#chronic ritalin

[tymkrs]

Ritalin and Dopaminergic System Part 6

Following article courtesy of @lmcomie:

http://www.plosone.org/article/fetchObject.action?uri=info%3Adoi%2F10.1371%2Fjournal.pone.0033693&representation=PDF

Sadasivan, S., Pond, B.B., Pani, A.K., Qu, C., Jiao, Y., & Smeyne R.J. (2012) Methylphenidate Exposure Induces Dopamine Neuron Loss and Activation of Microglia in the Basal Ganglia of Mice. PLOSONE, 7(3).

Last post about this article!  I finally found some time to go through the materials and methods.  So St. Jude Children's Research Hospital was where this research was done.  The mice were 3 weeks old and maintained on a 12h light/dark cycle (this is normal).  Starting when they were 28 days (this is about young adulthood/just off weaning), they were given injectsion of saline, 1mg/kg or 10mg/kg Ritalin at 5pm - 1 hour before their active nocturnal phase at 6pm.

Doses were chosen sbased on previous studies in rodents suggesting that Ritalin doses of < 5mg/kg mirror those that are used in clinical practice, as opposed to recreational and narcoleptic use = dose of 10mg/kg.

Ritalin injections were given 5 days a week and at the end of the 12 weeks, the animals were allowed a washout period of a week.

After the week of washout, the mice were anesthetized and euthanized.  The method they describe is fairly common for brain analysis.  And then the brain was sliced to 10um thick for analysis.  I can't tell if they had to count neurons by eye or not, but they mentioned using a "optical fractionator method" - it may be some sort of estimating tool developed with a specific microscope in mind.

For MPTP treatment, the final concentration was 5mg/ml and each animal was given 4 injections of 20 mg/kg MPTP - one every 2 hours for 8 hours.  All mice then stayed around for a week after the injections, before euthanization.

The rest of the article describes the methods they used for measuring mRNA and expression signals - unfortunately not my forte - but still a well documented method in many different studies.

I think one thing to note is that the n levels (number of subjects) that they tested throughout these experiments is relatively low compared to how many mice are usually put through an experiment.  The n numbers are anywhere from 3 to 8.  This is not very high.  IE. They're using the results from 3 mice to come to this conclusion.  The research would have been much stronger had there been at least 10 mice per category.  The logistics, however, would have been difficult.  (Why? Cuz mice don't like injections anymore than humans do)

@atdiy/@tymkrs

[tymkrs]

Ritalin and Dopaminergic System Part 5

Ritalin and Dopaminergic System Part 5

Following article courtesy of @lmcomie:

http://www.plosone.org/article/fetchObject.action?uri=info%3Adoi%2F10.1371%2Fjournal.pone.0033693&representation=PDF

Sadasivan, S., Pond, B.B., Pani, A.K., Qu, C., Jiao, Y., & Smeyne R.J. (2012) Methylphenidate Exposure Induces Dopamine Neuron Loss and Activation of Microglia in the Basal Ganglia of Mice. PLOSONE, 7(3).

The discussion section is like the summary section with a "why did we get the results we got" added in.  So if a lot of this looks familiar, it's probably because it is!

Alright, so they do a study looking at the effects of an ADHD and Narcolepsy dose of Ritalin.  They found that giving both doses chronically makes dopaminergic neurons in the substantia nigra, a location in the brain involved in reward, addiction, and Parkinson's, more susceptible to further stress.  And it does it by increasing the number of inflammatory factors that are activated/created and decreasing the factors that are usually responsible for creating new neurons, more dopamine, etc.

Specifically, they used a 3 month Ritalin schedule that spans the developmental period in mice and corresponds to the pre-adolescent through young adult period in humans, during which Ritalin's usually used.

Usually Ritalin works by increasing the amount of dopamine and norepinephrine in the brain by blocking the proteins that take back dopamine and norepi once they've finished their actions.  In the study, they saw that there was a significant increase in dopamine levels at the ADHD dose, one that was not seen at the narcolepsy dose.  Other small doses that have been studied have also seen this increase in dopamine levels.  

The lack of change in dopamine at the higher level may be because of chronic dosing of the drug, or maybe some sort of compensatory change in the production of dopamine due to there being fewer neurons.  So they measured the ratio of dopamine to dopaminergic neurons.  Interestingly, when examined as a ratio, both the 1 and 10mg/kg doses showed a significant increase in the dopamine:neuron ratios - 150% in 1mg/kg and a 132% increase in 10mg/kg Ritalin.  This suggests that both doses increase dopamine levels, not just that of 1 mg/kg.

