Can anyone make sense of this to explain to a layperson?

July 31, 2017

dm, I've looked and can't find that mice study of increased numbers of glutamate receptors. It was many years ago when I read it.

July 31, 2017

dm123, I don't know if this is helpful or just confusing since it doesn't focus on glutame and I can remove this post if you like.

There is much more than glutamate or gaba ... (not even talking about clonazepam!)

https://www.ncbi.nlm.nih.gov/pubmed/1358120
Cholinergic mechanisms in physical dependence on barbiturates, ethanol and benzodiazepines.

And more generic, the part of GABAA receptors likely isn't that interesting: https://www.hindawi.com/journals/aps/2012/416864/

Hi liberty, i will take a look look, please keep them posted. Right now I am focusing on mGluRs which are metabotropic Glutamate receptors (counterpart is Glutamate receptors),and are members of the family of receptors called G protein coupled receptors or GPCRs, and are extremely diverse in their effects on the Glutamate system. In fact, they can do things to the ionic channels and the action potential that fit my model well, for early stages of interdose withdrawal. Once tolerance develops to the benzodiazaphine, this will greatly exasperate the issue. They are also related to LTP as well.

Interestingly enough I ran across the metatropic counterpart for the acetylcholine receptor and thought about you. They are called mAchRs or muscarinic acetylcholine receptors. Both mGLuRs and mAchRs are GPCRs.

I hadn't realized that muscarinic receptors fell into this class, i.e. GPCRs

https://en.m.wikipedia.org/wiki/Muscarinic_acetylcholine_receptor

EDIT add:

I read through the first link very carefully. Ironically the muscarinic receptors seem to be a critical part of the research they did. They saw an increase in muscarinic receptors on withdrawal, implicating cholergenic hypersensitivity. The link I gave you above indicates that this atropine is a very powerful antagonist of all 5 muscarinic receptors M1-M5. Is the term "barbitol" just referring to barbiturates or to both benzos and barbs??. I ask because all the long term findings use the word barbitol and alcohol, but I noticed benzos were not explicitly stated....

July 31, 2017

'Is the term "barbitol" just referring to barbiturates or to both benzos and barbs. I ask because all the long term findings use the word barbitol and alcohol, but I noticed benzos were not explicitly stated....'

Its'a specific barbiture, and it's barbital: https://en.wikipedia.org/wiki/Barbital The text may be a bit confusing, but both at the beginning and the end benzodiazepines are used in the same sentence as alcohol and barbiturates. Keep in mind it's an abstract.

Note: I looked around for a bit. Hard to find something specific for benzodiazepines, without paying. It may also depend on the benzo/dose/duration of use.

My impression is that physical dependence on many drugs is mediated by cholinergic neurons. Here is one about opiates:http://www.eurekaselect.com/77918

I wish I could take a muscarinic antagonist ! I can only think of one or two other solutions, but the response of the average doc would likely be 'can't you taper off the clonazepam first' 'I didn't learn to do my job that way'/

August 1, 2017

Thanks for the additional link to expand the horizon of research. I like the tolerance GABAa one , section on Glutamate

Here they are failing to recognize that interdose withdrawal constitutes a very slow form of kindling. It took me 2.5 years. They use the term sensitization, but don't follow up on how the sensitization occurs. This is what I've found on a lot of studies. Also they tend to focus on discontinuation here. I'm focused on interdose wd and interdose tolerance wd. We know LTP can set up after abrupt discontinuation, but LTP, as good as it is, can't really be applied strictly to the glutamate changes we see interdose.

" The basis of benzodiazepine tolerance could then lie in sensitization of the glutamatergic system—a putative process that could account for the withdrawal symptoms after chronic benzodiazepine discontinuation [5, 110]. Such sensitization is reminiscent to adaptive glutamatergic processes as seen in kindling experiments, although it should be noted that kindling only occurs with intermittent and not after continuous treatment [111]

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This is interesting

"Although glutamate possesses lower affinity for the AMPA receptor compared to NMDA receptors, faster excitation-inducing kinetics are present at the AMPA receptor.

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This is for wd as well but interesting

"Relevant to this review, a study showed that AMPA receptor desensitization was caused by a rupture of a domain interface which allowed the ion channel to close, providing a simple yet elegant explanation [117].

There is mention of metabotropic, but nothing on it . I'm going to continue work on that for interdose Withdrawal.

Quote....

metabotropic receptor types.

End quote

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This is good

In rodents, the development of tolerance to the sedative effects of the classical benzodiazepines diazepam and chlordiazepoxide was prevented by coadministration of the NMDA receptor antagonists CPP, dizocilpine, MK-801, and ketamine [119–121]

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Conflicting data

This suggests that the mechanism underlying tolerance to the anxiolytic effects of diazepam is different from that underlying tolerance to the sedative effects. Increases in cortical mRNA of NMDA NR1 and subunits have been reported in rats tolerant to diazepam [124, 125], which were prevented by concomitant treatment with the NMDA receptor antagonist MK-801 [126]. However, another study showed decreases in hippocampal subunits after chronic flurazepam treatment, even though the total amount of NMDA receptors was unchanged [127].

In support, after long-term (but not acute) lorazepam treatment, no differences were found in the affinity or density of NMDA receptors, even though increased in vitro glutamate release and NMDA-induced cGMP efflux in the hippocampus was reported [128]. Together, these data suggest that NMDA-dependent mechanisms contribute to the development of benzodiazepine tolerance. However, as anxiolytic tolerance was not blocked by NMDA receptor antagonism, the NMDA system could also play a differential role in tolerance depending on the specific behavioral effects [123]. Moreover, a straightforward glutamate sensitization may be an oversimplification, as tolerance to the sedative effects of lorazepam after 21-day treatment correlated with a decreased rather than an increased sensitivity for glutamate (glutamate binding) [129].

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This is something I found in my research, and it's pretty consistent. The metabotropic Glutamate receptors type 1 especially. I found classic kindling studies implicating mGluR type 1 upregulation and type 2 and 3 downregulation . I'm not sure they are referring to metabotropic here, because this section is titled ionotropic.

From this article I don't think mGluR here refers metabotropic

They don't discuss metabotropic

"Specifically, significant reductions of mGLuR1 (cortex and amygdala) and mGluR2 mRNA (amygdala) were reported in rats treated chronically with diazepam, even though the effects were complex and dependent on treatment route (subcutaneous or intraperitoneal injections).

Their conclusion in this are is lack of universal support for a Glutamate hypothesis and I agree for ionotropic receptors and interdose wd.

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"Mechanistically, BDNF-induced suppression of GABAergic signaling was hypothesized to stem from altered GABAA receptor composition, increased GABAA receptor phosphorylation, decreased subunit synthesis, or increased postsynaptic receptor internalization or diffusion [139]. Interestingly, all these proposed mechanisms were already discussed in this paper. Thus, neurotrophin-induced changes may not be an independent mechanism, but be a player in a causal chain of events. Again, to our knowledge, no studies exist on the effects of chronic benzodiazepine treatment on neurotrophic expression and functionality.

Very interesting

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GPCRs with dopamine ,ACh, and serotonin receptors to GABAa. I will look into this more.

There is ample evidence that the serotonin, dopamine, and acetylcholine receptor systems can modulate the GABAA receptor functionality [143–146] (Figure 3). For example, the receptor for activated C kinase (RACK-1) potentiated PKC-dependent phosphorylation of GABAA receptors mediated by the activation of muscarinic acetylcholine receptors [145], and serotonergic neurotransmission inhibited GABAergic signaling via GABAA receptor PKC-dependent phosphorylation, again with involvement of RACK-1 [144]. Altogether, these neurotransmitter systems act via G-protein-coupled receptors to activate protein kinases (PKA and PKC) and scaffold proteins that may subsequently modulate GABAA receptor β and γ2 subunit phosphorylation (Figure 3) [95].

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This is good but we've known there are codependencies between the two systems here. Cortisol should be included here, but technically is not a neurosteroid.

"Adding to the complexity of the putative involvement of neurosteroids in benzodiazepine tolerance, factors such as GABAA receptor subunit composition, phosphorylation mechanisms, and ((extra)synaptic) localization—which are all factors that were already found to be involved in tolerance development—influence the specific dynamics of neurosteroid activity.

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Very very true:

"However, in vivo pharmacodynamic potency and pharmacokinetic half-life differences could greatly impact on tolerance processes [7].

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Very interesting about Librium in the conclusion, about induced seizures

"Surprisingly, chlordiazepoxide did not lead to any precipitated seizures, even though a comparable GABAA receptor occupancy was obtained. Therefore, the assumption that classical benzodiazepines act as a homogeneous class probably complicates the interpretation of the current literature.

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"

However, because these studies do not specifically address tolerance development, the rather general conclusion from these studies is that partial or selective modulation of the GABAA receptor results in a reduced liability for physical dependence. Thus, it is important to note that, even though zolpidem does not seem to engender any obvious tolerance development, zolpidem can lead to withdrawal symptoms that are comparable to those seen after chronic classical benzodiazepine treatment [29, 77]. Thus, tolerance and withdrawal symptoms may constitute separate entities in benzodiazepine dependence.

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This is well said

"From the large variance in the studies, it appears that either different (simultaneous) tolerance mechanisms occur depending on the benzodiazepine effect, or that one tolerance-inducing mechanism depends on the activated GABAA receptor subtypes. This is not unlikely, given that tolerance is a heterogeneous process that occurs at different rates for the various effects and also depends on the profile of the (subtype selective) benzodiazepine. Adaptations could then occur on different time scales depending on the receptor subtype and brain region involved. In line with this hypothesis, tolerance develops relatively quickly for the sedative and anticonvulsant actions of benzodiazepines, whereas tolerance to anxiolytic and amnesic effects most probably do not develop at all. It is intriguing that anxiolytic effects of classical benzodiazepines may not decline during prolonged treatment. In addition to subtype selectivity, additional factors may be important for a (subtype-selective) benzodiazepine to cause tolerance, including GABAA receptor potency (efficacy) and in vivo receptor occupancy over time. The finding that partial agonists with an overall but comparable lower efficacy at all α subunits of the GABAA receptor such as bretazenil did not result in anticonvulsant tolerance raises the possibility that chronic clinical use of these compounds is associated with a lower tolerance.

August 1, 2017

This model for interdose wd and interdose tolerance wd will have to be a hybrid kindling model with the following

-the regular GABAa receptor tolerance stuff

- GPCRs to Glutamate ionic channels. These would be(so far in my research) metabotropic Glutamate receptors (mGluRs), group 1 upregulation and/or groups 2 and 3 downregulation

These changes would model the depolarization and repolarizarion action potential changes that i mentioned in earlier addendums very well

- these GPCRs for GABAa ionic channels: ACh (mAchRs or muscarinic receptor dysregulation). I see the paper above references some for serotonin and dopamine as well, and these would need to be investigated all relative to kindling or an interdose wd model.

These changes would further model the dyregulation that we see in the hyperpolarization phase of the post synaptic membrane potential, and would effect the "effort" required to depolarize the neuron.

(These GPCRs in the presence of their respective neurotransmitters would facilitate PKA and PKC phosphorylation of the various GABAa receptor sub units. According to the short summary in the reference above the functional effects of these phosphorylations could lead to inhibitions or enhancements of the GABAa receptor's' activity....)

