In this study, we introduce the new image-guided registration system (IGRS) to interpret the dynamics and distribution of neuronal activities on temporal and spatial scales. Our device is designed to integrate microelectrode array (MEA) and optical coherence tomography (OCT) in single-body upright microscopy, which enables imaging of volumetric brain anatomy and measurement of neuronal activity at multiple sites simultaneously. To evaluate the performance of IGRS, the neuronal activities of the hippocampal region in the brain slice were monitored and the corresponding spatial and temporal map was intuitively visualized.
In the field of stimulation research, several optical methods have recently been introduced as an alternative technique due to its spatial resolution, multiple stimulation and non-invasive manner. Among optical stimulation methods, single-photon stimulation based on caged glutamate is one of the well-known techniques using photolysis to release neurotransmitters such as glutamate. Based on this, we introduce a ball-lens probe, which has several advantages in stimulating neurons.
And it is useful to apply for tissue sample and multi-stimulation due to probe type. In summary, the IGRS and bead-tipped probe would be a very effective tool to investigate and stimulate neuronal activities and connectivity in different areas.
Motivation
Image-guided recording system for spatial and temporal mapping of neuronal
Introduction
Image-guided recording system for spatial and temporal mapping of neuronal activities in brain slices. Finally we evaluate its performance as we apply it to record the spatial and temporal mapping of neuronal activities in the hippocampal region of the brain slice.
Methods and Materials
- Swept Source Optical Coherence Tomography
- Extracellular Recording
- Acute Brain Slice Preparation
For neuronal recording, we used a 60-channel extracellular recording system (USB-ME64, Multichannel Systems, Germany) mounted on the vertical microscope stage. In this program, we set the 60 Hz high-pass filter by default due to the exclusion of AC frequency from the voltage source. For AP analysis, a 300 Hz high-pass filter was added to detect it, and these signals were negative-amplitude thresholded.
The experimental setup was built around an inverted microscope (IX70 Olympus, Japan), including a customized culture chamber (Live cell instruments, Korea), a 37 °C temperature controller, and a humidified 5% CO2 gas supply to enhance viability and stabilization of the brain. slice. In the electrophysiological recording section of the device, a pMEA (perforated microelectrode array) was placed on the microscope stage to obtain the extracellular signals of the neurons in the brain slice. In addition, the pMEA has many holes that allow the sample to be clearly attached to the surface of the electrodes when the vacuum was applied by the syringe pump.
If the sample did not fully contact the electrodes, the space between the sample and the electrodes could become filled with solution. After stabilizing the sample, a coronal brain slice was placed on the surface of pMEA with oxygenated ACSF.
Results
- Images of Hippocampus with Thickness
- Action Potentials
- OCT images of brain slice on MEA
- Spatial and temporal mapping protocol of IGRS
- Measurement of neuronal activity using IGRS
- Application for Electrical Stimulation
As shown in Figure 1-3, the data was extracted as much as 1000 ms from original one in channel 58 and noise level was about ±10 μV. Then all APs are sorted and overlapped to check the shape of spikes in Figure 1-3 (B). The common way to observe the structure of brain slice is to use the upright microscope which can clearly visualize the surface of brain tissue as shown in Figure 1-4 (A).
In a feasibility study, a 50 mm focal length lens was used for comparison with a bright field microscope image, while a whole brain slice was displayed on the MEA in Figure 1-4 (B). In addition, the volumetric OCT image clearly showed the hippocampal area covered in 2 mm ´ 2 mm by the 4-inch objective lens, while providing the electrode distribution as shown in Figure 1-4 (C, D). We developed IGRS interpretation software for spatial and temporal mapping of neuronal signals in OCT images using MATLAB, as shown in Figure 1-5.
Since OCT image of electrodes with brain tissue in Figure 1-4 (C, D) looks blurry, we initially acquired pMEA image using OCT to provide clear electrode image before loading brain tissue. Top view OCT image related to pMEA well visualized both the perforated holes and multiple electrodes with 2 mm by 2 mm as shown in Figure 1-5 (A). Since our OCT image identifies the location of electrodes, we were able to confirm and match the exact position of brain tissue on the electrodes.
