This is the text from a paper that I wrote as an undergrad about different factors that affect neural activity while doing experiments in Dr. Luis Gonzalez-Reyes‘s lab. The experimental process was fun, but we didn’t find anything interesting enough to publish.
Abstract— Abstract— Transient receptor potential vanilloid 3 (TRPV3) is a cation-permeable ion channel found abundantly in the hippocampus, which is the main epileptogenic region in the brain. Previous studies have looked at this channel in response to heat eliciting chemicals and also how the agonist, 2-aminoethoxydiphenyl borate (2-APB) activates and sensitizes the ion channel. However, in this study, we observed the role of TRPV3 channels in the hippocampus of mice brains utilizing an in vitro model. We used 4-aminopyridine (4-AP) to trigger the epileptic-like seizures and then increased the temperature and administered 2-APB in 20 minute episodes in hopes of increasing excitability within the granular and pyramidal cell layers. To our dismay, our results did not show us definitive evidence that increasing the temperature or adding an agonist increased excitability. Furthermore, while we had expected that the agonist would increase the excitability of the cells in terms of amplitude, frequency, and power of the peaks, we noticed instead that the amplitude decreased while the frequency increased. Because of this characteristic, we also calculated the power, but there was not enough statistical evidence to conclude a trend.
The hippocampus in mammals is known to be an important area of study for clinical epilepsy. While TRPV3 channels are abundant within the hippocampus, its primary function is still widely unknown. In this study, we test to see if activating these channels via 4-AP, temperature, and an agonist, 2-aminoethoxydiphenol borate (2-APB) lead to the excitability of the granular cell layer and the CA3 pyramidal cells. We hypothesized that adding each of these solutions in order would increase the excitability of the cells in respect to frequency and amplitude.
Mice were anesthetized via isoflurane inhalation and then decapitated. The brains were quickly removed and placed in sucrose-rich artificial cerebrospinal fluid (S-aCSF). Brains were sliced utilizing a vibrotome (VT1000S, Leica, Nusslock, Germany) while the tissue was bathed under 3-4 oC and oxygenated with S-aCSF consisting of (mM): sucrose 220, KCL 3, NaH2PO4 1.25, MgSO4 2, NaHCO3 26, CaCl2 2, and dextrose 10. The resulting slices were then placed in an oxygenated S-aCSF buffer consisting of (mM): NaCl 124, KCL 3.75, KH2PO4 1.25, MgSO4 2, NaHCO3 26, CaCl2 2, and dextrose 10 for 3 hours under room temperature.
Pipette and Electro-physiology rig Preparation:
To prepare the micropipettes, a capillary tube was pulled using a Sutter Instrument P-97 micropipette puller. These glass pipettes were then infused with 150 mM NaCl using a syringe and Pt electrodes were inserted.
A transversal brain slice was then transferred to interface-recording chamber (aCSF, 33±2oC, bubbled with 95% O2/5% CO2). The DG and CA3 cells were connected to Channels 1 and 2, respectively. Each population was stimulated with a pulse (100 uS, 50-350 uA, 0.05-0.1 Hz), and the extracellular field recordings were obtained.
The two populations of cells were put under three different scenarios. First, a solution of 100 uM 4-AP was administered to the cell layers to produce a spontaneous epileptiform activity, which was recorded for 20 minutes. Second, the temperature of the solution was increased to 40oC, and the cells were again recorded for another 20 minutes. Finally, the agonist 2-APB was added to measure for any additional excitability. This again was recorded for 20 minutes.
After the experiment had run to completion, we used LabChart to identify all of the peaks. Each peak was classified as any aberration in a data point more than 4 standard deviations above the baseline. Then each group was analyzed for 2 minute epochs to calculate the average amplitude and frequency of the peaks. The analyzed data was then exported to Excel to create graphs and charts for figures.
Figure 2: The pictures above are screen shots of our raw data. The red graphs correspond to channel 1, which was DG, and blue corresponds to CA3. The top two graphs are from our control, the middle two graphs are from the heated test, and the bottom two graphs are from the agonist.
CA3 Cell Layer Attributes
The cells in this area of the hippocampus changed their action potentials in terms of amplitude, frequency, and power (Figure 1). For amplitude, we saw that as the solution was increased to 40 oC, there was an increase in amplitude from 0.065V to 0.112V (n=10, ANOVA, p=0.010, Fig. 1). However, as the agonist was added, the peak amplitude decreased to 0.051V (n=10, ANOVA, p=~0, Fig. 1). There was no conclusion that could be made about the 4-AP solution peaks and the 2-APB peaks because the p-value was 0.288, which is greater than our α value of 0.05 (Table 1).