August 4, 2017

Hi

I had a fellow BB ask a question regarding differences in the terms "subsensitivity", insufficiently sensitive, downregulation, tolerance. These are mostly relative to terms that are used when discussing what happens to our GABAa receptors during benzodiazaphine chronic use and tolerance. Since the introduction of a paper that I am working on already had this info, I am putting it here as an ADDENDUM 6. I could not post the images even with the image tag. If someone knows how to do this let me know..... One image is from Pers' paper and is not referenced via URL. So it can't be viewed. The other figures are referenced by URL., and can be viewed.

It's from an introduction of a paper, hence it's a bit piecemeal.....cut and paste.

A lot of people are already familiar with this stuff, but I think the terminology needs clarification.

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ADDENDUM 6:Downregulation, Upregulation, Sensitization, and Tolerance

Dependent states are when the nervous system undergoes changes that create symptoms when the person stops taking the benzodiazaphine (i.e. Withdraws from the drug). The tolerance state develops when the drug at it’s current dose fails to “work” (i.e. Withdrawal symptoms emerge), and the dose must be increased to alleviate these symptoms. It is agreed upon in the scientific literature that tolerance develops over time, but in some individuals can develop quite rapidly, depending on variations in the type of benzodiazaphine that is used (half life being very important) as well as variations in genetics, metabolism, etc.

Since interdose withdrawal is the main focus of this paper, short and moderate half life benzodiazaphines are inherently provocative in this respect. These drugs include clonazepam, lorazepam, xanax, etc.

Downregulation and Upregulation

A very important a note. The terms “downregulation” and “upregulation” will be used quite frequently in this paper. Sometimes these terms are used quite loosely to imply a sensitization or desensitization of the receptor itself. However, it should be noted that these terms, in the strict sense, imply only an increase in the density population of the receptor, in the case of upregulation, and conversely a decrease in the density population of the receptor, in the case of down regulation. Thus, the change in the receptor population causes an increase or decrease in the sensitivity of the receptor population for a particular extracellular ligand.

In the strict sense, these terms do not involve changes in the composition of the receptor itself (i.e., conformational changes) that reduce or increase the sensitivity of each receptor itself to a particular ligand or PAM. These later types of changes involving changes in gene expression, can also occur to receptors, but don’t fall under the strict definitions of the terms upregulation and downregulation. The references below explain the strict definitions of upregulation and downregulation further:

Background

References:

https://en.m.wikipedia.org/wiki/Downregulation_and_upregulation

Quote

An example of downregulation is the cellular decrease in the number of receptors to a molecule, such as a hormone or neurotransmitter, which reduces the cell's sensitivity to the molecule. This is an example of a locally acting(negative feedback ) mechanism. [homeostatic response]

An example of upregulation is the response of liver cells exposed to such xenobiotic molecules as dioxin. In this situation, the cells increase their production of cytochrome P450 enzymes, which in turn increases their degradation of these molecules.

…

Receptors are created, or expressed, by the DNA of the cell, and they can be increased, or upregulated, when the signal is weak, or decreased, or downregulated, when it is strong.[1]

End quote

To further explain:

http://meandmymastcells.com/explain-to-me-receptor-upregulationdownregulation/

Quote

Receptors are born in the cell. The cell's DNA contains the instructions to create each type of

receptor. The cell runs the DNA program, which results in the creation of a receptor protein, and that receptor protein is then pushed out to the cell boundary and embedded in the cell's membrane. This is called ‘expression' of the receptor. Once receptors have been expressed, the cell can receive and respond to messages the receptor is tuned to.

But the receptors have a life cycle.

They can be taken out of the membrane and recycled into the cell. So fewer receptors are left on the cell membrane and therefore the less notice is being given to that specific message. So a decrease in the number of receptors to a message sited on the cell membrane reduces the cell's sensitivity to the message.

That's called down-regulation.

Similarly if the cell receives a weak signal, it can up-regulate by pumping out more receptors such as to increase the sensitivity to the weak message. So an increase in the number of receptors to a message sited on the cell membrane increases the cell's sensitivity to the message.

That's called up-regulation.

So this is a very flexible and changeable environment; the cell is constantly monitoring what's coming in to its receptors and adjusting their quantities accordingly.

End quote

To understand the proteins that are actually involved in this absorption (i.e., downregulation ) process, we can look at one specific example and one way the cell can mediate the absorption of receptors, in this case insulin receptors.

This example with insulin and insulin receptors offers an excellent example of the downregulation process itself.

Endocytosis and downregulation

Quote

Elevated levels of the hormone insulin in the blood trigger downregulation of the associated receptors.[2] When insulin binds to its receptors on the surface of a cell, the hormone receptor complex undergoes endocytosis and is subsequently attacked by intracellular lysosomal enzymes.[3]

The internalization of the insulin molecules provides a pathway for degradation of the hormone as well as for regulation of the number of sites that are available for binding on the cell surface.[4] At high plasma concentrations, the number of surface receptors for insulin is gradually reduced by the accelerated rate of receptor internalization and degradation brought about by increased hormonal binding.[5] The rate of synthesis of new receptors within the endoplasmic reticulum and their insertion in the plasma membrane do not keep pace with their rate of destruction. Over time, this self-induced loss of target cell receptors for insulin reduces the target cell's sensitivity to the elevated hormone concentration.[5]

End quote

https://en.m.wikipedia.org/wiki/Receptor-mediated_endocytosis

Quote

Receptor-mediated endocytosis (RME), also called clathrin-mediated endocytosis, is a process by which cells absorb metabolites, hormones, other proteins – and in some cases viruses – by the inward budding of plasma membrane vesicles containing proteins with receptor sites specific to the molecules being absorbed (endocytosis).

End quote

https://en.m.wikipedia.org/wiki/File:Itrafig2.jpg

Mechanism of clathrin-dependent endocytosis. Clathrin and cargo molecules are assembled into clathrin-coated pits on the plasma membrane together with an adaptor complex called AP-2 that links clathrin with transmembrane receptors, concluding in the formation of mature clathrin-coated vesicles (CCVs). CCVs are then actively uncoated and transported to early/sorting endosomes.

https://en.m.wikipedia.org/wiki/Endocytosis

Quote

Clathrin-mediated endocytosis is mediated by the production of small (approx. 100 nm in diameter) vesicles that have a morphologically characteristic coat made up of the cytosolic protein clathrin. Clathrin-coated vesicles (CCVs) are found in virtually all cells and form domains of the plasma membrane termed clathrin-coated pits. Coated pits can concentrate large extracellular molecules that have different receptors responsible for the receptor-mediated endocytosis of ligands, e.g. low density lipoprotein, transferrin, growth factors, antibodies and many others.[4]

End quote

Internalization is endocytosis

Upregulation and exocytosis

https://en.m.wikipedia.org/wiki/Cell_surface_receptor

https://en.m.wikipedia.org/wiki/Exocytosis

https://en.m.wikipedia.org/wiki/Receptor_(biochemistry)

Note, as with downregulation it’s a locally acting feedback mechanism and a homeostatic response if due to an external ligand or PAM.

Quotes from Pers’ “How GABAa receptors are created”

Quote

….Once modification and sorting of the proteins is complete, the subunits are encapsulated into transport vesicles that bud off from the trans-cisterna region of the Golgi apparatus and then delivered and inserted into the cell membrane where they form receptors. (43) The picture below shows the side and top view of a receptor inserted into the cell membrane.

The receptors may be inserted directly into their final location in the cell membrane (e.g. postsynaptic (in the center of the postsynaptic terminal), perisynaptic (on the side of the postsynaptic terminal), or extrasynaptic (on the cell body outside of the postsynaptic terminal), or they may laterally move through the plasma membrane (diffuse) into that location after membrane insertion elsewhere. (28) Some researchers have reported that GABARs are inserted into and removed from the membrane solely at extrasynaptic sites, which would make lateral diffusion (depicted in the picture below) an important part of getting receptors to their final locations. (37)

End quote

https://www.hindawi.com/journals/aps/2012/416864/fig2/

See figure on next page

Quote

Receptor trafficking and associated proteins. GABAA receptors are assembled from individual subunits in the endoplasmatic reticulum (ER) where the chaperones BiP and Calnexin assist in quality control. Unassembled GABAA receptor subunits that are to be targeted for ER-associated degradation are ubiquitinated and degraded in the proteasome (dm123: endocytosis).

The ubiquitin-like protein PLIC can interact with GABAA receptors thereby inhibiting their targeting for proteasomal degradation. Assembled pentameric GABAAreceptors exit the ER and bind the guanidine exchange factor brefeldin-A-inhibited GDP/GTP exchange factor 2 (BIG2) in the Golgi. Here they also interact with the palmitoylase transferase GODZ and Gamma-aminobutyric acid receptor-associated protein (GABARAP). GABARAP interacts with the NEM sensitive fusion (NSF) protein, as does the GABAA receptor β subunit, and this association may facilitate transport of the receptor complexes to the cell surface. GABAA receptors are inserted at extrasynaptic sites and can diffuse along the plasma membrane in and out of synaptic domains. At synapses they are stabilized by an interaction with the scaffolding protein Gephyrin. The interaction of the GABAA receptor intracellular loops with the μ2 subunit of the adaptin complex AP2 is important for GABAA receptor internalization. GABAA receptors are delivered by a clathrin-mediated pathway to early endosomes where they can be targeted for degradation in the lysosome or for recycling upon binding of Huntington-associated protein (HAP1). Reprinted by permission from Elsevier, reprinted from [101].

End quote

Quote

Cells can increase (upregulate) or decrease (downregulate) the number of receptors to a given hormone or neurotransmitterto alter their sensitivity to different molecule. This is a locally acting feedback mechanism.

End quote

(1) GABAa receptor changes in benzodiazaphine tolerance

Input stimuli causing this effect: the benzodiazaphine as a PAM of the GABAa receptor.

Some very solid papers have been published about GABAa receptor downregulation, uncoupling, altered subunit configuration and other changes in gene expression, as a way to account for benzodiazaphine tolerance.

Please reference Pers’s papers on Glutamate, LTP and Withdrawal, and Nuclear Mechanisms of PWS.

