the local delivery channel.
The position of the retina with respect to the local delivery channel is another crucial factor in this experiment. When transferring the retina into the well, a filter paper with a hole cut open in the middle is used to hold the retina. The optical nerve head (ONH) is placed in the open hole region and visible under the microscope. If the ONH is placed near the local delivery channel, as shown in Figure 4.4c and 4.4d, the uneven structure near the ONH [106] will prevent intimate contact between the retina and polyimide and leave a gap in between. This gap leads to a poor sealing of the etched holes on the local delivery channel, causing most of the medium to be withdrawn from the top well. On the other hand, when the position of the ONH is farther away from the channel, as shown in Figure 4.4e and 4.4f, the local delivery channel is mainly filled with medium from the supply reservoir, which is labeled with blue food dye. This is because the tissue becomes flatter as the distance from the ONH increases, allowing for a better sealing of the etched holes on the local delivery channel. It is essential to carefully control the flow of the medium and avoid introducing bubbles to the local delivery channel, as the width of the channel is only about 200 µm. Any bubble introduced to the local delivery path may block the local delivery channel and stop the continuous flow of the media. If an attempt is made to remove the bubble by increasing the negative pressure to the channel, it may damage the tissue as the retina is fragile. To address these issues, a two to one port is used to switch between the Ames media with and without a highK+ concentration. This allows for more precise control over the flow of the medium and helps to avoid introducing bubbles to the local delivery channel.
Figure 4.4: (a) The optical image of theµpMEA device mounted on a metal holder under an inverted microscope. The red and silver tubes represent the perfusion and suction cannulas, respectively. The polyimide film is clamped to a zero-insertion-force connector, which is a lab-made printed circuit board adapted to an Intan chip to record the electrical activity of RGCs. (b) The schematic of the media flow (indicated by the black arrow) inside the local delivery channel when retina is placed on top of µpMEA device. (c) Optical image of the optical nerve head (ONH) located near the local delivery channel. The blue food dye has been mixed into the source media to highlight the media flow inside the local delivery channel. The uneven structure near the ONH leads to a poor sealing of the etched holes on the polyimide substrate, resulting in only partial media flow through the local delivery channel. (d) Corresponding fluorescent image in (c). The red dotted line depicts the boundary of the local delivery channel. (e) Optical image of the ONH located far away from the local delivery channel. In this case, the retina creates a good sealing to the etched holes on top of the local delivery channel, allowing the media to flow successfully underneath the retina. (f) Corresponding fluorescent image in (e). [8]
the local delivery channel is switched from Ames to Ames with additionalK+ (22 mM) with a duration of 90 s while the supply to the top well remains to be the baseline Ames media. During the electrophysiological measurements, the chemical stimulation paradigm was systematically repeated up to three times per retina to ensure the reliability and repro- ducibility of the observed responses. Previous research has shown that when RGCs are exposed to high potassium media, their firing rate initially increases due to high potassium induced depolarization, but then experiences a cessation due to voltage-gated channel reg- ulation. Interestingly, in our experiments it was observed that theK+ stimulated response was not only detected by electrodes located right on top of the delivery channel, but also by electrodes located outside of the delivery channel (Figure 4.5b and 4.5c). This suggests that highK+ media in the delivery channel can not only stimulate neurons on top of the delivery channel, but also those far away from the delivery channel.
Figure 4.6 presents the diverse responses detected by the electrodes located away from the delivery channel when exposed to 22 mMK+ media. Responses were categorized as strong, mild, or weak based on their similarity to a standard response to highK+media, as described before. A significant number of electrodes located outside of the delivery chan- nel detected strong firing responses, similar to those located inside the delivery channel, suggesting that RGCs can respond to highK+stimulation even without direct contact with the highK+ medium. We observed the spread of depolarization block in cells located on electrodes spanning the entire sensing region, and the response to highK+stimulation was not attenuated even at a distance of over 600 µm from the delivery channel. To ensure a robust comparison of the activity correlated between electrodes inside and outside of the delivery channel, we only considered the strong responses from electrodes outside of the delivery channel (Figure 4.6a). To measure the correlated activity, we tracked the time when firing ceased and resumed (Figure 4.7). A statistical analysis was conducted using data from seven recordings of distinct retinas, which revealed an average time delay of 10.3 seconds for the cessation of activity between two electrodes positioned 160µm apart
Figure 4.5: (a) Optical image of a retina placed on top of µpMEA device with chemical stimulation in the local delivery channel. The scale bar is 200µm. Number 1 and number 2 represent the electrode 1 located inside and electrodes 2 located outside of the local deliv- ery channel, respectively. Electrode 2 is 160µm away from the local delivery channel. (b) Spiking response of the electrode 1 and (c) Electrode 2 with the global flow of Ames media in the well and local highK+ media to the retina. The green window defines the period of flowing local highK+ media. (d) Spiking response of electrode 1 and (e) Electrode 2 with the global flow Ames media containing 100µM of CBX in the well and local highK+me- dia to the retina. (f) The number of electrodes located outside of the local delivery channel with correlated response under global Ames media and under CBX blocker media. [8]
inside and outside of the delivery channel, respectively. It was observed that the electrodes located outside of the local delivery channel displayed a shorter refractory period, on av- erage by 25.2 seconds, in comparison to the electrode situated inside the delivery channel.