Of course, increased extracellular dopamine may be a problem.  Oxidation of dopamine can produce a whole bunch of superoxides and in turn radicals.  Neurotoxins abound!  And as mentioned before, they hypothesized that chronic Ritalin would cause the neurons to be more sensitive to a later stress.  So they gave MPTP, an agent that is known to induce oxidative stress (one that normally does not induce any stress on the particular strain of mouse they had).  They found that chronic exposure to both doses increased the sensitivity of the neurons to oxidative stress - based on the fact that both doses lost neurons when given Ritalin and MPTP as compared to saline.

They also found that there was a significant increase in Ritalin-induced microglia - so they think that perhaps, an increase in radical formation from increased dopamine levels + a neuroinflammatory response (increase in microglia) increases the sensitivity of the dopamine neurons to a later oxidative challenge.

They wanted to see if there were any specific explanations for why the neurons were more sensitive to oxidative stress after having been given Ritalin.  Through genetic analysis, they saw that there were a number of genes that changed their level of expression after both 1 and 10mg/kg doses.  These were mostly related to inflammation and cell damage and repair pathways.  They found a decrease in expression of genes involved in dopamine synthesis and handling - again after both doses of Ritalin.

They found that short term exposure to higher doses of Ritalin increased the expression of inflammatory genes in the striatum.  Surprisingly they did not find an increase in inflammatory gene expression after chronic administration of Ritalin, though there was still an increase in activated microglia.  This suggests that sometime during the course of the chronic Ritalin, there may be some sort of repression of inflammatory gene expression.

Unknown if the gene repression after chronic treatment with Ritalin is permanent or if it can be later re-induced.  If this is the case, then the activated microglia may potentially play a modulatory role in inducing oxidative stress.  Or perhaps the microglia that are activated are not able to return to their pre-inflamed state.

So all together, this study suggests that chronic Ritalin in mice results in a reduced expression of neurotrophic factors (creation/healing), increased inflammation, and a loss of dopamine neurons.

Tomorrow, the methods! (Arguably one of the most important sections)

@atdiy/@tymkrs

[tymkrs]

Ritalin and Dopaminergic System Part 4

Following article courtesy of @lmcomie:

http://www.plosone.org/article/fetchObject.action?uri=info%3Adoi%2F10.1371%2Fjournal.pone.0033693&representation=PDF

Sadasivan, S., Pond, B.B., Pani, A.K., Qu, C., Jiao, Y., & Smeyne R.J. (2012) Methylphenidate Exposure Induces Dopamine Neuron Loss and Activation of Microglia in the Basal Ganglia of Mice. PLOSONE, 7(3).

The wording keeps getting more and more jargony! Please be sure to check out parts 1, 2, and 3! 

Alterations in Gene Expression following Acute and Chronic MPH Exposure in SN:

They wanted to see whether there were any changes to gene expression and see what the genes were for.  They found that expression of 115 genes had changed between saline and the 1mg/kg Ritalin dose and expression of 54 genes had changed between saline and the 10mg/kg Ritalin dose.  Of these genes, 23 were expressing differently between the lower and higher Ritalin dose.

Since the larger changes in neuronal number and microglia (cells involved in fixing the brain after traumatic brain injury) occurred with the higher dose, they wanted to look at the gene expression in mice with only this dose.  They looked at specific genes associated with basal ganglia toxicity (note: the basal ganglia consists of structures involved in control of voluntary motor movements, procedural learning, routine behaviors or "habits", eye movements, cognition and emotion) including:

brain derived neurotropic factor (bdnf): support survival of neurons and growth of new ones

glial derived neurotopic factor (gdnf): promotes survival and differentiation of dopaminergic neurons

tyrosine hydroyxlase (th): creates precursors of dopamine (L-DOPA specifically)

dopamine transporter DAT1 (slc6a3): recycles dopamine

vesicular monoamine transporter VMAT2 (slc18a2): membrane protein that transports neuropeptides like dopamine

They found significant decreases in mRNA expression in gdnf, th, slc6a3, and slc18a2 after both acute and chronic administration of 10 mg/kg Ritalin while bdnf was only reduced after chronic 10 mg/kg Ritalin. 

Note - mRNA turns into proteins.  It's like a template that proteins get copied off of.  If there's less mRNA, there're fewer proteins that it's making.

Again, I think it would have been much more interesting had they also included the 1mg/kg dose and those results, even if they were no different than the saline mice.

Evidence for inflammation associated with acute doses of MPH:

They also wanted to see if since there was an increase in activated microglia following the higher dose of Ritalin, they wanted to see whether inflammatory genes had changed expression as well.  Specifically:

il-6: signaling protein that is usually activated in burn/tissue damage traumas

tnf-alpha: regulates immune cells by inducing fever or even cell death

cox-2: causes fevers in response to immune system attack

il1b: involved in cell proliferation, cell differentiation and cell death

They found that there were significant increases in mRNA expression of tnf-alpha and il-6 in those given a single dose of 10mg/kg MPH as compared to saline-injected mice.