See this reference as well

http://downloads.hindawi.com/journals/aps/2012/416864.pdf

In summary, the GABAa receptors are affected in the following ways:

Main Quote

1) Changes in subunit composition of the GABAA receptor through gene expression to reduce sensitivity to BZs. (1, 2, 18). (I.e., desensitization)

(Dm123: the literature supporting this is not certain)

http://downloads.hindawi.com/journals/aps/2012/416864.pdf

Quote

Of all subunits, α, β, and γ subunits have been mostly examined. This paper confirms that both for mRNA and protein subunit levels, the available evidence leads to a divergent and sometimes conflicting picture, although the majority of the studies essentially do not show any significant difference in subunit expression [102]. Furthermore, a lack of consistency appears for subunit changes in different specific brain areas. Moreover, the length and method of chronic treatment seem relevant since differences in GABAA receptor subunit mRNA levels after chronic diazepam treatment in rats can depend on whether diazepam is administered as daily systemic injections or via osmotic minipumps [103]. Binding studies also generally report no changes in benzodiazepine binding after chronic treatment [92, 93, 104]. Together, GABAA receptor expression (both mRNA and protein levels) is not consistently and robustly altered after various long-term treatment regimens. Thus, a general central downregulation or even consistent region-specific changes in GABAA receptor expression after chronic benzodiazepine use are not supported by the literature. Even though methodological differences (e.g., treatment regimen, species, route of administration, and applied drug) may account for some conflicting findings, the results seem overall inconsistent. Moreover, molecular results are often not combined with behavioral tests, preventing a direct correlation between behavioral tolerance and molecular changes. Clinical studies applying in vivo binding or postmortem GABAA receptor expression after chronic benzodiazepine treatment are to the knowledge of the authors lacking

End quote

2) Phosphorylation, in which a phosphate may be added or removed to the GABAA receptor to either turn it on or off respectively (GABAA receptors are phosphorylated by various protein kinases and dephosphorylated by phosphatases). (2) (dm123: uncoupling)

(Dm123: Note that this is thought to be how GABAa receptor uncoupling occurs. This is thought to be the most likely explanation of these 3 to account for GABAa tolerance to the benzodiazaphine . Benzodiazaphines are PAMs because their binding alters the GABAa receptor confirmation with an increased capacity to bind GABA itself, leading to an increased channel opening frequency, and a greater chloride influx into the post synaptic neuron, and to (more frequent) hyperpolarization. GABAa receptor uncoupling is defined as a decreased ability of benzodiazaphines to enhance the GABA-induced IPSPs at the GABAa receptor. It’s hypothesized that chronic usage of benzos , i.e. Tolerance leads to this uncoupling effect on the GABAa receptor. Decreased coupling in the broader sense may develop as a result of changed GABA subunit composition(see above), alterations to the receptor itself via phosphorylation (i.e., this section) or the receptors second messenger ligands, or any process affecting the conformational state of the receptor. The uncoupling hypothesis is attractive because it does not assume direct changes in subunit expression and ligand binding. This uncoupling has been shown to reduce the effects that the benzodiazaphine has on the receptor very significantly, even when there are no significant changes in benzodiazaphine binding site density or affinity, i.e. No downregulation or desensitization. Coupling can be affected through an altered GABAa receptor assembly process resulting in point 1 above (gene expression is altered) from subunit replacements. Coupling can also be affected by phosphorylation.(see below what this means). This could directly affect the inhibitory synaptic strength, with changes in chloride channel openings. We don’t know if these changes in allosteric coupling are relevant to the development of tolerance in vivo)

Quote

Note- Phosphorylation (previously noted) is a tool the cell uses to control levels by turning proteins on and off as needed. This is accomplished by adding or removing phosphate molecules. Phosphates act as molecular switches. When they are present the protein is on and when they are not present the protein is off. The protein is turned on when a protein kinase transfers a phosphate molecule to the protein (the protein is then considered to be ‘phosphorylated’- kinase uses ATP to do this). The protein is turned off by an enzyme called phosphatase, which removes the phosphate molecule (the protein is then considered to be ‘dephosphorylated’).

(Dm123: some examples of protein kinases that would directly be involved are cAMP-dependent protein kinase A (PKA), and Ca2+/phoppholipid-dependent protein kinase C (PKC). You may see these terms in some of the references throughout this paper. )

End quote

3) Down-regulation of GABAA receptors, in which GABAA receptors are absorbed back into the neuron through endocytosis, (see Introduction and figures above) thereby reducing their number at the synapse. (3)

Reagarding downregulation by benzodiazaphines, it’s a complex process. Referring to the introduction in section (0) and quote below:

https://www.hindawi.com/journals/aps/2012/416864/

Quote

The processes that control the assembly, membrane trafficking, and synaptic accumulation of GABAA receptors are complex (for review, see [100]). In short, GABAA receptors are assembled from individual subunits out of the endoplasmic reticulum within minutes after their translation, with amino acid sequences in the N-terminus influencing the GABAA receptor subtype (Figure 2). Then, receptor trafficking to the plasma membrane takes place, facilitated by diverse helper GABAA receptor-associated proteins (among that GABARAP, BIG2, PRIP, gephyrin, and radixin). Ultimately, (clathrin-dependent) endocytosis occurs after receptor dephosphorylation, after which degradation or recycling may ensue (Figure 2). If prolonged activation of the GABA system leads to receptor downregulation, then this could be established by interfering at multiple steps of the dynamic GABAAreceptor life cycle. These include decreased subunit mRNA transcription, subunit degradation in the endoplasmic reticulum (e.g., by ubiquitylation), decreased expression of GABAA receptor-associated helper proteins, and alterations in the endocytosis of specific GABAA receptor subtypes. The finding that the protein synthesis inhibitor cycloheximide and the RNA synthesis inhibitor actinomycin D blocked the effects of chronic diazepam exposure in recombinant cells expressing receptors indicates that GABAA receptor synthesis is of at least some importance [87].

End quote

Note that 1 and 3 can be completely independent, i.e. Even if 1 is unlikely, 3, or downregulation can still occur, and vise versa.

Quote

Changes in rates of GABAA receptor endocytosis, receptor membrane insertion, intracellular trafficking, and association with helper GABAA receptor-associated proteins could still play a role, leading to a reduction in membrane surface receptors without affecting overall subunit protein expression (e.g., see [105])

…..

Moreover, it cannot be excluded that particular subunits play a role in the development of tolerance after chronic treatment in the absence a direct up- or downregulation.

End quote

Main end quote

4) in addition to the 3 items listed below, there is also a possibility of tolerance and a loss of inhibitory synaptic function occurring due to lateral diffusion away from clustering of the receptors at the synapses. See the 2 figures in the Introduction

Quote

Another interesting suggestion is that a possible loss of synaptic function after chronic exposure could be due to a shift to a perisynaptic or even an extrasynaptic localization of GABAA receptors, away from clustering of GABAAreceptors at synapses (Figure 2) [106]. At least in alcohol research, such dynamic changes in plasticity at inhibitory synapses have been shown [107]

End quote

It’s important to note that three of these 4 major types of changes are thought to occur during benzodiazaphine administration as tolerance is developed, and may persist for a time after the drug is tapered, as the body heals. I don’t think the body can recover fully in this area until the drug is completely stopped, but healing in this area can start during the taper itself as the drug dosage is lowered. I don’t know if here are any clinical studies in this particular area of tapering and GABAa receptor recovery.

These are neuroplastic homeostatic changes to the receptor, as the receptor is bombarded by the benzodiazaphine which positively modulates it.

From Pers’ paper on Nuclear Mechanisms of PWS

Quote

Ashton’s statement:

“When these receptors are slowly reinstated after drug withdrawal, they may return in a slightly altered form. They may not be quite so efficient as before in increasing the actions of GABA, the natural 'calming' neurotransmitter.”

The ‘slightly altered form’ refers to a change in gene expression. The prolonged presence of the drug will prompt the neuron to transcribe the receptors subunits from different genes than previously to replace the old receptors with ones that are not sensitive to benzo binding. The neurons do this to counteract the action of the drug and to restore the equilibrium.

When benzos bind to the receptors they enhance the action of GABA. We have natural endozepines in our bodies that normally bind to these sites. So when the receptors are replaced with ones less sensitive to benzos, they may be less sensitive to our natural endozepines as well, resulting in a decrease in the action of GABA. This is why she stated “They may not be quite so efficient as before in increasing the actions of GABA.”

This is a neuroplastic homeostatic change and should be reversible over time. There are no long-term studies saying one way or another, however the anecdotal success stories support a reversal.

End quote

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For those of you who are interested, I have provided some references from Pers' papers on how the GABAa receptors are affected by benzodiazaphines. Many have read this info already and it's included here as a supplement to the above.

(See the conclusion at the end of this post if you wish to skip this section)

Quote

First- let’s look at what Ashton said in her 2011 supplement to the Ashton Manual:

“Long-term effects of benzodiazepines

One mechanism which might be involved in long-term (and possibly permanent) effects of benzodiazepines is an alteration in the activity of benzodiazepine receptors in brain GABA neurones. These receptors down-regulate (become fewer) as tolerance to benzodiazepines develop with chronic use. Such down-regulation is a homeostatic response of the body to the constant presence of the drugs. Since benzodiazepines themselves enhance the actions of GABA, extra benzodiazepine receptors are no longer needed, so many are, in effect, discarded. These down-regulated receptors are absorbed into neurones where, over time, they undergo various changes including alterations in Gene expression. When these receptors are slowly reinstated after drug withdrawal, they may return in a slightly altered form. They may not be quite so efficient as before in increasing the actions of GABA, the natural 'calming' neurotransmitter. As a result, the brain may be Generally less sensitive to GABA and the individual is left with heightened central nervous system excitability and increased sensitivity to stress. Molecular biologists point out that changes in Gene expression can be very slow, or even unable, to reverse.” (6)

The Up and down regulation of the GABA receptors are a form of Gene Regulation. Gene Regulation is the modulation of any of the stages of Gene expression by the cell and is a homeostatic response. Gene regulation is essential for survival as it increases the versatility and adaptability of cells in our body by allowing the cells to express or deactivate proteins as needed. It determines which Genes will be expressed, when, and for how long. Through Gene Regulation, a cell can increase or decrease production of Gene products (proteins or RNA) which gives the cell flexibility to adapt to environmental changes, external signals, hormone levels, etc.

Gene Regulation and receptor internalization are a normal bodily functions and even occur in response to hormone levels in our body. For instance, elevated levels of Insulin will cause down-regulation of insulin receptors reducing sensitivity to the hormone. Exercise and diet can reverse this by increasing sensitivity. (13) Up and down regulation of certain GABA receptors occurs during a woman’s estrous cycle in response to changing levels of Progesterone. (12) Alterations in GABAA subunit expression also occur during anxiety, and chronic stress (17, 23). There are numerous circumstances in which the body utilizes Gene Regulation to maintain and restore equilibrium:

“GABAARs are not static entities on the neuronal cell surface but are believed to cycle continuously between the plasma membrane and intracellular compartments. The relative rates of receptor exo- and endocytosis are therefore key determinants in controlling the size of the postsynaptic pool accessible to GABA and GABAergic compounds and thus the strength of synaptic inhibition.” (37)

In the previous excerpt from the Ashton 2011 supplement, Ashton stated “down-regulated receptors are absorbed into neurons where, over time, they undergo various changes including alterations in gene expression” and “When these receptors are slowly reinstated after drug withdrawal, they may return in a slightly altered form.” What the precise changes were was not specified. However, there may be two separate issues here.

