The buspirone challenge test clearly distinguishes ME/CFS patients from healthy controls: why is it not being developed and deployed?

I know increased BBB permeability is a bit of a neuroinflammation meme at this point, but it did occur to me as another potential confounder for the buspirone results. If there doesn’t seem to be evidence of differences in drug metabolism, this would be another explanation for both these findings and the sensitivity to lots of different drugs that pwME commonly report.

Even though buspirone normally crosses the BBB, it would reach higher concentrations at lactotrophs if the drug could get in through diffusion as well as through transporters. (And it would have to be only a slight increase in permeability, just enough for small drug molecules, otherwise we would have noticed other obvious signs of the problem). Another related possibility is upregulation of the specific transporter proteins that buspirone uses to cross the BBB fhe to some local signaling in the brain. Unfortunately we usually don’t know which transporters are responsible for what

I’m not sure off the top of my head if there are easy ways to test this and rule it out. Maybe it’s already been assessed in ME/CFS. Presumably there are tracers that normally don’t have good CNS penetrance and can show a difference between ME/CFS and healthy controls. @SNT Gatchaman would be happy to hear your thoughts on this.
 
ScoutB said:
I had the impression lactotrophs were outside the bbb? Could be a confounder anyway if it's working via some effect on the brain itself as you say.
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Sorry for my lack of clarity, was writing quickly before bed. Yes you’re right I believe. I was also making th assumption that the relevant dopamine/serotonin circuits would start inside the brain but that might not be true upon reflection
 
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After some quick googling it seems clear that buspirone does probably act locally at the pituitary, since studies like this one show an effect on cultured lactotrophs exposed to drugs.

journals.plos.org

Dopamine-Induced Apoptosis of Lactotropes Is Mediated by the Short Isoform of D2 Receptor

Dopamine, through D2 receptor (D2R), is the major regulator of lactotrope function in the anterior pituitary gland. Both D2R isoforms, long (D2L) and short (D2S), are expressed in lactotropes. Although both isoforms can transduce dopamine signal, they differ in the mechanism that leads to cell...
journals.plos.org journals.plos.org

Though there’s still a possibility of an additional effect from other circuits beyond the pituitary in-vivo.

so maybe the interesting question is not “Whats going on in the lactotrophs?” but “what circuits have far enough reach to affect lactotrophs in addition to other cell types that could mediate ME/CFS symptoms?”

GRH-releasing cells also seem to be regulated by dopamine from a quick google, so theoretically the same circuit could explain both. Or, alternatively, it’s not a circuit at all but the same soluble factor floating between parts of the brain. If we take the CRH neuron findings from the autopsy study at face value, that does give a hint as to the range of areas affected.

Maybe an additional clue would be other symptoms: light and sound sensitivity, some of the visual disturbances noted in the SPOT test thread? We know that hypothalamic signaling can affect all sorts of things, though I’m at a loss for problems originating from the hypothalamus or pituitary that are known to affect sensory systems like that.
 
I created a new version of the prolactin response document. I mainly wanted to get the tables in, since I agree that it would help to more easily interpret it. I also added a couple sections, and made minor changes in various places.

I've attached it, but someone suggested it would be good to also host it on github, since people who aren't logged in to S4ME can then access it as well, so here is a link to that: https://raw.githubusercontent.com/r...se/main/prolactin-response-mecfs-overview.pdf

I'll just copy the main sections I added in this version, so no one has to reread the whole thing if they're interested to see what changed.

Neuroendocrine challenge testing
A number of studies have been undertaken to investigate the functioning of the nervous system through the use of neuroendocrine challenge tests. These tests often consist of measuring the effect of a drug on pituitary hormonal release. As the pituitary gland is regulated by the brain, this testing strategy can be considered a relatively non-invasive “window” into central nervous system functioning (3).

In support the ability of these tests to detect altered neural functioning, researchers have demonstrated changes in prolactin response following neurotoxic exposure. For example, studies have shown altered prolactin response to serotonin-related drugs following 5-HT neuron-damaging toxin exposure in monkeys (4) and rats (5,6).
Controlling for confounders
To ensure interpretable results, and to increase the likelihood of detecting real differences, one must be mindful to control for potential confounders when studying prolactin response in ME/CFS.

One of the most important factors to consider in studies of prolactin response is the sex of participants. As summarized above, females demonstrate larger prolactin responses than males (19). Thus, at minimum, case and control groups should have the same ratio of females to males. However, it may be wise to also control for sex in an appropriate statistical model or analyze males and females separately to reduce the influence of variance associated with sex.

