“Now they help, now they don’t!” - A mysterious story about how L-type calcium channels help dopamine be released…but only sometimes!
An accessible summary of Brimblecombe et al (2023) by lead author Dr Katherine Brimblecombe
Please note that underlined words are fully described in the accompanying glossary
The big picture
Spot the difference!
We know that people with Parkinson’s don’t release enough of the chemical “dopamine”. Our speciality is measuring dopamine, and understanding the rules and machinery that controls how dopamine is normally released, with the aim of finding out what goes wrong in Parkinson’s.
My approach to investigating the causes of Parkinson’s disease is a game of “spot the difference”: We know that dopamine-producing brain cells die in Parkinson’s, but not all dopamine-brain cells are the same. One group of dopamine-brain cells are especially at risk in Parkinson’s, whereas their neighbours are relatively spared. I look at what is different between these groups of dopamine-brain cells. Another approach is to take something that is known to be different between groups of people with and without Parkinson’s and see how it affects dopamine cells (“Spot the difference Cartoon”).
A bit of background
Calcium is needed for nearly every process that keeps cells alive and is the trigger for cells to send messages to their neighbours. Understanding how calcium affects the way brain cells live and talk to each other is extremely tricky. One of the ways that calcium can get into brain cells is via the “L-type calcium channel”. One difference that has previously been found between brain cells that die in Parkinson’s and those that don’t, is that vulnerable brain cells use “L-type calcium channels”.
Why do cells need calcium? Imagine brain cells communicate by email: calcium is the “send button”. Without calcium coming into the cells, no messages get sent. Calcium isn’t just the trigger to send messages to other cells, it also is a way the cell sends messages to itself. Therefore, calcium can signal how much energy it needs, which parts of it needs repairing and how loud it’s outgoing messages need to be (a bit like how emailed meeting invites will often sync to your online-calendar). When it works well, this system is a great way to balance its internal needs to what the system is asking of it. When it goes wrong, the calcium messages get in a muddle, and the cell can’t balance it’s messaging system and maintain its own health. To stop this from happening, how calcium comes in and where it is stored must be tightly controlled. Imagine the carnage it would create if your emails were sent at random times, or to random people! I hope you can see that calcium is completely necessary for sending messages, but you can’t have it coming and going all over the place without causing huge amounts of damage! One of the ways calcium comes into a cell is by the “L-type calcium channels”. But how this calcium is used and the way this route is gated is not well understood.
We previously found that stopping “L-type calcium channels” with a drug called isradipine decreased the amount of dopamine released from the brain cells that dies in Parkinson’s. When we measured dopamine release from cells that don’t in Parkinson’s, “isradipine” didn’t change how much dopamine came out, what a mystery!...But we only looked in male mice (doi:10.1113/jphysiol.2014.285890).
What we tried to find out
One of the mysteries surrounding “L-type calcium channels” is that they appear to be made by both the dopamine-brain cells that die and those that survive in Parkinson’s, but only appear to act in the vulnerable cells, why would this be?
Calcium comes in through L-type calcium channels and helps dopamine be released… but not always
The tools we used
My research is interested in what controls dopamine release, and in particular how this differs from brain cells that die in Parkinson’s and cells that are resistant (spot the difference!). Our speciality is measuring how much dopamine comes out of mouse-brain when we stimulate with small bursts of electricity. We measure dopamine using a technique called “fast-scan cyclic voltammetry (FCV)”. For this paper, we placed our electrodes in the area we were interested in and measured how much dopamine was released (we call this the “control condition”); we then added a drug that stops “L-type calcium channel” from opening, this drug is called isradipine and tested if it changed the amount of dopamine that’s released (“Testing “L-type calcium channels” cartoon”). If Isradipine decreases the peak amount of dopamine that comes out after electrical stimulation, we say that “L-type calcium channels” control dopamine release in that situation. In each brain slice, we measure the amount of dopamine that comes out when we stimulate with electricity in “control conditions”, we then add “isradipine” and measure dopamine again. We take an average of the measurements of dopamine in each condition.
We repeated these experiments in at least three brain slices from three different mice.
A) we take a thin (300 µm) slice of mouse brain and place our electrodes in “the striatum”. We connect our electrodes to a computer so we can give small precise stimulations and measure how much dopamine comes out. We take a measurement every 0.125 s and plot how much dopamine there is over time. When we stimulate dopamine quickly comes out, reaches a peak and then gradually returns to baseline in a curve. We call these lines “dopamine transients”
B) We can stop L-type calcium channels from working with a drug called isradipine.