For the frequency of peaks, we could not determine a relationship between when the solution increased in temperature because the p-value was 0.854 (Table 1). However, we noticed that when the agonist was added, the frequency increased from 0.696 Hz to 0.892 Hz (n=10, ANOVA, p=0.0138, Fig. 1). Furthermore, the change from the original 4-AP solution to the 2-APB solution increased the frequency from 0.713 Hz to 0.892 Hz (n=10, ANOVA, p=0.0007, Fig. 1).
Lastly the power of the peaks was also calculated to determine a more reliable measure for determining excitability of the cells. The power from the 4-AP solution to the 40 oC solution increased from 0.0462 V/sec. to 0.0760 V/sec. (n=10, ANOVA, p=0.03, Fig. 1). Furthermore, the power decreased from 0.0760 V/sec. to 0.0468 V/sec when 2-APB was added (n=10, ANOVA, p=0.017, Fig. 1).
DG Cell Layer Attributes
Again, the cells in the granular cell region were influenced by the different types of solutions utilized, and this was measured by amplitude, frequency, and the power of the action potentials. In terms of the amplitude of the peaks, there was a decrease from 0.021 V to 0.0182 V (n=10, ANOVA, p=0.03, Fig. 1). Between the 4-AP solution and the 2-APB added solution, there was an overall decrease in amplitude from 0.0334 V to 0.0182 V (n=10, ANOVA, p=0.03, Fig. 1).
In terms of frequency of the spikes, there were no note-worthy measurements because all of the ANOVA tests provided us with p-values greater than our confidence level (Table 2).
Finally, the power of the peaks had two relevant measurements. First, the increase in temperature decreased the power from 2.482 V/sec. to 1.677 V/sec. (n=10, ANOVA, p=0.0022, Fig. 1). Second, the addition of the agonist also decreased power from 1.677 to 1.58 V/sec. (n=10, ANOVA, p=0.015, Fig. 1)
TRPV3 channels are abundant within the hippocampus. Thus we studied how these channels lead to the excitability of DG and CA3 cells when placed in various conditions. In general, we found that the agonist 2-APB increased the frequency of neuron firing, and decreased the amplitude of these firings. The significant differences are diagrammed in the results section.
In Xu et al., TRP channels located in the skin, tongue, and nose were studied in how they respond to carvacrol, thymol, and eugenol, which are major components in plants. By utilizing immuno-staining, they found that TRPV3 is sensitized and activated by these compounds (Xu, Delling, Jun, & Clapham, 2006). Similarly, our experiment also looked at the heat-sensitive TRPV3 channels. However, instead of being located on the epithelial layers of the skin, tongue, and nose, the channels were in abundance in the hippocampus. Furthermore, Xu et al. found that when the channels were exposed to the muscarinic agonist carbachol, the effect of the compounds enhanced. Our experiment found that the agonist generally led to a significant increase in activity of the channels with the single exception being between 4AP and 2-APB in our CA3 measurements.
In Chung et al., TRPV3 activity was measured via whole cell patch clamping. The study found that TRPV3 channels were only excited in conditions of heat with the agonist 2-APB. When only the agonist was administered, no significant change was noted (Chung, Lee, Mizuno, Suzuki, & Caterina, 2004). Similar to this study, we looked at the heat-gated TRPV3 channel. However, instead of looking at single cells with this channel, we studied the DG and CA3 cell layers in the hippocampus. Our experiment found that the amplitude was significantly different between the heated test and the 2-APB test in both tissue layers, and the power of the peaks.
Hence, from our experiment, we do not have definitive enough information to verify our hypothesis. With the exception of the previously noted 4-AP/heat power test, and the heat/4-AP amplitude test, we never actually had significant differences in both of our cell layers. Specifically, the hypothesis that increased agonist concentration would increase neuron activity is not confirmed. Most surprising is that an increase in temperature did not always cause significantly more frequent firing or larger amplitude spikes because the TRPV3 channels are known to the heat-sensitive (Xu, Delling, Jun, & Clapham, 2006). One reason that temperature did not have a profound effect could have been that we did not test at 40oC. In order to make sure that the temperature is at high enough, we could in a future test have a small temperature inserted next to the slice. Furthermore, we were also concerned with the data analysis method. Because we had broken up the data into two minute chunks, we obtained a result where artificial smoothing had occurred, and the standard deviations were very different depending on how we broke up the data into the two minute groups. Hence, this experiment could be tested in the future in a more controlled environment at which point the data may result in a different conclusion. If this were the case, we could move on to how the channels respond at the single cell level by utilizing patch-clamping.
Chung, M.-K., Lee, H., Mizuno, A., Suzuki, M., & Caterina, M. J. (2004). 2-Aminoethoxydiphenyl Borate Activates and Sensitizes the Heat-Gated Ion Channel TRPV3. The Journal of Neuroscience , 24 (22), 5177-5182.
Xu, H., Delling, M., Jun, J. C., & Clapham, D. E. (2006). Oregano, thyme and clove-derived flavors and skin sensitizers activate specific TRP channels. Nature Neuroscience , 9 (5), 628-635.