex Goldman-Fox Scissors	Fisher Scientific	12-460-517                        MILTEX INC                           No.:5-SC-320	This scissors is used to cut the skull of the mice.
Miltex Hysterectomy Forceps	Claflin Medical equipment	CESS-722033-00001	This Forceps is used to peel the cut skull to expose the brain
Micro Spatula	Cardinal Health		This micro spatula is used to tranfer the whole brain of a semisphere into the recorering chamber.
Frey Scientific Stainless Steel Semi-Micro Spatula	Cardinal Health		this semi micro spatula is used to tranfer the unfolded hippocampus into the glucose aCSF in the recovering chamber.
small paint brush	Lowe's	tem #: 105657                  Model #: 90219	The one with the smallest size in a normal paint brush package
Fire polished glass help tool	N/A	N/A	This tool was fire polished and made from the regular Pasteur glass pipettes.
Custom made glass needle	N/A	N/A	This tool was fire polished and made from the regular Pasteur glass pipettes.
Custom made glass tool with a metal wire loop	N/A	N/A	This tool was fire polished and made from the regular Pasteur glass pipettes with a reshaped metal wire loop.
Custom made glass solution dropper	N/A	N/A	This tool was  made from the regular Pasteur glass pipettes with its tips cut and a rubber head attached with the cut end.
Custom made tissue anchor	N/A	N/A	Nylon fiber mesh was glued on a insulated copper wire ring. The tissue anchor was hold by an micromanipulator.
Custom fabricated microelectrode array	N/A	N/A	More detail about the array please refer to  Kibler, et al, 2011.
Custom made filter and amplifiers circuits for the array	N/A	N/A	More detail about the array please refer to  Kibler, et al, 2011.
Data acquisition processor 3400a	Microstar Laboratories	N/A	This is a complete data acquisition system with A/D converter.