C) We compare how much dopamine is released before and after adding isradipine
What we found
1) Do factors that increase people’s risk of getting Parkinson’s affect if “L-type calcium channels” control dopamine release?
SEX! (now I’ve got your attention…) after age, being male is the biggest risk factor for developing Parkinson’s. We don’t know the reasons for this and to be honest there has been surprisingly little research to find this out! Possible reasons could be genetic, hormonal, environmental or even that women present differently with Parkinson’s and so are under or mis-diagnosed. I will not be answering the question of “why men are more at risk of getting Parkinson’s than women” here, but hope by including data from both male and female mice, we can gain insights that will help all people with Parkinson’s.
Scientists haven’t published as many direct comparisons between male and female mice as you might expect. It is only recently that most researchers started to use female mice at all. Here we found that the peak amount of dopamine released was the same in both males and female mice, but that the dopamine is taken away more quickly in females (Picture 1 A). We were surprised when we saw that dopamine is taken away was slightly faster in female mice than males. The differences are small and we still don’t know how much these differences would change dopamine signalling as a whole.
As we have shown previously, in male mice, isradipine decreased how much dopamine came out after we electrically stimulated the brain. But this time, we also looked in female mice and found that in the female mice, isradipine had no effect on the amount of dopamine released (picture 1B).
Picture 1: A) The wavelike lines are called “Dopamine transients”. They show the amount of dopamine we are measuring before, during and after we use an electric pulse to make the mouse brain release dopamine. The black arrow is when we give an electric pulse. You can see a flat baseline (purple dashed line) before we give a pulse that goes up quickly and then slowly goes back to the baseline in an arc. We compare the highest points of these lines to see how much dopamine is released and the steepness of the curved line going back to baseline to see how quickly the dopamine is taken away. We compare these two measurements in male mice (Black lines) and the female mice (Grey lines). We measure the amount of dopamine in different mice to get an “average”. The shaded bit around the lines shows “the error”. The graph with lots of little circles shows the peak amount of dopamine for each measurement we take. We can measure how steep the curved line going back to the baseline is using an “exponential decay curve”. When the curve is very steep and the line goes back to baseline very quickly you have a big k number, when the curve is shallow, and it takes a long time to go back to baseline you have a small k number. Female mice have steeper curves and go back to baseline faster than male mice. We try to show our measurements in lots of different ways, especially at the start so it’s clear (especially to other scientists) how we got our measurements. The *s show that the difference in dopamine amounts we measure are “statistically significant”.
B) “Dopamine transients” from male and female mice, before (black line) and after we add isradipine (red line). You can see that in male mice, isradipine makes the peak of the dopamine transient smaller, showing less dopamine has come out. In the female mice the transients over-lap showing that isradipine hasn’t changed the amount of dopamine coming out. I’ve also drawn a little cartoon to remind you that isradipine stops “L-type calcium channels” from opening, meaning no calcium can come through them. When isradipine decreases the amount of dopamine being released we think this shows that “L-type calcium channels” were helping dopamine be released.
Genetic risk factors: We also know that there are many genetic risk factors for developing Parkinson’s. Here we looked at two genetic risk factors: “α-synuclein” (pronounced alpha-synuclein) and “GBA”. We know that having a lot of α-synuclein make it more likely that people will get Parkinson’s [Disclaimer: Why α-synuclein is a risk factor in Parkinson’s is still hotly debated and probably depends on a lot of factors, saying people with a lot of α-synuclein are at risk of Parkinson’s is probably an oversimplification]. Here we found that in mice with a lot of “α-synuclein”, “isradipine” decreases dopamine release in male but not female mice (i.e the same as in our normal “wild-type” mice) (Picture 2A). However, in mice with no α-synuclein, isradipine had no effect in male mice either (Picture 2B).
Next, we compared the effect of “isradipine” in female mice that have normal levels of “GBA” with those that have less “GBA”. Having less GBA is a risk factor of Parkinson’s [Disclaimer: this is also an over simplification]. We found that in female mice with half the amount of GBA, isradipine decreases dopamine release. By looking at how factors that make people more at risk of getting Parkinson’s we found that the effect of isradipine on dopamine release is variable. The three risk factors we have looked at here (male sex, α-synuclein and less-GBA), increase the chance of isradipine being able to decrease dopamine release.
Picture 2: A) “Dopamine transients” from male and female mice with lots of α-synuclein, before (black line) and after we add isradipine (red line). You can see that in male mice, isradipine makes the peak of the dopamine transient smaller, showing less dopamine has come out. In the female mice the transients over-lap showing that isradipine hasn’t changed the amount of dopamine coming out. You can compare this to picture 1B and see that both in normal mice and mice with lots of α-synuclein isradipine decreases the amount of dopamine released in males but has no effect in female mice.
