Rat Brains
As seen in Figure 1, imaging of rat brains is commonly used to view the effects of lead and the figure is one example of what the imaging could look like. For lead experiments, many researchers use rats as their subjects. This is because rats are small, easy to take care of, and have a short life span.55 Rat brains can be comparable to human brains because the brains of rats and humans are anatomically similar. In addition, rat and human brains both express high levels of 5-HT6 mRNA.56 Rats are preferred over mice in experiments dealing with cardiovascular research, behavioral studies, and toxicology.55 In addition, rat brains are larger, so there is better spatial resolution. Another advantage of using rats is they can be trained to sit during imaging procedures. This means that rats do not need anesthesia,56 which is useful because anesthesia can interfere with brain activity.56 Cells in the brain can be analyzed for the density of receptors and the effects of lead on different receptors.33 Experimenters also test the viability of the brain cells in the rats after exposure to lead.33 Animals are used rather than humans due to ethical guidelines to prevent harm to humans.
Nuclear Magnetic Resonance (NMR) Spectroscopy
NMR Spectroscopy is a method that can be used to detect lead binding to domains to which it does not usually bind. Two such domains are the C2 domains in Synaptotagmin I (See Lead and Calcium for more information about Synaptotagmin I).13 Briefly, NMR is a spectroscopy method that provides insight into structure by reporting on the electronic environments and quantity of specific atoms (typically protons, Carbon-13, or Nitrogen-15) in a species. Chemical shifts provide information about functional groups, and coupling constants and splitting provides information about which atoms (e.g. protons) neighbor each other.
Two techniques of NMR are Heteronuclear Single-Quantum Correlation Spectroscopy and ZZ exchange NMR spectroscopy. Both of which are explained further in this page. The study High affinity interactions of Pb2+ with Synaptotagmin I by Katti et al. utilize both to analyze and expand their data on leads high affinity interactions.
Heteronuclear Single-Quantum Correlation Spectroscopy (HSQC) is an NMR technique used to correlate between two different types of nuclei.57 Typically, it uses 1H with 13C or 15N. 1H-15N HSQC can be used to monitor lead binding to domains.57 In Katti et al., the 1H-15N HSQC monitored Pb2+ binding to [Uridine-15N] enriched Synaptotagmin I C2 domains.13 Lead at different concentrations was tested and a spectrum was generated. This spectrum was was analyzed to obtain Pb2+ binding curves. Figure 1 shows an example of what the binding curves would look like when determining ligand binding from HSQC NMR. Figure 1 shows an example of 15N-1H HSQC NMR-detected binding curves generated in experiments.
NMR Spectroscopy can also be used to detect mixed species as well. For example, in Katti et al. it was used to detect a mixture of Pb2+/Ca2+.13 In this case, the binding sites were equilibrated with lead to sufficiently populate site 1 with lead which previously was shown to be the site lead binds to leaving two others vacant. 13 Then, aliquots of calcium solution were added to the samples to observe how the spectrum changes and if there were any noticeable differences in peak sizes and placements.13 This was to detect lead inhibition on calcium binding in site 1 as well as the vacant sites of 2 and 3. This can also be done with other mixed species pertaining to lead, such as Pb2+/Zn2+.
In an experiment, information on binding can be expanded using ZZ exchange NMR spectroscopy. ZZ exchange NMR is a specific 2D NMR experiment in which the kinetics of the interconversions between molecular states are measured.58,59 In regard to studying lead, this experiment allows researchers to find the kinetic parameters of lead binding to the sites of interest. This technique is typically repeated at various temperatures. Figure 2 displays an example of ZZ exchange NMR and how it measures the binding behavior between molecular states. In Katti et al., the technique was used specifically to measure the kinetic parameters of lead binding to [U-15N] enriched Synaptotagmin I C2 domains.13
Other Methods
(These will not be elaborated on, but are useful to answer the question)
- Assays
- Phospholipid binding assays
- Terbium metal binding assays
- Protease protection assays
- Comet assays
- Radiolabeling assays
- Cell viability assays
- Absorption Spectrometry
- Zebrafish brains
- Immunofluorescence
- Electron spin resonance spin trapping (to visualize free radicals)
- Immunocytochemical staining
- X-Ray Crystallography
- Cryostat Microtome
Is there a reason you selected these 2 methods in particular? If you don’t that’s fine but maybe a small paragraph at the beginning explaining why you chose to focus on these techniques rather than the others would be nice. You could also have a brief summary how other techniques are used if you want to go a little further into detail but if you think it would become oversaturated with information don’t worry about it. As for the content of the page, I don’t think you need to mention the last sentence about why rats are used instead of humans and maybe go a bit more into the distinction of why rats are chosen over mice.
I’m a little confused by your first figure and the information leading up to the first figure. The paragraph starts with you talking about Heteronuclear Single-Quantum Correlation Spectroscopy (HSQC). Is this another form of NMR? I know it’s in the NMR section but maybe clarify to the reader that this is also an NMR technique. In the same paragraph, for figure 1, you stated that the image was ligand binding from NMR. Is the image of ligand binding from HSQC or just normal NMR? Also for figure 1, were there any relevant bindings curves in the literature for lead or calcium ligand binding instead of lactose? I feel like it would be more effective to show the reader the relevant curves than to leave things to the imagination. The same follows with your figure showing ZZ exchange NMR spectroscopy. Also, this may be my personal stylistic preference, but the logic of the page may flow better if you talk about the mice before talking about the spectroscopy techniques. Other than that, I thought the webpage was well-written :).
I agree with Bob’s comment that it’s unclear whether HSQC is another form or sub-technique of NMR; a sentence clarifying exactly how it relates to NMR could be helpful. It may also be helpful to include a sentence briefly introducing the Katti et. al study, as it is mentioned multiple times but never formally introduced (just a brief statement that they were looking at lead binding and that they used these techniques to do so). I liked the description of why rats are specifically used over mice for the imaging experiments; did not expect for there to be so many differences. A sentence clarifying that the technique being used on the rats is some sort of brain imaging and stating that figure 3 is what this imaging could look like would tie this section together nicely.