To clearly visualize the anatomy of brain tissue morphology, color-coded neuronal signal map was adjusted by thresholding and transparency values (Figure 1-5 (G)). C) 3D OCT image visualizing the volumetric hippocampus with electrodes (D) OCT projection image representing the hippocampus morphology and extracted electrode (E) Neuronal activities recorded by pMEA in 60 channels. In Figure 1-6 are action potential signals obtained from MEA in 60 channels and mapping images based on these data in normal, 4-AP and TTX treatment.
Local field potential signals (LFP) and waveforms obtained from the DG, CA3 and CA1 region of the hippocampus by MEA in 60 channels, as shown in Figure 1-7. At the same time, positive and negative signals began to be generated in CA3, as shown in Figure 1-8 (B3). IGRS made it easy to identify defects in a brain slice because our system was based on a 3D OCT image.
Using IGRS, we could find sample defects such as holes in the red rectangular boxes as shown in Figure 1-9 (A, B). Also, the depth change was more obvious, but also the exact depth values based on the pixel calculation as shown in Figure 1-9 (D).
Discussion
Introduction
Methods and Materials
- Photolysis of caged glutamate in single-photon stimulation
- Primary Cell Culture
After anesthesia treatment with carbon dioxide (CO2), primary hippocampal neurons were isolated from rats of embryonic day 17 - 18. The rat brains were removed from the embryos and their hippocampal neurons were rapidly dissected from the cortex at 4 °C in HBSS. After being dissociated in a 0.25% trypsin-EDTA solution in a water bath at 37°C for 15 min, DMEM containing 10% horse serum was added to the solution to stop the effect of trypsin.
![Figure 2-1. The mechanisms of glutamatergic synapses in the axon terminals [47]](https://thumb-ap.123doks.com/thumbv2/123dokinfo/10522718.0/32.892.124.759.175.794/figure-2-mechanisms-glutamatergic-synapses-axon-terminals-47.webp)
Results
- Fiber Optic Simulation & Measured Data
- Ball Lensed Stimulator Manufacturing
Figure 2-3 shows a schematic diagram of the production step and image of ball lens fiber. There are two types of materials to build a ball lens fiber: single-mode fiber (SMF) and coreless silica fiber (CSF). SMF (core/cladding diameter: 3.3 μm / 125 μm) is an optical fiber designed to transport light directly only along the fiber in the transverse axis and CSF is used to increase the beam diameter as a spacer.
The wire stripping machine must first remove the SMF outer sheath and cladding, as SMF consists of a fiber core, a glass cladding, an intermediate coating and an outer sheath. Then, the end of the fiber was cut with a fiber splitter (Fiberoptic, China) to cut it neatly. It was placed on a fusion splicer stage (Fitel, Japan) and the residue cleaning step was processed under an arc strength of 10 mV and a duration time of 50 ms.
In the second step, CSF, which had also been cleanly cut off by a fiber cutter, was loaded into the opposite phase of the SMF. After another debris clearing step, the end of SMF and CSF was straight aligned by controlling the x- and y-axis motor. Then, two types of fibers were fused under an arc power of 110 mV and a duration of 950 ms.
Then SMF-CSF fused fiber was cut by fiber cutter at the point where CSF length could be 1000 µm as we simulated in Figure 2-3. Finally, residual fiber between two discharge probes was aligned to be fully affected by it. Arc discharge under 90 mV arc power and 650 ms duration was applied to produce optical fiber with ball lens.
The hardware of stimulator was designed using SolidWorks (Dassault System, France) and built by 3D printer. Fully developed fiber and piezo motor (Faulhaber, Germany) to control the focal point is assembled with hardware of stimulator and its actual image as shown in Figure 2-5 (C).
Discussion
Conclusion
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