”GABAARs can be delivered to the cell surface either as newly assembled channel complexes via a de novo secretory pathway or reinserted following internalization.” (37)

Receptors that have been internalized (aka- down-regulated, or endoscytosed) during tolerance would most likely not be recycled if the drug is still present for an extended period of time. These internalized receptors would therefore be subject to ubiquitination, and thus would not be recycled to the cell surface:

“Endocytosed GABAARs that fail to be recycled are targeted for lysosomal degradation…” (36)

Researchers observed a change in subunit configuration after chronic benzodiazepine use that appeared to originate in the expression of new receptors. In this study the neuron apparently swaps out receptors with subunit configurations sensitive to benzodiazepine binding with ones that are not:

“In rats given benzodiazepines chronically, the common α 1 γ2 sub-units are down-regulated, while rarer sub-units are elevated proportionately (Holt et al, 1999). It is suggested that transcription of the Gene cluster on Chromosome 5 (which encodes for α1 β2 γ2 sub-units) is inhibited on chronic benzodiazepine administration, while the transcription of the Gene cluster on Chromosome 15 is upregulated (Holt et al, 1999). In certain brain regions, the Chromosome-5-encoded receptor sub-unit proteins are replaced by those encoded in Chromosome 15, which show less sensitivity.” (7)

In this situation, the neurons apparently switched out receptors that had high sensitivity to benzodiazepines for ones with low sensitivity to counteract the effect of the drug. Changes to receptor subunit configuration and phosphorylation have been suggested to be possible causes of receptor uncoupling:

“Long-term treatment of rats with BZs results in so-called “uncoupling,” a decrease in the ability of BZs to potentiate the action of GABA on GABAA receptors and in a decrease in the ability of GABA to potentiate BZ binding (Gallager et al., 1984; Marley and Gallager, 1989; Tietz et al., 1989). This uncoupling might be due to changes from BZ-sensitive to -insensitive receptor subtypes (changes in receptor subunit combination) and/or changes in receptor function without changes in receptor subtype.” (18)

“A decreased coupling may develop as a result of changed GABAA receptor subunit composition, alterations to the GABAA receptor itself (including phosphorylation) or its second messenger ligands, or any process affecting the conformational state of the GABAA receptor.” (45)

It is assumed after the long term benzodiazepine treatment of the rats that there was no examination of what happened with these changes after withdrawal recovery, since the animals were most likely destroyed in order to examine these changes while still in the state of tolerance. However, it stands to reason that these changes could reverse to the pre-benzo state after the drug was discontinued for the same reason they ensued in the first place- to restore homeostasis- since neuroadaptations the body makes to counteract the effects of benzos are functional, or plastic changes:

“Benzodiazepine tolerance is considered to constitute an adaptive mechanism following chronic treatment, and it may thus be regarded as an example of neuronal plasticity.” (45)

Glutamate receptors can become up-regulated during benzodiazepine usage to compensate for benzodiazepine induced enhancement of inhibition. (18) Changes to Glutamate receptors have an enormous effect on cell function:

“…glutamate-gated channel opening of NMDA receptors enables calcium (Ca2+) influx into the dendritic spine, which initiates a cascade of signaling events involving the stimulation of the Ca2+/calmodulin-dependent protein kinase (CaMK) as well as the extracellular signal regulated kinase (ERK). The stimulation of CaMK and ERK triggers the phosphorylation-induced activation of a myriad of cellular targets including ion channels and transmembrane receptors, which in turn modifies their conductance properties.” (51, 52)

Activation of these signaling pathways can regulate the activity of nuclear factors triggering changes in gene transcription, and perhaps activate transcription regulators. (51) Adding to the difficulty of sorting it all out, there are many stages in gene expression where changes in GABAAR subunit expression might occur:

“The precise mechanisms that account for alterations in GABAAR and GABABR subunit Gene expression are only now beginning to be revealed. Exciting new discoveries indicate that GABA-R subunit expression is controlled by multiple levels ranging from subunit Gene expression to control of protein turn over by cell-signaling pathways. Several mechanisms that underlie regulation of subunit expression include: chromatin remodeling, transcription initiation, alternative splicing, messenger RNA (mRNA) stability, translation, post translational modification, intracellular trafficking, and protein degradation.” (22)

“If prolonged activation of the GABA system leads to receptor downregulation, then this could be established by interfering at multiple steps of the dynamic GABAA receptor life cycle. These include decreased subunit mRNA transcription, subunit degradation in the endoplasmic reticulum (e.g., by ubiquitylation), decreased expression of GABAA receptor-associated helper proteins, and alterations in the endocytosis of specific GABAA receptor subtypes.” (45)

Success stories offer anecdotal evidence that reversal neuroadaptive changes can and do happen anywhere from months to years after benzodiazepine discontinuation. When studying benzodiazepines, what most research studies consider ‘long term’ benzodiazepine treatment only tends to be anywhere from 7-32 days, and, the effects in neurons after withdrawal are examined only anywhere from 6 hours to 7 days after withdrawal has been induced. (18) To my knowledge there have not been any long term studies examining reversals of neuroadaptations after benzo withdrawal. Researchers make assumptions as to the reversibility of these changes based on their observations, and without long term studies, any claims regarding irreversibility of changes to gene expression would be unsubstantiated, and perhaps, reckless.

Researchers have made associations between susceptibilities to tolerance and SNPs. ( 8 ) These discoveries may help explain why some people can take benzodiazepines without experiencing tolerance or withdrawal and others cannot.

End quote

Conclusion: Final comments and summary on downregulation and sensitization, GABAa receptors, and chronic benzodiazaphine use

When something is upregulated , the receptors that are created, are not necessarily more sensitized(than their “original peers”) to the endogenous ligand that binds to them. They might be, but this does not have to be the case. Physiologically this can occur, but conceptually, receptor sensitivity and upregulation/downregulation are separate processes.

(Upregulation or downregulation) vs. (sensitization or desensitization) should be thought of as 2 different conceptual processes. However, I do understand that all processes like these are interrelated physiologically , especially during Benzo tolerance and recovery. However, if one does not clearly define the terms, discussions can get very confusing.

For simplicity, upregulation and downregulation should be thought of in terms of receptor population at the postsynaptic membrane. If a receptor population is upregulating to an antagonist drug, one does not know for sure how sensitive the new receptors (that are popping up) are in terms of their response to endogenous ligand(GABA, in this case). If a receptor population is downregulating to an agonist or PAM (benzos!), one does not know how long the person can be on the drug (agonist or PAM) before the receptors that have be endocytized (absorbed) have any chance of coming back, and if they are recycled, how sensitive they will be. One will see this is the case in section (1) below, when a review of GABAa receptors during benzodiazaphine tolerance is done. The receptors can be destroyed as well (see the nice figure in one of the references). This chart illustrates how the receptors that are endocytized can be either destroyed or recycled.

https://www.hindawi.com/journals/aps/2012/416864/fig2/

We don’t know enough about this HAP1 protein that recycles the receptor , to access how sensitized the receptor is when it comes back, or if it can even do things like that.

Quote

GABAA receptors are delivered by a clathrin-mediated pathway to early endosomes where they can be targeted for degradation in the lysosome or for recycling upon binding of Huntington-associated protein (HAP1).

End quote

https://en.m.wikipedia.org/wiki/Huntingtin-associated_protein_1

This HAP1 interacts with vesicular trafficking (like in the diagram). One can note in the list below GABAaRs are included, but no other receptors are listed. This might relate to why it’s so difficult to repopulate GABAa receptors after they have been downregulated ……..

Quote

The role of HAP1 in HD pathogenesis may involve aberration of cell cycle processes, as high immunostaining of HAP1 during the cell cycle has been observed. It may have a part in spindle orientation, microtubule stabilization or chromosome movement. More importantly, HAP1 may also disrupt endocytosis, as it has been detected on vesicles involved in the early stages of this process. It is possible that the non-pathogenic activity of HAP1 is intracellular trafficking and that this is perturbed following its association with mHtt. HAP1 also interacts with proteins other than Htt and it is likely that their function is altered in HD pathogenesis.

….

HAP1 also interacts with other factors involved in vesicular trafficking including GABAA receptor, Rho-GEF, and HGS.

End quote

Alternatively, the cell can create new receptors from scratch via exocytosis, after the benzodiazaphine is discontinued. Whether or not they are “normal” we don’t know. We don’t have studies in this area.

An important point is that even though the individual receptors may not have changed in sensitivity, when a population increases (upregulation), the CELL’S sensitivity to the ligand effectively increases, and when a population decreases (downregulated) , the CELL’S sensitivity to the ligand decreases. This makes sense because the up/downregulation are inherently cellular modulation processes.

In the same way, one can think of sensitization and desensitization as conceptually separate from downregulation and upregulation. One can think of sensitization and desensitization as things that happen to the receptor itself. Changes in subunit composition involving changes in gene expression, and phosphorylation (uncoupling is thought to occur as a result). Uncoupling is the most promising theory .

Number 4 in the list, lateral diffusion of the receptors , to reduce sensitivity(move out) or increase sensitivity (move in) is more related to a population of receptors, rather than changes to the receptor itself. In this way, if I had to choose, I would put it in the downregulation / upregulation category.

Physiologically these separate conceptual terms of course overlap. For example, the theory is that the receptors are downregulated, then once the benzodiazaphine is cleared from the system, the receptors are repopulated, and may come back with different sensitivity to the GABA ligand.

The research in tolerance and interdose withdrawal, is very sparse. That’s why I’m focused in this area, because the majority of us are in this grey zone, and suffering. Furthermore , the research in long term studies, as Pers indicated are very lacking. Both of these areas are required to answer the additional questions that we have in this area. Until then, we are dealing with some facts and some theory.

Where does this leave us? We know how we feel, and it’s safe to assume that some of the theories are probably correct, and I’ve read of success stories on here where people recover years after their last dose. So it’s safe to assume that in some of us, the process is slow, but nevertheless the body does heal….

August 4, 2017

I read all the theory ...

If I were to keep it very simple, 'Receptors are created, or expressed, by the DNA of the cell, and they can be increased, or upregulated, when the signal is weak, or decreased, or downregulated, when it is strong.[1]

Downregulation of receptors happens when receptors have been chronically exposed to an excessive amount of neurotransmitters, whether endogenous or drugs. This results in ligand-induced desensitization or internalization (absorption) of that receptor. It is usually exhibited by various hormone receptors. Upregulation of receptors, on the other hand, results in super-sensitized receptors especially after repeated exposure to the drug.'

It should come back fine after you're off the drug. So what's the problem ? (after reading that twice, they would just be sensitized). Many people believe that time off the drug heals all, no matter what. What most doctors believe.

Then there is that other theory that receptors would come back in altered form ... I'm not so sure it's that simple but you wrote that already, at least partially.

I, for one, never fully recovered from the time I was on lorazepam. Still on clonazepam, of course.

And do those receptors grow back, no matter what ? If the intructions are just in the DNA ? What if they grow back to get things just functioning on a minimal level ?

Good work.

One would wonder if 'super-sensitized receptors' are somehow linked to cholineric hyper/supersensitivity. Dependence mediated by cholineric neurons.

And as a side note to the last statement and that is a bit personal, chronic stress could cause cholinergic hyper/supersensitivity (?) https://www.researchgate.net/publication/19869551_Effects_of_stress_on_muscarinic_mechanisms https://www.researchgate.net/publication/19419860_Stress_induces_supersensitivity_of_a_cholinergic_system_in_rats etc.)

One wonders what that would do ...

August 5, 2017

I read all the theory ...

If I were to keep it very simple, 'Receptors are created, or expressed, by the DNA of the cell, and they can be increased, or upregulated, when the signal is weak, or decreased, or downregulated, when it is strong.[1]
Downregulation of receptors happens when receptors have been chronically exposed to an excessive amount of neurotransmitters, whether endogenous or drugs. This results in ligand-induced desensitization or internalization (absorption) of that receptor. It is usually exhibited by various hormone receptors. Upregulation of receptors, on the other hand, results in super-sensitized receptors especially after repeated exposure to the drug.'

It should come back fine after you're off the drug. So what's the problem ? (after reading that twice, they would just be sensitized). Many people believe that time off the drug heals all, no matter what. What most doctors believe.

Then there is that other theory that receptors would come back in altered form ... I'm not so sure it's that simple but you wrote that already, at least partially.

I, for one, never fully recovered from the time I was on lorazepam. Still on clonazepam, of course.

And do those receptors grow back, no matter what ? If the intructions are just in the DNA ? What if they grow back to get things just functioning on a minimal level ?

Good work.

One would wonder if 'super-sensitized receptors' are somehow linked to cholineric hyper/supersensitivity. Dependence mediated by cholineric neurons.

And as a side note to the last statement and that is a bit personal, chronic stress could cause cholinergic hyper/supersensitivity (?) https://www.researchgate.net/publication/19869551_Effects_of_stress_on_muscarinic_mechanisms https://www.researchgate.net/publication/19419860_Stress_induces_supersensitivity_of_a_cholinergic_system_in_rats etc.)
One wonders what that would do ...