B) “Dopamine transients” from male mice that have no α-synuclein before (black line) and after we add isradipine (red line). You can see that in male mice with no α-synuclein, the transients over-lap showing that isradipine hasn’t changed the amount of dopamine coming out. You can compare this to picture 1B and 2A, and see that in order for “L-type calcium channels” to help dopamine be released in male mice, there needs to be at least some α-synuclein.
C) “Dopamine transients” from female mice that have either normal or half the amount of “GBA” before (black line) and after we add isradipine (red line). You can see that female mice with normal amounts of “GBA”, the transients over-lap showing that isradipine hasn’t changed the amount of dopamine coming out. Whereas in the female mice with half the amount of “GBA” isradipine has made the peak amount of dopamine go down. You can compare this to picture 1B, and see that in female mice “GBA” is stopping “L-type calcium channels” from helping dopamine to be released.
2) Do factors that differ between cells that die in Parkinson’s and those that don’t, affect if “L-type calcium channels” control dopamine release?
The first is a factor called “calb1”. It is basically a calcium sponge (although we are finding it probably has more complicated functions, but for now think of it as a calcium sponge that cleans up any calcium spills after a message has been sent). It’s been known for ages that cells that don’t have much calb1 die in Parkinson’s. We know that the dopamine-cells that survive in Parkinson’s have lots of calb1, and “L-type calcium channels” don’t help dopamine release. Here we found that isradipine can decrease dopamine, when we take away calb1 (Picture 3A).
B) When DAT is stopped working with cocaine, isradipine no longer decreases dopamine release. When DAT is boosted in mice without α-synuclein, isradipine now decreases dopamine release
C) When D2-receptors are stopped from working with a drug, in the area of the brain resistant to Parkinson’s, isradipine decreases dopamine release. Stopping D2-receptors in the sensitive area of the brain, doesn’t change how much isradipine decrease dopamine release.
The next factor we tested was dopamine transporter (DAT). (see previous post for more info about how DAT works). Cells that die in Parkinson’s have more DAT. If we stop the DAT from working with “cocaine”, isradipine doesn’t decrease release anymore (Picture 3B). In addition to stopping “L-type calcium channels” from working by stopping the DAT, we can also make “L-type channels work by boosting the DAT. We boost the DAT by adding cholesterol to brain slices from mice with no α-synuclein (isradipine doesn’t normally change dopamine release in these mice, see Picture 2B). We don’t really understand how DAT could be doing this. (see box below, if you are interested, but don’t worry if it’s a bridge too far!)
This is our best guess at the moment for why L-type calcium channels stop working when we stop the DAT: One thing we know DAT does, is make it easier for messages to be sent (called depolarising the membrane), L-type calcium channels only open when the cell is sending messages, so we think that DAT is turning on “L-type calcium channels”. Therefore, if DAT is stopped, it’s harder for messages to be sent and “L-type calcium channels” aren’t being turned on. Another confusing thing is that isradipine doesn’t like to stick to “L-type calcium channels” when the cell can’t send messages (called hyper-polarised membrane, i.e the opposite of what DAT does), so it’s possible that when we see an effect of isradipine its not telling us what the “L-type calcium channel” is doing at all, rather just if isradipine can stick to it. To test this, we used a different drug called CP8 (made by us by our friends in the chemistry department!) which, can stick to the “L-type channel” whether the cell wants to send messages or not. CP8 did the same thing as isradipine, so we think when isradipine has an effect it’s telling us about what “L-type channels” are doing, rather than how likely the cell is to send a message (but we haven’t completely ruled it out because these types of experiments only give clues not definite answers)
The last factor is called a “D2-receptor”, these “receptors” are how the dopamine message is read by cells. Lots of cells have “D2-receptors” including dopamine cells themselves (so they can remember the messages they sent). We found that if we stop “D2-receptors” from working, then isradipine can decrease dopamine release from dopamine cells that survive in Parkinson’s. To be honest, this is a very confusing part of the story, and we included it so other researchers can help us understand why and how this could work. We had thought that the D2-receptors would explain how DAT and isradipine talks to each other, but it didn’t! But we wanted to share it with other researchers even though we can’t quite make sense of it.