And these were the results!  Tomorrow we'll start on the discussion section - which is usually where the researchers talk about why they think they saw what they saw.

@atdiy/@tymkrs  

[tymkrs]

Ritalin + Dopaminergic System Part 2

I'm currently reading through the following article courtesy of @lmcomie:

http://www.plosone.org/article/fetchObject.action?uri=info%3Adoi%2F10.1371%2Fjournal.pone.0033693&representation=PDF

Sadasivan, S., Pond, B.B., Pani, A.K., Qu, C., Jiao, Y., & Smeyne R.J. (2012) Methylphenidate Exposure Induces Dopamine Neuron Loss and Activation of Microglia in the Basal Ganglia of Mice. PLOSONE, 7(3).

We just went through the introduction yesterday, and now for results! (Usually methods comes next, but the authors chose to go results first).

Chronic MPH administration affects SNpc DA neuron number:

So we finally hear what they found out.  They looked at the substantia nigra pars compacta, a region of the brain that has been associated with reward, addiction, and degeneration of which is linked to Parkinson's Disease.  They looked to see what would happen in this region of the brain with chronic exposure (90 days) to saline vs 1mg/kg (ADHD dosage) vs 10 mg/kg (narcolepsy/recreational dosage) - specifically the number of dopaminergic neurons.

They found that for the mice treated with 1mg/kg there was not any difference from the saline (control group) but that in those treated with 10mg/kg for the 90 days had a 20% reduction of dopaminergic neurons in the substantia nigra.  And that it was the neurons towards the back of the substantia nigra that was affected more than those in the front.  The significance of that is (as far as I can tell from a cursory search), still being studied.

The red arrows highlight dopamine neuronal counts for mice with just saline, 1mg/kg, and 10mg/kg Ritalin.  The second graph shows the distance from front to back of the substantia nigra and how many neurons were found in those different sections.

Chronic MPH exposure results in microglia activation in the SNpc:

So as we've mentioned a couple of times while reading this study, excess dopamine causes the brain to be in an inflamed and stressed out state.  The scientists wanted to see if chronic Ritalin could induce a reaction similar to that of excess dopamine by measuring the number of resting and active microglia.  

Remember that excess dopamine causes release of signaling pepties (cytokines/chemokines) that cause microgliosis (scar formation, new neuron formation, but also production of toxic factors.

What they found was that giving the 1mg/kg (ADHD dose) Ritalin didn't change any microglia numbers.  And while giving 10mg/kg (narcolepsy dose) Ritalin did not change the number of resting microglia, it did cause a significant increase of activated microglia.  This suggests that there was some form of stress/inflammation occurring, otherwise those microglia would not have activated.

The red arrows point to saline vs 1mg/kg vs 10mg/kg Ritalin.  On the left graph are the resting microglia and the right graph are the activated microglia.  No difference in resting, but statistically significant difference with the 10mg/kg dose of Ritalin.

Dopamine and dopamine turnover affected following chronic MPH dosing:

Then they wanted to see if chronic administration of Ritalin resulted in changes in total striatal dopamine levels or dopamine turnover.

Note: The striatum feeds the basal ganglia, all of the parts of the brain that are responsible for control of voluntary motor movements, procedural learning, routine behaviors, eye movements, cognition, and emotion

Note: Classically, dopamine turnover is defined as the ratio between dopamine metabolites and dopamine itself.  I think of it as dopamine's lego-building blocks as opposed to the dopamine itself.  From a quick glance around other articles, an increase in dopamine turnover has been hypothesized to occur early in Parkinson's disease (PD) as a compensatory mechanism for dopaminergic neuronal loss.  So not a great thing to have, but maybe one of the first lines of defense.  Get rid of the excess dopamine by changing it to its metabolites, save the neuron!

They found that long-term 1mg/kg increased total striatal dopamine compared to saline but that long-term 10mg/kg did not.  And there was a significant increase in major dopamine metabolite named DOPAC at both Ritalin doses.  Luckily (or maybe not) only the 10mg/kg MPH dose saw an increase in dopamine turnover.  So 1mg/kg did not seem to be enough to trigger the dopamine becoming all of its metabolites although there was an increase in DOPAC. (See note after graph)

In looking at the graphs, we can see % dopamine did increase in the 1mg/kg dose but not the 10mg/kg dose.  And that in both 1mg/kg and 10mg/kg doses, there's an increase in DOPAC.  But it seems that the scientists may have misread graph C because according to this graph, it seems that both 1mg/kg and 10mg/kg MPH have significantly increased turnover as compared to Saline.

I may have to write them asking them! More tomorrow!  (Also this is why it's important to read the graphs, and not just what is written about them!)

@atdiy/@tymkrs