Hi liberty I removed this portion of the Wikipedia quote in my post. I don't like it and it's not footnoted. It mixes sensitization and up/down regulation ,which is very confusing to the reader. There are other references that are quoted that make this distinction much clearer.

REMOVED quote

Downregulation of receptors happens when receptors have been chronically exposed to an excessive amount of neurotransmitters, whether endogenous or drugs. This results in ligand-induced desensitization or internalization (absorption) of that receptor. It is usually exhibited by various hormone receptors. Upregulation of receptors, on the other hand, results in super-sensitized receptors especially after repeated exposure to the drug.

End

Regarding your post

Great points!

Reason why I removed that quote above from the Original post:

I’m not sure if when something is upregulated , the receptors that are created, are more sensitized(than their “original peers”) to the endogenous ligand that binds to them. They might be.

(Upregulation or downregulation) vs. (sensitization or desensitization) should be thought of as 2 different conceptual processes. However, I do understand that all processes like these are interrelated physiologically , especially during Benzo tolerance and recovery. However, if we don’t clearly define the terms, discussions can get very confusing.

For simplicity, I like to think of upregulation and downregulation in terms of receptor population at the postsynaptic membrane. If a receptor population is upregulating to an antagonist drug, we don’t know for sure how sensitive the new receptors (that are popping up) are in terms of their response to endogenous ligand(GABA, in this case) If a receptor population is downregulating to an agonist or PAM (benzos!), we don’t know how long the person can be on the drug (agonist or PAM) before the receptors that have be endocytized (absorbed) have any chance of coming back, and if they are recycled, how sensitive they will be(as the quotes above state). They might be destroyed as well (see the nice figure in one of the references). I really like this chart

https://www.hindawi.com/journals/aps/2012/416864/fig2/

I don’t know enough about this HAP1 protein that recycles the receptor , to access how sensitized the receptor is when it comes back, or if it can even do things like that.

Quote

GABAA receptors are delivered by a clathrin-mediated pathway to early endosomes where they can be targeted for degradation in the lysosome or for recycling upon binding of Huntington-associated protein (HAP1).

End quote

https://en.m.wikipedia.org/wiki/Huntingtin-associated_protein_1

This HAP1 interacts with vesicular trafficking (like in the diagram). I notice the list below includes GABAaR, and the list is very short, i.e. Not a lot of other receptors there  This might relate to why it’s so difficult to repopulate GABAa receptors after they have been downregulated ……..

Quote

The role of HAP1 in HD pathogenesis may involve aberration of cell cycle processes, as high immunostaining of HAP1 during the cell cycle has been observed. It may have a part in spindle orientation, microtubule stabilization or chromosome movement. More importantly, HAP1 may also disrupt endocytosis, as it has been detected on vesicles involved in the early stages of this process. It is possible that the non-pathogenic activity of HAP1 is intracellular trafficking and that this is perturbed following its association with mHtt. HAP1 also interacts with proteins other than Htt and it is likely that their function is altered in HD pathogenesis.

….

HAP1 also interacts with other factors involved in vesicular trafficking including GABAA receptor, Rho-GEF, and HGS.

End quote

Alternatively, the cell can create new receptors from scratch via exocytosis, after the Benzo is docontinued. Whether or not they are “normal” we don’t know. We don’t have studies in this area.

An important point is that even though the individual receptors may not have changed in sensitivity, when a population increases (upregulation) the CELL’S sensitivity to the ligand effectively increases, and when a population decreases (downregulated) the CELL’S sensitivity to the ligand decreases. This makes sense because the up/downregulation are inherently cellular modulation processes.

--------------------

In the same way, I like to think of sensitization and desensitization as conceptually separate from downregulation and upregulation. I think of sensitization and desensitization as things that happen to the receptor itself. Changes in subunit composition involving changes in gene expression, ‘and phosphorylation (uncoupling is thought to occur as a result). Uncoupling is the most promising theory .

Number 4 in the list, lateral diffusion of the receptors , to reduce sensitivity(move out) or increase sensitivity (move in) is more related to a population of receptors, rather than changes to the receptor itself. In this way, if I had to choose, I would put it in the downregulation / upregulation category.

You bring up great questions, but the fact is, the research in tolerance and interdose withdrawal, is very sparse. That’s why I’m focused in this area, because the majority of us are in this grey zone, and suffering. Furthermore , the research in long term studies, as Pers indicated are very lacking. Both of these areas are required to answer your excellent questions. Until then we are dealing with some facts and some theory.

Regarding ACh, I think its related to the GABAa system via the mAChR (muscarinic) receptor, and you have posted several great studies to get us started in that direction of research. It’s part a larger group of GPCRs that I believe are dysfunctional with long term benzodiazaphine use.

Where does this leave us? We know how we feel, and it’s safe to assume that some of the theories are probably correct, and I’ve read of success stories on here where people recover years after their last dose. So it’s safe to assume that in some of us, the process is slow, but nevertheless the body does heal….

August 5, 2017

Thanks again for these posts! Somehow knowing a bit about brain neuro-plasticity and the information here, I truly find hope that we all can fully recover in time. It's all helpful to know.

August 5, 2017

dm123,

That's so much text ... and I'm not at my best right now.

Quoting myself:

'And as a side note to the last statement and that is a bit personal, chronic stress could cause cholinergic hyper/supersensitivity (?) https://www.researchgate.net/publication/19869551_Effects_of_stress_on_muscarinic_mechanisms https://www.researchgate.net/publication/19419860_Stress_induces_supersensitivity_of_a_cholinergic_system_in_rats etc.)

One wonders what that would do ...'

Here is one: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1993356/ depression/stress 'Although it is tempting to speculate on the use of cholinergic hypersensitivity as a trait marker (cause) vs. a state marker (consequence), the data suggest that the mechanism for this hypersensitivity is likely an early event in the onset of the disease, and these earlier events are mediated, at least in part, by nicotinic rather than muscarinic mechanisms. These data further suggest that the development of specific nicotinic agonists selectively targeting the β2 subunit of the receptor will have therapeutic benefits in depressive disorders.' I read a lot of that years ago, it's one of the older theories (before the monoamine hypothesis) of biological psychiatry. 'sensitized neurons'

I was wondering if that chronic stress (not directly benzo related), assuming it would cause cholinergic hyper/supersensitivity, would affect the GABAA receptor, possible the ''benzodiazepine receptor' !

If that falls beyond the scope of what you are doing, fine. I was curious about that. Sorry, I'm not having my day.

August 5, 2017

dm123,

That's so much text ... and I'm not at my best right now.

Quoting myself:
'And as a side note to the last statement and that is a bit personal, chronic stress could cause cholinergic hyper/supersensitivity (?) https://www.researchgate.net/publication/19869551_Effects_of_stress_on_muscarinic_mechanisms https://www.researchgate.net/publication/19419860_Stress_induces_supersensitivity_of_a_cholinergic_system_in_rats etc.)
One wonders what that would do ...'

<too bad I can't insert PDF files I found>

Here is one: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1993356/ depression/stress 'Although it is tempting to speculate on the use of cholinergic hypersensitivity as a trait marker (cause) vs. a state marker (consequence), the data suggest that the mechanism for this hypersensitivity is likely an early event in the onset of the disease, and these earlier events are mediated, at least in part, by nicotinic rather than muscarinic mechanisms. These data further suggest that the development of specific nicotinic agonists selectively targeting the β2 subunit of the receptor will have therapeutic benefits in depressive disorders.' I read a lot of that years ago, it's one of the older theories (before the monoamine hypothesis) of biological psychiatry. 'sensitized neurons'

I was wondering if that chronic stress (not directly benzo related), assuming it would cause cholinergic hyper/supersensitivity, would affect the GABAA receptor, possible the ''benzodiazepine receptor' !

If that falls beyond the scope of what you are doing, fine. I was curious about that. Sorry, I'm not having my day.

Hi liberty, hope you feel better soon. I incorporated some changes to the original ADDENDUM 6 to clarify things further based on your comments. When you feel better you can just reskim it over.

Regarding your question, the short answer is yes. But I have to do more research in this area of GPCRs. The dopamine,serotonin, and ACh have all been tied to GABAa receptors. It's thought that these types of receptors modulate GABAa beta and gamma2 subunit phosphorylation via protein kinases (PKA and PKC, for those interested). In context of your question, I think the mAchR(muscarinic)receptor would apply. The references that I have to go through are below. Once I go through them I can read your links that you've sent regarding cholinergenic hypersensitivity and stress.

Note that last link you supplied I s nicotinic and not muscarinic.

Nicotinic receptors are not GPCRs.

Here are the references. Hope you feel better soon

The first 5 are the important ones

J. T. Kittler and S. J. Moss, “Modulation of GABAA receptor activity by phosphorylation and receptor trafficking: implications for the efficacy of synaptic inhibition,” Current Opinion in Neurobiology, vol. 13, no. 3, pp. 341–347, 2003. View at Publisher · View at Google Scholar · View at Scopus

143-149

1. Z. Yan, “Regulation of GABAergic inhibition by serotonin signaling in prefrontal cortex: molecular mechanisms and functional implications,” Molecular Neurobiology, vol. 26, no. 2-3, pp. 203–216, 2002.View at Publisher · View at Google Scholar · View at PubMed · View at Scopus

2. J. Feng, X. Cai, J. Zhao, and Z. Yan, “Serotonin receptors modulate GABAA receptor channels through activation of anchored protein kinase C in prefrontal cortical neurons,” Journal of Neuroscience, vol. 21, no. 17, pp. 6502–6511, 2001. View at Google Scholar · View at Scopus

3. N. J. Brandon, J. N. Jovanovic, T. G. Smart, and S. J. Moss, “Receptor for activated C kinase-1 facilitates protein kinase C-dependent phosphorylation and functional modulation of GABAA receptors with the activation of G-protein-coupled receptors,” Journal of Neuroscience, vol. 22, no. 15, pp. 6353–6361, 2002.View at Google Scholar · View at Scopus

4. X. Wang, P. Zhong, and Z. Yan, “Dopamine D4 receptors modulate GABAergic signaling in pyramidal neurons of prefrontal cortex,” Journal of Neuroscience, vol. 22, no. 21, pp. 9185–9193, 2002. View at Google Scholar · View at Scopus

5. D. J. Nutt, P. J. Cowen, and M. Franklin, “The effect of diazepam on indices of 5-HT function in man,” Pharmacology Biochemistry and Behavior, vol. 24, no. 5, pp. 1491–1495, 1986. View at Google Scholar

6. A. Khan and D. J. Haleem, “Tolerance in the anxiolytic profile following repeated administration of diazepam but not buspirone is associated with a decrease in the responsiveness of postsynaptic 5-HT-1A receptors,” Acta Biologica Hungarica, vol. 58, no. 4, pp. 345–357, 2007. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus

7. A. A. Hegarty and W. H. Vogel, “The effect of acute and chronic diazepam treatment on stress-induced changes in cortical dopamine in the rat,” Pharmacology Biochemistry and Behavior, vol. 52, no. 4, pp. 771–778, 1995. View at Publisher · View at Google Scholar

August 6, 2017

Wonderful video but it does not take into account we are all missing a few billion of those receptors now or that maybe they just changed the way they operate called "CONFORMATIONAL BRAIN CHANGES" (Google it)

How it works is great to know if you have any left over receptors to fix. Luckily the brain is "PLASTIC" and it can make other adjustments to reach a balance like lowering the amount of glutamate it needs to release in order to operate but it's a very slow process. Which is why taper speed should not exceed healing speed.