Additionally, as females exhibit variance in prolactin response throughout the menstrual cycle (19,65), researchers should consider controlling for this factor as well. Some of the clearest differences between ME/CFS and healthy participants occurred in studies of females tested during the luteal phase (9,12), while a study that included females tested during the follicular phase showed a smaller group difference (16). While one can not make strong conclusions about menstrual phase effects from such limited data, this may suggest that the optimal time for testing females is during the luteal phase. However, if females in different stages of the menstrual cycle are tested within the same study, this variable should be controlled for.

It may also be worthwhile to control for the effects of additional placebo-like factors on prolactin response (88). As stress can cause changes in prolactin secretion (89,90), this factor could potentially play a role in ME/CFS findings, for example through the stress associated with venipuncture. Though, at least one study has tested prolactin response to placebo in ME/CFS, and found that patients did not exhibit an abnormally increased prolactin response following the placebo test (29).

Sedentariness associated with ME/CFS may also play a role in abnormal prolactin response. One study suggested that sedentary individuals exhibit an increased prolactin response to buspirone, when compared to endurance-trained athletes (91), while another study found no difference between groups in a d-fenfluramine prolactin challenge (92). To decrease the potential influence of sedentariness on results, researchers may wish to include non-ME/CFS controls matched for physical activity level.

Further, researchers should ensure that other significant medical conditions which could influence prolactin response are not present in the study cohort. For example, liver and kidney diseases affect metabolism of buspirone (93), and this may alter prolactin response to this drug. Even if such disorders are excluded, it may still be worthwhile to test for plasma concentrations of neuroendocrine probes to avoid confounding due to differences in drug metabolism. Also, as some studies have suggested blunted prolactin response in depression (27,59), it may be beneficial to exclude participants with comorbid depression, to reduce the likelihood of masking an ME/CFS-associated increased prolactin response.
Reviving a forgotten thread
Almost all studies of abnormal prolactin response in ME/CFS were conducted in the 1990s, with no studies directly testing prolactin response in ME/CFS in over 15 years. The literature does not appear to suggest that anyone has argued that ME/CFS findings were misleading or false positives.

Duval et al. argue that neuroendocrine challenge testing, at least in the field of psychiatry, was largely abandoned in recent years for a few reasons, including the difficulties of controlling for all relevant confounding influences on hormones, difficulty in recruiting drug-naive participants, and the lack of interpretability afforded by readily available but non-specific probes (94). One can imagine that prolactin response research in ME/CFS may have been a casualty of the pivot away from neuroendocrine research in other fields.

Considering the massive burden of ME/CFS, both in terms of prevalence as well as the substantial suffering imposed on individuals, the importance of continuing this research is clear. Through careful consideration of study design and consultation with experts in endocrinology and neurology, future research into prolactin response may uncover important insights into the pathophysiology of ME/CFS, a condition which has, so far, eluded explanation.

Edit: I made a version 4 with just very minor changes like adding my email address and making the figure larger, and replaced the attachment here. All previous versions can be seen on GitHub.
 

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jnmaciuch said:
Or, alternatively, it’s not a circuit at all but the same soluble factor floating between parts of the brain.
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While acknowledging that there are probably a million other things it could be, and with not much certainty about it, my top contender would be estradiol as the soluble factor. One of the papers I read said it can affect dopaminergic circuits in other parts of the brain as well (quoted at end of this linked post). It's such a tantalizing connection to the female bias in ME/CFS.

Of course, the big problem is that sex hormones seem normal in most studies so far. But maybe it's from estradiol produced in small amounts in the brain itself. (paper claiming that can happen in animals in this post).
 
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forestglip said:
What would cause increased estradiol, I don't have a clue.
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One of the signals that also gets triggered in the aftermath of brain injury, it seems like:

Neuroprotective Actions of Brain Aromatase - PMC

The steroidal regulation of vertebrate neuroanatomy and neurophysiology includes a seemingly unending list of brain areas, cellular structures and behaviors modulated by these hormones. Estrogens, in particular have emerged as potent ...
pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
 
(Edit: ah seems you’ve already covered estradiol effects on the pituitary specifically)

It seems like estradiol upregulates PRLR and differentially increases the long form receptor. So there could be something there in some cells?

I’m a bit unclear on how this varies between different cells though and a lot of the literature seems to focus on cancer cells and implications. But given how widespread the receptor seems to be, could be something going on even locally given extrapituatary prolactin production.