Finally, for every experiment we did, we told a computer, if isradipine decreased dopamine release or not, and all of the information about that experiment (i.e. the sex of the animal, the type of mouse, which type of dopamine-cell we were measuring dopamine release from, and any other drugs we had included). We then asked the computer to sort the data based on whether isradipine decreased dopamine release to see if there was a pattern, this is called a “decision-tree”. The computer did find a pattern, and it was the same pattern we saw when we looked at the data piece-by-piece, so this gave us confidence that the way we sorted the data matters. We took all this information and put it into a little cartoon showing a seesaw with “L-type Calcium channels” (LTCC) as the pivot point, showing how their role is not fixed and can be tipped based on many factors, which broadly split into risk or protective factors against Parkinson’s.
Picture 4: A) A decision tree sorting all of the experiments into whether isradipine decreased dopamine release or not. B) Cartoon showing how protective factors against Parkinson’s limit “L-type calcium channels” (LTCC) from controlling dopamine release, and risk factors promote them.
What does it all mean?
We think it’s important to share that the effect of isradipine on dopamine release is not always the same, and changes depending on how you do your experiment. This can help the science be more reproducible. The reviewers agreed, but encouraged us to think how these findings can help with understanding Parkinson’s. So here are some of our thoughts!
We still don’t understand how “L-type calcium channels” help send dopamine messages. The way they let calcium in isn’t thought to be in the right time or place to actually “press the send button” so it’s likely that the calcium coming in through “L-type calcium channels” is helping dopamine cells coordinate its internal signals: balancing its energy needs with the incoming signals and out-going messages (it’s a hectic life being a brain cell!). But we need to do a lot more work to understand the specifics. Other researchers have found that “L-type calcium channels” are important for it getting enough energy to send messages. We don’t have any information to either agree or disagree with it, but we do think it’s something to test in the future.
In the future we want to know, if the conditions when isradipine decreases release (I,e, associated with risk factors of Parkinson’s), the cells are tired and hungry and need the L-type channels to keep going (like a marathon runner eating jelly babies to keep going); or if the “L-type calcium channels” are making the cells tired and hungry (like someone with an unbalanced diet, relying on a diet of sweets and crisps, which makes them feel unwell and lethargic). i.e. is the “isradipine effect” the canary in the coalmine: signalling which cells are exhausted, or are the “L-type calcium channels” causing the damage.
This is important, because if we try to protect cells buy stopping “L-type calcium channels” with isradipine, but they are just signalling they need extra energy, it won’t actually help the cells (and may even hurt them), and they will continue to suffer in silence. If we can understand what dopamine cells are using the calcium from “L-type calcium channels” for, we can try to bypass it to keep them healthy
We hope by sharing these findings it will help the scientific community consider if “L-type calcium channels” are causing vulnerability to Parkinson’s or acting as signals of being vulnerable. Lastly, we hope that by using both sexes of mice we are moving beyond box-ticking, towards mechanistic insights for Parkinson’s that can benefit the broader Parkinson’s community.
What the reviewers said
We submitted this paper to European journal of neuroscience (EJN) as part of their special edition following the (rescheduled) Dopamine 2022 conference in Montreal. I presented much of this data at the conference and therefore wanted to contribute to this edition. The reviewers were very positive and we were grateful for their enthusiasm. The reviewers wanted us to expand on what we think the data mean for People with Parkinson’s and we tried to follow through with this. The reviewers also asked us to expand the GBA dataset. We agree that sharing more information about GBA is important, but asked them to be patient and wait for us to publish a separate paper dedicated to this question in the future. The reviewers also asked us to clarify and justify how we had analysed some of the data, in particular the number of times we repeated each experiment (known as a replicate). So, we explained our decision making and did some additional analyses to ensure our approach clear and consistent. When working with mice, we need to balance using as few mice as possible whilst producing robust and repeatable dataset. We hope we have struck the right balance. The peer-reviewed manuscript is now available: http://doi.org/10.1111/ejn.16180
Who paid for it
This project wasn’t specifically funded as a project grant, and I’ve been collecting bits and pieces of data alongside many wonderful students for many years. Some of the data was collected during my PhD, funded by Parkinson’s UK. Since then I’ve been funded by MRC, Parkinson’s UK, John Fell fund and most recently by Wellcome trust and ASAP (Aligning Science Across Parkinson’s through the Michael J Fox foundation), my travel to the Dopamine 2022 conference was funded by Guarantors of Brain (https://guarantorsofbrain.org/). We couldn’t buy the drug “CP8”, so we asked our colleagues Prof. Angela Russell and Dr Carole Bataille in the chemistry department to make it for us. We were so grateful for their help, one of the best things about being at the University of Oxford, is the amount of incredibly talented people who are just an email away!