August 7, 2017

For much more detail on GABAaR endocytosis and exocytosis, as well as lateral diffusion and clustering, GABAa membrane trafficking, and post-endocytic GABAa sorting see sections V, VI, VII, VIII, IX of the document below. Figures and tables are at the end of the PDF.

(Also see https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709246/ Or PDF

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709246/pdf/nihms83460.pdf

; it’s an earlier article)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4382539/pdf/nihms675517.pdf

Or

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4382539/

August 7, 2017

For much more detail on GABAaR endocytosis and exocytosis, as well as lateral diffusion and clustering, GABAa membrane trafficking, and post-endocytic GABAa sorting see sections V, VI, VII, VIII, IX of the document below. Figures and tables are at the end of the PDF.
(Also see https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709246/ ; it’s an earlier article)
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4382539/pdf/nihms675517.pdf
Or
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4382539/

Interesting that GabaA receptors effect schizophrenia. I felt schizophrenic during fast withdrawal and had classic schizophrenia behavior too so my close friends told me.

we highlight a number of neurological disorders, including epilepsy and schizophrenia

August 8, 2017

For much more detail on GABAaR endocytosis and exocytosis, as well as lateral diffusion and clustering, GABAa membrane trafficking, and post-endocytic GABAa sorting see sections V, VI, VII, VIII, IX of the document below. Figures and tables are at the end of the PDF.
(Also see https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709246/ ; it’s an earlier article)
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4382539/pdf/nihms675517.pdf
Or
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4382539/

Interesting that GabaA receptors effect schizophrenia. I felt schizophrenic during fast withdrawal and had classic schizophrenia behavior too so my close friends told me.

we highlight a number of neurological disorders, including epilepsy and schizophrenia

Section 6 of the older article has very interesting comments on this, as well as alchohol and benzodiazaphine use. It's no wonder why a drug that modulates GABAa receptors causes so many psychiatric issues upon discontinuation . This receptor should have never been tinkered with via man made drugs,

August 8, 2017

In continuing the homeostatic theme, I thought that this part of the 2008 reference was very interesting. Neuronal stimulation . So it's good to try to use your mind as much as possible, stimulate it without too much stress by learning new things, distraction, and even exercise, etc.

From section 2. Controlling GABAaR assembly

Section 2

Quote

Activity-dependent GABAAR ubiquitination The ER is responsible for the retention and degradation of misfolded or unassembled subunits and, accordingly, homomeric unassembled GABAARs subunits have been shown to be degraded in this organelle14,18. ER-associated degradation (ERAD) involves protein ubiquitination and degradation via the ubiquitin-proteasome system (UPS)19. GABAAR subunits have recently been shown to be ubiquitinated in an activity-dependent manner20. Chronic blockade of neuronal activity dramatically increased the levels of GABAAR ubiquitination within the ER, resulting in decreased insertion at the plasma membrane20. Correspondingly, increasing the level of neuronal activity resulted in a decrease in the level of GABAAR ubiquitination and an enhancement of receptor cell surface expression20. Thus, neuronal activity can regulate the ubiquitination of GABAARs in the ER, affecting their rate of degradation via the UPS. This may be one mechanism neurons use to homeostatically regulate synaptic inhibition.

End quote

August 8, 2017

More on HAP1 alluded to in ADDENDUM 6 above

HAP1 and recycling….

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709246/

Section 5.

Quote

Once endocytosed, most internalized GABAARs recycle back to the plasma membrane over short time frames; however over longer time periods they are targeted for lysosomal degradation88. Clearly the fate of internalized GABAARs will therefore play a critical role in controlling cell surface receptor levels and hence the efficacy of synaptic inhibition. Huntingtin associated protein-1 (HAP1)98 is a GABAAR associated protein that binds the intracellular loop of β subunits in vitro and in vivo88. HAP1 is a cytoplasmic protein with several central coil-coiled domains that are likely to regulate

protein-protein interactions. Overexpression of HAP1 in neurons inhibits GABAAR degradation and consequently increases receptor recycling88. Furthermore, HAP1 overexpression increased steady state surface levels of GABAARs and produced a 63% increase in mIPSC amplitude, showing a dramatic functional effect of increased surface receptor number88. The mechanism underlying post-endocytic GABAAR sorting remains to be elucidated, and HAP1’s specific role in this process is also an area of active research. The impact of HAP1 regulation of GABAARs was recently shown in the hypothalamus, where down-regulation of HAP1 levels resulted in decreased GABAAR levels, causing decreased food intake and loss of body weight99. An unresolved issue is whether HAP1 acts to promote recycling of GABAARs or prevents their lysosomal degradation

End quote

August 9, 2017

Hi

I wanted to continue ADDENDUM 6 and expand on item 4 of the first part above: namely, present more detail on the GABAa receptor trafficking, lateral diffusion, and clustering, how this actually occurs in terms of specialized proteins, and finally present why it's important for us to understand this process.

One of the main questions one might have after reading ADDENDUM 6 is: ok, we are downregulated during benzodiazaphine use, we were hitting tolerance , and now we are tapered off. In what form do the receptors come back, and is the subunit configuration of these receptors in a given cluster similar to what we had before? The discussion below might shed light on one possible scenario that can occur based on alcohol studies.

ADDENDUM 6 continued: part II: lateral diffusion, postsynaptic targeting (i.e., where they end up on the membrane) of GABAa receptors, and clustering of GABAa receptors

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709246/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4382539/

Where the GABAa receptors end up is very important. The location can be either synaptic or extrasynaptic, and this is primarily dictated by their subunit composition. As indicated above, once in place, the GABAa receptors can move laterally. It has been found that GABAa receptors located synaptically are benzodiazaphine sensitive, whereas those that are benzodiazaphine insensitive are mostly located extra synaptically. The synaptically located GABAa receptors typically have an (alpha 1,2,or3)(beta 1,2,or 3)(gamma, typically 2) subunit configuration. The extrasynaptically located receptors typically have a (alpha 5)(beta)(gamma) or (alpha 4)(beta)(delta) or (alpha 6)(beta)(delta) configuration. Why particular subunit receptors move to a particular location is something that is dictated by specialized proteins. This will be explained below.

The synaptically located receptors are found to regulate the phasic mode of inhibition, i.e. Short lasting during an action potential, whereas the extra synaptically located receptors are found to regulate the long lasting tonic mode of inhibition. The lateral diffusion of the receptors out of the synaptic location as a means to achieve homeostasis during benzodiazaphines use, makes sense, when viewed in this simplistic manner.

Quote

After navigating their way through the secretory pathway, GABAARs are inserted into the plasma membrane, where they are able to access inhibitory postsynaptic specializations or extrasynaptic sites, depending on subunit composition (Fig. 3). The mechanisms that facilitate these distinct subcellular fates are described below.

GABAARs on the neuronal cell surface exist as diffuse receptor populations or synaptic or extrasynaptic clusters. Lateral diffusion within the plasma membrane allows for continual exchange between these receptor populations55,56. GABAARs that can bind bungarotoxin have been used to examine the subcellular sites of GABAAR insertion into the neuronal membrane. These studies have demonstrated that most receptors are delivered to extrasynaptic locations in the plasma membrane. Over time, diffusion and trapping increases the population of synaptic receptors57.

Heteromeric GABAARs retain distinct cell surface expression patterns dependent on subunit composition. Most surface receptor clusters of γ2 receptor subunits are synaptic, whereas β3-containing GABAARs have a higher proportion of diffuse and/or extrasynaptic receptors55,58. α5-containing receptors are predominantly extrasynaptic8,9. Other receptor subunits, such as δ, appear as diffuse populations on the neuronal surface59,60 and are exclusively located outside the synapse at perisynaptic and extrasynaptic locations57,61. These extrasynaptic α5- and δ- containing GABAARs are considered the main receptors mediating tonic inhibition.

End quote

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709246/figure/F1/?report=objectonly

Quote

GABAA receptor structure and neuronal localization

(A) GABAA receptors are members of the ligand-gated ion channel superfamily. Receptor subunits consist of four hydrophobic transmembrane (TM1–4) domains, where TM2 is believed to line the pore of the channel. The large extracellular N-terminus is the site for the binding of the neurotransmitter GABA, as well as containing binding sites for psychoactive drugs, such as benzodiazepines (BZ). Each receptor subunit also contains a large intracellular domain between TM3 and TM4, which is the site for various protein interactions as well as the site for various post-translational modifications that modulate receptor activity. (B) Five subunits from 7 subunit subfamilies (α,β,γ,δ,ε,θ,π) assemble to form a heteropentameric chloride-permeable channel. Despite the extensive heterogeneity of GABAA receptor subunits, the majority of GABAAreceptors expressed in the brain consist of 2α, 2β, and 1γ subunit, where the γ subunit can be replaced by δ, ε or π. Binding of the neurotransmitter GABA occurs at the interface between the α and β subunits and triggers the opening of the channel, allowing the rapid influx of chloride ions. BZ-binding occurs at the interface between α(1,2,3 or 5) and γ subunits and potentiates GABA-induced chloride flux. © GABAA receptors composed of α(1–3) subunits together with β and γ subunits are thought to be primarily synaptically localized, whereas α5βγ receptors are located largely at extrasynaptic sites. Receptors composed of the aforementioned subunits are benzodiazepine-sensitive. In contrast, receptors composed of α(4,6)βδ are benzodiazepine-insensitive, and are localized at extrasynaptic sites.

End quote

Phasic vs. tonic inhibition

http://www.scholarpedia.org/article/Neural_inhibition

Quote

In contrast, during postsynaptic inhibition, the inhibitory transmitter receptors are located on the postsynaptic neuron. Activation of the receptors leads to an increase in postsynaptic conductance, a change in the membrane potential of the postsynaptic neuron, or a combination of both. Ultimately these effects can lead to the inhibition of action potential generation in the postsynaptic cell. Second, inhibition can be "phasic" or "tonic". Phasic inhibition is a short-lasting inhibition typically generated by the activation of GABAA receptors following action potentials in a presynaptic interneuron. However, there are also more long-lasting forms of inhibition. One form is the activation of GABAB receptors by spillover of GABA caused by GABA release from specialized interneurons. A second form is the "tonic" GABAA conductance activated by ambient GABA in the extracellular space (Farrant and Nusser, 2005). This form of inhibition is mediated by molecularly and functionally specialized GABAA receptors. These receptors contain alpha6 or delta subunits, which display a high affinity for GABA binding. Furthermore, a long-lasting form of inhibition can be mediated by asynchronous or spontaneous vesicular release of GABA.

End quote

Quote

Differences in subunit composition between synaptic and extrasynaptic receptors are reflected in a differential modulation of phasic and tonic signaling by a number of compounds of therapeutic importance. The most frequently cited example of this is the role of the α subunit in defining receptor affinity for benzodiazepines. GABAARs incorporating either an α4 or α6 subunit renders receptors insensitivity to fumctional modulation by benzodiazepines (Benson et al., 1998) as does elimination or substitution of the γ2 subunit. This difference can be attributed solely to the presence of a conserved arginine residue in α4 and α6 subunits, which in α1–3 and α5 subunits is a histidine (Wieland et al., 1992). Thus benzodiazepine site ligands selective for α1–3 subunits largely influence phasic signaling, whereas those selective for α5 subunits are capable of primarily modulating tonic conductance.