Estrogen regulates PRLR transcription through the preferentially utilized PIII promoter via a non-classical ERE independent mechanism in target cells. The protein association induced by estradiol of estrogen receptor α (ERα) with DNA-bound Sp1 (constitutive) and C/EBPβ (recruited by the ERα-SP1 complex) is essential for human prolaction receptor gene transcription (figure 1C).
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https://atlasgeneticsoncology.org/gene/42891/

I think this is the original paper https://www.sciencedirect.com/science/article/pii/S0021925818348932
This seems more up to date https://pubmed.ncbi.nlm.nih.gov/16651265/
Also see https://www.sciencedirect.com/science/article/abs/pii/S096007600200184X
 
I wrote a brief blog article on the buspirone findings to help highlight the findings. It links to this S4ME discussion and the excellent overview by forestglip.
mecfsscience.org

Increased prolactin response to buspirone - ME/CFS Science

Multiple studies have found an increased response of the hormone prolactin to the drug buspironeContinue readingIncreased prolactin response to buspirone
mecfsscience.org mecfsscience.org
 
jnmaciuch said:
One of the signals that also gets triggered in the aftermath of brain injury, it seems like:

Neuroprotective Actions of Brain Aromatase - PMC

The steroidal regulation of vertebrate neuroanatomy and neurophysiology includes a seemingly unending list of brain areas, cellular structures and behaviors modulated by these hormones. Estrogens, in particular have emerged as potent ...
pmc.ncbi.nlm.nih.gov pmc.ncbi.nlm.nih.gov
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Interesting. Something to keep in mind.

The review says that brain damage of seemingly any type increases aromatase expression around the damaged area, leading to increased estrogen levels. The estrogens are produced primarily in reactive astrocytes and radial glia. Estrogens appear to help protect against and repair damage.
Upon the induction of brain damage via excitotoxicity or mechanical injury, however, aromatase expression was not only induced in many brain areas including the hippocampus, striatum, cortex and corpus callosum, but was localized to reactive astrocytes.
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Damage to the vertebrate brain also induces aromatase expression in radial glia as well.
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To the best of our knowledge, aromatase induction in astroglia seems to occur following damage to the vertebrate brain regardless of the nature of this perturbation.
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Estrogens are protective under numerous types of stressors, including oxidative stress [167;171;172], glutamate excitotoxicity [173; 174], chemical lesions [175], traumatic or mechanical injuries [169], ischemia [135;176], iron toxicity [168;177], glucose or serum deprivation [178;179], and specialized disease related pathogens such as Aβ[180;181], and HIV proteins [182;183].
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Upon MCAO [middle cerebral artery occlusion] in male rats, plasma estradiol concentration remains constant, while estradiol levels in the parabrachial nucleus and the central nucleus of the amygdala are increased [137;199].
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(Study: Neuroprotective Actions of Brain Aromatase, 2010)
 
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It is a great writeup and hopefully will help get more attention on this.

I do wonder if extrapituitary prolactin could be at play. Do we need to ask which of the cells which produce extrapituatary prolactin could be affected directly by buspirone?

Or could a prolactin increase triggered by buspirone acting on the hypothalamus or pituitary then cause a secondary increase in other cell groups and its that that we are seeing?

What if what we’re after here is the immune system and T cells, B cells, NK Cells or macrophages? Or maybe the CNS and some neurons in the hypothalamus or hippocampus themselves or some nearby astrocytes?

I’m keen to look at ways we can try to reproduce the core findings here but also then narrow down on the mechanism. And that may take thinking well outside the original study. I’m not sure quite where or how mind you!
 
hotblack said:
I do wonder if extrapituitary prolactin could be at play. Do we need to ask which of the cells which produce extrapituatary prolactin could be affected directly by buspirone?
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Could be, though I'm not sure we would expect buspirone and d-fenfluramine, drugs that act on neurotransmitter receptors, to similarly increase prolactin from non-neural cells like lymphocytes.

hotblack said:
Or could a prolactin increase triggered by buspirone acting on the hypothalamus or pituitary then cause a secondary increase in other cell groups and its that that we are seeing?
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At least in the pituitary, prolactin is regulated through negative feedback, where increased levels cause decreased secretion. So we'd probably need evidence that prolactin in other cells can be regulated by positive feedback.

hotblack said:
What if what we’re after here is the immune system and T cells, B cells, NK Cells or macrophages? Or maybe the CNS and some neurons in the hypothalamus or hippocampus themselves or some nearby astrocytes?

I’m keen to look at ways we can try to reproduce the core findings here but also then narrow down on the mechanism. And that may take thinking well outside the original study. I’m not sure quite where or how mind you!
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Good thought experiment. I'm not sure how you could prove it's extrapituitary prolactin.
 
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These are all good questions @V.R.T. And @forestglip Rather than being able to clearly answer them or trying to argue a particular point, because my honest answer is “I don’t know” my hope is that others can look at some of the literature and see what they think. I posted some papers in this thread. It does seem prolactin can be produced as well as there being receptors for it in many places though. Including those cells I mentioned.

I suppose also given these papers all measure levels in the wider bloodstream my question would be why we should focus narrowly without clear evidence to do so? Closing doors and making assumptions seems to be how this idea was lost in the first place.
 
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