This variation could be attributed to the constant exposure of RGCs located outside of the delivery channel to the baseline Ames’ medium, which requires some time to replace the highK+ media inside the delivery channel.
Figure 4.6: The responses recorded by electrodes located away from the delivery channel can be classified into three distinct categories: strong (a), mild (b), and weak (c) under the local 22 mM K+ stimulation. This categorization is based on the level of similarity ob- served in comparison to the standard activity elicited by highK+ stimulation, as illustrated in Figure 6c. [8]
As the retina comprises a network of interconnected neurons, when a portion of neurons are excited, signals from the stimulated neuron can propagate within the connected network and trigger response from other neurons. We posited that the intercellular communication facilitated by the fluidic isolation provided by our device (depicted in Figure 4.5a) played a crucial role in the correlated depolarization block triggered by highK+. To support our hypothesis, previous research has revealed that various neuronal and glial processes that govern firing and refractoriness can coordinate correlated activity [127; 128]. In the con- text of the retina, RGC networks have exhibited a substantial level of correlated activity, accounting for more than half of all RGC activity [129; 130]. We identified gap junctions, which oversee correlated firing in RGCs and K+ buffering among astrocytes [131; 132;
133], as a compelling candidate to investigate as the potential source of correlated depo- larization block. To verify our understanding of the connectivity of neurons in the retina,
Figure 4.7: Time analysis of the response to locally deliveredK+ stimulation, comparing the neuronal activity of electrodes located both inside and outside (160µm away from) of the delivery channel. The results indicate that the depolarization block occurs 12.5 seconds earlier and the reappearance of firing activity occurs 17 seconds later for the electrode inside the local delivery channel compared to the outside counterparts. The cessation and recovery of action potentials are depicted by the red and green lines, respectively, representing the electrodes located inside and outside the delivery channel. [8]
we introduced the gap junction blocker carbenoxolone (CBX) to the retina tissue. Previous research has shown that 50-100 µM of CBX can effectively eliminate electrical coupling through gap junctions in the retinal network [134]. First, we identified and counted the number of electrodes located outside of the local delivery channel showing correlated re- sponse with the electrodes on the delivery channel when retina is globally bathed with Ames media from the top but stimulated with local highK+ media in the delivery channel.
After this stimulation, the retina was recovered with spontaneous spiking in the bath of Ames media from both the global and local delivery paths. We then switched the global baseline Ames media to Ames media containing 100µM of CBX. To ensure that all neu- rons were fully exposed to the gap-junction blocker, the flow process lasted for 5 minutes before we introduced high K+ media from the local delivery channel. In this case, we observed that only the electrodes located on the local delivery channel showed the typical potassium-stimulated increased firing pattern, while the electrodes outside of the channel no longer showed correlated response (Figure 4.5d and 4.5e). After repeating the exper-
iment with multiple retinas, Figure 4.5f illustrates that there were 23 electrodes located outside of the local delivery channel that displayed correlated responses under the global bath of normal Ames media, while only 1 electrode located outside the delivery channel still showed correlated response when bathed with Ame’s media mixed with CBX gap-junction blocker.
Furthermore, we compare the waveforms between global and localK+stimulation. For global chemical stimulation, in which the stimulation reagents are applied to the retina from the top well, Figure 4.8a and 4.8b illustrates that the median spiking waveform of the action potential. Under the global highK+ stimulation, the waveform becomes wider compared to that under the global Ames bath [135]. However, when the chemical stimulus is applied through the local delivery channel, the median spiking waveform extracted from both the spontaneous spiking and stimulated spiking periods does not show significant difference (Figure 4.8c and 4.8d). The same observations also occurred with the exposure to the global CBX blocker media (Figure 4.8e and 4.8f). One possible explanation for this is that, during the local delivery of a chemical stimulus, only a few RGCs are believed to be in direct contact with the stimulant. Additionally, most components in the retina, including photoreceptors and bipolar cells, are still immersed in the global Ames or CBX blocker media. On the other hand, global chemical stimulations to the retina, with the whole retina exposed to a high K+ environment, may affect the signal sensed by the electrodes due to the response of upstream elements of RGC receptive fields, leading to an increased action potential half band width.