End quote

The diagram below illustrates the receptor lateral mobility in more detail and the anchoring proteins involved in scaffolding the receptors to the synaptic site(gephyrin), and extrasynaptic sites (radixin). Gephyrin plays a critical role in anchoring and lateral diffusion of newly inserted receptors into the synaptic sites. The newly inserted receptors are on the right side called “Diffuse receptors”. The extrasynaptic cluster is on the left side and is anchored via radixin which directly binds F-actin, a modeling protein.

Figure

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709246/figure/F3/?report=objectonly

Quote

One protein strongly implicated in regulating the clustering of GABAARs at inhibitory synapses is the multifunctional protein gephyrin, which was first identified by its association with glycine receptors62. Gephyrin binds directly with the intracellular domain of the β subunit of glycine receptors, which stabilizes these proteins at inhibitory synapses in the spinal cord63–67. Gephyrin is also widely expressed in non-neuronal tissues65. In the brain, it is found in neurons, and is enriched at postsynaptic specializations that contain GABAAR subtypes composed of α(1–3), β(2,3) and γ2 subunits68.

End quote

Quote

The highly selective subcellular location of GABAaR subtypes implies that subunit composition plays a major role in the postsynaptic targeting and

clustering of these receptors. While the exact molecular mechanisms that govern the accumulation of GABAARs at inhibitory synapses are not yet fully understood, it involves a number of receptor-associated proteins and cytoskeletal elements that are concentrated at postsynaptic densities (PSDs).

End quote

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709246/figure/F3/?report=objectonly

Quote from figure above

Dynamic regulation of receptor lateral mobility at the GABAergic synapse

GABAA receptors are inserted into the plasma membrane at extrasynaptic sites, where they can then diffuse into synaptic sites. Lateral diffusion (black arrows) within the plasma membrane allows for continual exchange between diffuse receptor populations and synaptic or extrasynaptic receptor clusters, with anchoring molecules tethering or corralling moving receptors. The synaptic localization of α2-containing GABAAreceptors is maintained by direct binding to gephyrin, which binds to microtubules and actin interactors such as the GDP/GTP exchange factor collybistin72, Mena/ VASP (vasodilator-stimulated phosphoprotein)144 and profilins 1 and 2144,145. No direct interaction between gephyrin and the γ2 subunit has been demonstrated. However, gephyrin depletion increases γ2 cluster mobility, and loss of the γ2 subunit results in post-synaptic sites devoid of gephyrin. This suggests an unidentified intermediary interactor or a post-translational modification that could link γ2-containing receptors and gephyrin. Alternatively, clustering of γ2-containing receptors might occur via an independent mechanism. Gephyrin also displays local lateral movements (red double arrow), and removal or addition by microtubule dependent trafficking, contributing additional mechanisms to regulate synaptic transmission. Extrasynaptic localization of α5-containing GABAA receptors is controlled by binding to activated radixin, which directly binds F-actin.

End quote

Quote

GABAARs play a central role in mediating neuronal inhibition and mediating the effects of a broad range of anxiolytic, anticonvulsant, hypnotic and sedative agents. GABAARs mediate both phasic and tonic modes of inhibition, phenomena that are mediated via receptor subtypes with distinct molecular structures. Phasic inhibition is mediated largely by benzodiazepine-sensitive receptor subtypes that are highly enriched at synaptic sites and principally assembled from α1–3, β1–3 and γ2 subunits. A critical determinant of receptor number at inhibitory synapses is the steady-state cell levels of these receptors on the neuronal plasma membrane. This is likely determined by the relative rates of receptor exo- and endocytosis, processes that are intimately controlled by covalent modifications and interaction with specific binding partners. While the molecular details of these processes are being evaluated their significance for the efficacy of neuronal inhibition and ultimately for behavior remains to be established. Our comprehension of how GABAARs are selectively stabilized at inhibitory synapses is limited; however, a central role for gephyrin is emerging

End quote

Why is clustering subunit arrangement important to us?

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709246/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709246/figure/F5/?report=objectonly

Part B of the figure

From alcohol studies perhaps we can learn something.

-Significant changes in both the subunit composition and expression of the synaptic and extrasynaptic populations

-changes in phosphorylation of the receptor subunits and alterations of endocytosis for specific receptor subtypes.(phosphorylation is a means by which AP2, a protein that can bind to specific subunits in the GABAa receptor from inside of the membrane, is blocked. AP2 cannot bind to Subunits that are phosphorylated. AP2 binding to these subunits is necessary , if the receptor is to be absorbed , i.e. Endocytized. Phosphorylation can prevent this from happening and dephosphorylation permits the AP2 binding to occur, and for endocytosis to proceed)

-enhanced endocytosis (absorption) of alpha1 subunit synaptic receptors

-increased absorption of delta subunit extrasynaptic receptors

-most importantly, insertion of (alpha4,beta,gamma2) receptors at SYNAPTIC sites. These receptors don’t behave like the receptors we normally find in synaptic sites, and furthermore, they are benzodiazaphine insensitive!

Quote

Considerable evidence exists to support a role for GABAARs in mediating the addictive properties of drugs of abuse105,129. In particular, chronic use of alcohol or benzodiazepines, which are both allosteric modulators of GABAARs, can lead to drug tolerance, dependence and withdrawal symptoms following drug cessation. Changes in the mRNA and protein expression of various GABAAR subunits have been documented after alcohol and benzodiazepine administration in both cultured neurons and animal models130,131. However, the mechanisms responsible for these alterations have only recently begun to be elucidated. Significant alterations in the surface expression and composition of both synaptic and extrasynaptic GABAAR populations have been observed after a single intoxicating dose of alcohol in rats132 (Fig. 5B). These changes were found to be persistent after chronic alcohol administration and withdrawal132,133. This long-term plasticity in GABAARs is likely to involve changes in the phosphorylation of GABAAR subunits and alterations in the endocytosis of specific GABAAR subtypes. For example, the association of PKC with GABAAR subunits is altered after chronic ethanol exposure134. Increased associations between clathrin adaptor proteins and α1 subunits have also been demonstrated135, suggesting that enhanced clathrin-mediated endocytosis of α1-containing GABAARs contributes to changes in GABAAR trafficking after chronic alcohol use (Fig. 5B). Interestingly, the well-documented phenomenon of cross-tolerance to benzodiazepines after chronic alcohol use136 suggests that similar mechanisms may be responsible for tolerance to both of these drugs. Thus, understanding tolerance-inducing alterations in GABAAR trafficking should not only advance our

understanding of the disease process that leads to alcoholism, but also improve the development of drugs to treat insomnia and anxiety disorders without causing tolerance.

End quote

Dysregulation in GABAA receptor trafficking in neurological disease

Quote

(B) Alcohol-induced plasticity in GABAA receptors involves changes in both synaptic and extrasynaptic GABAA receptor populations. After alcohol administration, there is an increased internalization of δ-containing extrasynaptic GABAA receptors. There is also an increased internalization of α1-containing synaptic GABAA receptors via clathrin-dependent internalization. Insertion of distinct GABAA receptor populations at synaptic sites (ie. α4βγ2) have been hypothesized to serve a compensatory role at inhibitory synapses, however, these receptors differ in their physiological functions from normal synaptic GABAA receptor populations, as well as being benzodiazepine-insensitive.

End quote

August 9, 2017

dm123,

I doubt anyone has ever collected and integrated this knowledge the way you have !

I do have a question though.

[/i]

'Section 2

Quote

Activity-dependent GABAAR ubiquitination The ER is responsible for the retention and degradation of misfolded or unassembled subunits and, accordingly, homomeric unassembled GABAARs subunits have been shown to be degraded in this organelle14,18. ER-associated degradation (ERAD) involves protein ubiquitination and degradation via the ubiquitin-proteasome system (UPS)19. GABAAR subunits have recently been shown to be ubiquitinated in an activity-dependent manner20. Chronic blockade of neuronal activity dramatically increased the levels of GABAAR ubiquitination within the ER, resulting in decreased insertion at the plasma membrane20. Correspondingly, increasing the level of neuronal activity resulted in a decrease in the level of GABAAR ubiquitination and an enhancement of receptor cell surface expression20. Thus, neuronal activity can regulate the ubiquitination of GABAARs in the ER, affecting their rate of degradation via the UPS. This may be one mechanism neurons use to homeostatically regulate synaptic inhibition.

End quote'

I'm having a very hard time making sense of this. I did look up 'ubiquitination'. This blockade of neuronal activity, what kind are we talking about ? Inactivity, drugs, taking benzodiazepines ?

August 9, 2017

If I were to give information here to doctors and nurses, dm123, what in particular would I give them in order that they TRULY UNDERSTAND what goes on with having benzos in the system? And thank you again for all of your valuable research!

What would make the most impact, in other words, so finally they would GET IT!

August 9, 2017

Hi

I wanted to continue ADDENDUM 6 and expand on item 4 of the first part above: namely, present more detail on the GABAa receptor trafficking, lateral diffusion, and clustering, how this actually occurs in terms of specialized proteins, and finally present why it's important for us to understand this process.

Confessing, I haven't read any of this. But wondering if you could explain in dumbed down layman language why it's important for us to understand this process.

Thanks!

August 10, 2017

Thanks for all the great research you've done, dm123. This has been one of the best threads I have seen here in a long time. Good, solid research backed up with a lot of studies and references. I find it very useful myself as I go through this process

. Thanks again

August 10, 2017

dm123,

I doubt anyone has ever collected and integrated this knowledge the way you have !

I do have a question though.

[/i]
'Section 2
Quote
Activity-dependent GABAAR ubiquitination The ER is responsible for the retention and degradation of misfolded or unassembled subunits and, accordingly, homomeric unassembled GABAARs subunits have been shown to be degraded in this organelle14,18. ER-associated degradation (ERAD) involves protein ubiquitination and degradation via the ubiquitin-proteasome system (UPS)19. GABAAR subunits have recently been shown to be ubiquitinated in an activity-dependent manner20. Chronic blockade of neuronal activity dramatically increased the levels of GABAAR ubiquitination within the ER, resulting in decreased insertion at the plasma membrane20. Correspondingly, increasing the level of neuronal activity resulted in a decrease in the level of GABAAR ubiquitination and an enhancement of receptor cell surface expression20. Thus, neuronal activity can regulate the ubiquitination of GABAARs in the ER, affecting their rate of degradation via the UPS. This may be one mechanism neurons use to homeostatically regulate synaptic inhibition.
End quote'

I'm having a very hard time making sense of this. I did look up 'ubiquitination'. This blockade of neuronal activity, what kind are we talking about ? Inactivity, drugs, taking benzodiazepines ?

Hi liberty, the blockade of neural activity is extreme, As is the way they caused neuronal activity. However, consistent with the theme of this thread, this is a grand example of another homeostatic response at the cellular level. I hope this is not too much info, but I wanted to answer your question in every way. This next ADDENDUM 7 could be thought of as being part of the downregulation bucket, item 3 in our GABAa list from ADDENDUM 6

I could not do much formatting on this, due to time, but here it is. If anyone knows of a way to easily import word docs with the formatting intact, let me know

This one is definitely worth reading.

ADDENDUM 7: cellular regulatory homeostasis in action: Chronic changes in neuronal activity regulate the numbers of GABAAR expressed on the neuronal cell surface

~"You gonna see, but you ain't gonna believe." - Sergio Oliva

Here is the source reference cited in that quote above. This is the full text. You have great questions. I had a similar question way back when I started researching this area. This study gives us incredible insight into a very complex cellular homeostatic response to external stimuli, via The level of Neuronal activity in the postsynaptic neuron.

http://www.jneurosci.org/content/27/48/13341.long

The 2011 reference goes into much further detail about the ubiquitination process. Section X, Part B, and it references the same footnote as that in the 2008 article that I quoted above. I read the section B and it’s quite confusing, but here is the relevant part. We can then work backwards.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4382539/#!po=22.1154

Quote1

The regulation of GABAAR trafficking by the ubiquitin-related protein Plic1 suggests that GABAARs may also be direct targets for modification by the polypeptide ubiquitin. The covalent attachment of one or more copies of the 76-amino acid ubiquitin monomer to lysine residues of target proteins is referred to as ubiquitination. Monoubiquitination is reversible and serves as an active signal in diverse intracellular trafficking pathways, including as a trigger for endocytosis. In contrast, polyubiquitination is required for the translocation of proteins from the ER back into the cytosol, where they are degraded by the proteasome. Activity-dependent polyubiquitination of GABAAR β3 subunits has been shown to reduce the stability of newly-translated and assembled receptors in the ER via a mechanism dependent on the activity of the proteasome (Saliba et al., 2007).

Coincident with a loss of cell surface expression levels, chronic blockade of neuronal activity by tetrodotoxin (TTX) treatment reduced both the amplitude

frequency of mIPSCs (Saliba et al., 2007). TTX had no effect on the enhanced functional expression of GABAARs incorporating β3 subunits in which all twelve lysine residues within the ICD of this subunit had been mutated to arginines (β3K12R). These mutations did not alter GABAAR cell surface half-life or internalization rates but did significantly enhance receptor insertion into the plasma membrane (Saliba et al., 2007).

End quote1

We can start by figuring out some terms.

First, in reading the very confusing quote 1 above, we want to focus on polyubiquitination.

I’m sure you read the ubiquitination article on Wikipedia.

The important part is quoted below

https://en.m.wikipedia.org/wiki/Ubiquitin

Quote2

The addition of ubiquitin to a substrate protein is called ubiquitination or less frequently ubiquitylation. Ubiquitination affects proteins in many ways: it can mark them for degradation via the proteasome, alter their cellular location, affect their activity, and promote or prevent protein interactions.[4][5][6] Ubiquitination involves three main steps: activation, conjugation, and ligation, performed by ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s), respectively. The result of this sequential cascade is to bind ubiquitin to lysine residues on the protein substrate via an isopeptide bond, cysteineresidues through a thioester bond, serine and threonine residues through an ester bond, or the amino group of the protein's N-terminus via a peptide bond.[7][8][9]

End quote2

The key here is binding to lysine residues on the target protein substrate by ubiquitin = ubiquitination

Our target protein for the purposes of the study referenced above(the original 2008 quote and quote1 above ) are the beta3 subunits of the GABAa receptors.

Next, we need to find out what this substance is called tetrodotoxin or TTX, because that’s what they are using to cause a chronic blockade of all neuronal activity.

As suspected TTX is a potent neurotoxin

https://en.m.wikipedia.org/wiki/Tetrodotoxin

This is where our knowledge presented in the previous ADDENDUMs on action potentials comes in

Here is what TTX does

Quote3

Tetrodotoxin is a sodium channel blocker. It inhibits the firing of action potentials in neurons by binding to the voltage-gated sodium channels in nerve cell membranes and blocking the passage of sodium ions (responsible for the rising phase of an action potential) into the neuron. This prevents the nervous system from carrying messages and thus muscles from flexing in response to nervous stimulation.[1]

Its mechanism of action, selective blocking of the sodium channel, was shown definitively in 1964 by Toshio Narahashi and John W. Moore at Duke University, using the sucrose gap voltage clamp technique.[2]

End quote3

It completely blocks the voltage gated sodium channels in the post synaptic neuron. This will completely prevent any current from going down the axon, because even if threshold potential is reached via the ion channel influx of Na+, the action potential or AP , for short, won’t be triggered because the sodium channels won’t be able to open to let all of the voltage gated sodium in required for that massive depolarization that is needed to create an AP.

So this is their definition of blocking neuronal activity. So when they did this in that 2007 study, nothing was getting through in terms of AP, and they found that this dramatically increased the ubiquitination of the GABAaR subunits in the Cell. This resulted in a loss of cell surface expression levels of the receptor. The end result was lower frequency and amplitude of mIPSCs or miniature inhibitory post synaptic currents. Not good

From the quote above “polyubiquitination is required for the translocation of proteins in the ER or endoplasmic reticulum back into the cytosol, where they are degraded by the proteasome…..”. The ER contains unassembled GABAa receptor subunits. These are targeted for ER-associated degradation by being ubiquitinated and degraded by the proteasome. A proteasome is a protein that degrades another protein that has been tagged by or bound by ubiquitin.

They found the opposite was true when neuronal activity was increased, i.e. Ubiquitination was decreased and there was an enhancement of receptor cell surface expression. More receptors made it to the surface.…. More about how they increased neuronal activity later. (Note : don’t confuse Plic-1 and 2 ubiquitin like proteins with ubiquitin)

To test the hypothesis that ubiquitination was involved and that there was a loss of cell surface expression of GABAaRs due to ubiquitination, they created mutated variants of GABAaRs where all the lysine residues in the beta3 unit were mutated to arginines. As explained above ,the ubiquitin cannot bind to the beta3 subunit without lysine present, thus it can’t ubiquitinate the receptor subunits . When they did that the TTX had no effect on the expression of these mutated receptors!!!!!

Note, These mutations did not alter the receptor half life or internalization rates but increased receptor insertion into the plasma membrane because ubiquitination was not occurring.

So , in summary and in layman’s terms, lack of neuronal activity via this neurotoxin that completely knocks out the AP from occurring, increases ubiquitination, i.e. Degradation , of these subunits inside the cell, thereby decreasing the receptor surface expression. The opposite, increasing neural activity did the opposite. This was confirmed by blocking ubiquitination via the mutated beta receptor. This is what they called “activity dependent ubiquitination “

So what did they do to increase neuronal activity? In going through the original study from 2007, see quote 6 below. They used a GABA antagonist. It was not flumazenil, but this might be why flumazenil therapy is so effective in tapering recovery! Amazing.

Using the body’s homeostatic response to our advantage by increasing neuronal stimulation and activity. Homeostatically, this all makes sense. You try to put the neuron to sleep by excessive inhibitory potentials, and the neuron responds by producing less GABAa receptors that make it to the surface, You excite and stimulate the neuron, as painful as it is, and the neuron responds by slowing the degradation of subunits (inside the cell)thereby increasing the number of assembled receptors that make it to the surface. This gives a whole new appreciation to the term downregulation, and gives us insight into one of the mechansims that the cell uses to achieve this.

So now the original study should make sense. Here are some quotes

And here are some main points. What an awesome study:

-Chronic changes in neuronal activity regulate the numbers of GABAAR expressed on the neuronal cell surface

-Chronic changes in neuronal activity regulate the levels of GABAAR β3-subunit ubiquitination

-Identification of major sites for ubiquitination within the GABAAR β3 subunit

- Decreasing β3 subunit ubiquitination increases receptor accumulation on the neuronal cell surface

- Ubiquitination of the β3 subunit modulates GABAAR insertion into the plasma membrane

- The effects of neuronal activity on GABAAR cell surface expression levels are mediated via β3 subunit ubiquitination

-Chronic blockade of neuronal activity regulates the accumulation of GABAARs containing β3 subunits at synaptic sites

Quote 4

Treatment of cultures with 2 μm TTX for 24 h resulted in a significant decrease (to 47.8 ± 5.3% of control) (Fig. 1A) in cell surface levels of GABAARs containing β3 subunits. The total expression of receptor β3 subunits was also reduced by this treatment (to 45 ± 6.7% of control) (Fig. 1A). Similar statistically significant decreases in the cell surface expression levels of GABAAR α2 and γ2 subunits were also evident (supplemental Fig. 1, available at www.jneurosci.org as supplemental material), suggesting that chronic neuronal inactivity leads to the loss of functional heteromeric cell surface GABAARs. In addition TTX treatment also significantly decreased the number of cell surface GABAARs containing β3 subunits in 16 DIV hippocampal neurons

End quote

This is really interesting, with Glutamate antagonists to reduce neuronal activity.

Quote 5

To corroborate our experiments with TTX, we assessed the effects of blocking excitatory synaptic transmission on the cell surface levels of GABAARs with glutamate receptor antagonists, which have been established to reduce neuronal activity in culture (Rao and Craig, 1997; O'Brien et al., 1998). Blockade of glutamate receptors significantly decreased the cell surface expression levels of GABAARs containing β3 subunits to 45.8 ± 13.1% of control (Fig. 1B)

End quote

They used a GABAa antagonist, PTX to increase neuronal activity,

Quote 6

We also examined the effects of chronically increasing neuronal activity on the cell surface expression levels of GABAARs using picrotoxin (PTX) (40 μm), a GABAAR antagonist and has been shown to increase the activity of neuronal cultures (Rao and Craig, 1997; O'Brien et al., 1998). In contrast to the effects of TTX and glutamate receptor antagonists, treatment of neurons with PTX significantly increased both the cell surface (by 55 ± 2.3% of control) and total (by 47 ± 8.5% of control) expression levels of the GABAARs β3 subunit (Fi

End quote

So now the abstract should make sense.....

Quote 7

Abstract

GABA(A) receptors (GABA(A)Rs) are the major mediators of fast synaptic inhibition in the brain. In neurons, these receptors undergo significant rates of endocytosis and exocytosis, processes that regulate both their accumulation at synaptic sites and the efficacy of synaptic inhibition. Here we have evaluated the role that neuronal activity plays in regulating the residence time of GABA(A)Rs on the plasma membrane and their targeting to synapses. Chronic blockade of neuronal activity dramatically increases the level of the GABA(A)R ubiquitination, decreasing their cell surface stability via a mechanism dependent on the activity of the proteasome. Coincident with this loss of cell surface expression levels, TTX treatment reduced both the amplitude and frequency of miniature inhibitory synaptic currents. Conversely, increasing the level of neuronal activity decreases GABA(A)R ubiquitination enhancing their stability on the plasma membrane. Activity-dependent ubiquitination primarily acts to reduce GABA(A)R stability within the endoplasmic reticulum and, thereby, their insertion into the plasma membrane and subsequent accumulation at synaptic sites. Thus, activity-dependent ubiquitination of GABA(A)Rs and their subsequent proteasomal degradation may represent a potent mechanism to regulate the efficacy of synaptic inhibition and may also contribute to homeostatic synaptic plasticity.

End quote

August 10, 2017

Hi

I wanted to continue ADDENDUM 6 and expand on item 4 of the first part above: namely, present more detail on the GABAa receptor trafficking, lateral diffusion, and clustering, how this actually occurs in terms of specialized proteins, and finally present why it's important for us to understand this process.

Confessing, I haven't read any of this. But wondering if you could explain in dumbed down layman language why it's important for us to understand this process.

Thanks!

Hi abcd, my ultimate goal is to improve our understanding of interdose wd and interdose tolerance wd, so that we can create more effective tapering therapies, especially when things don't go as planned.

for example this clustering explains why we feel so bad and why it's important ones stay away from PAMs during taper and perhaps after wd. ADDENDUM 7 above was clearLy unexpected and unplanned, but in the process of writing it, I learned why flumazenil therapy is so effective. At least in part. Lastly, for me I just want to understand how 1 drug messed things up for me so much. I would like to get the drug rescheduled as class II, especially short acting benzos. The science is clearly pointing me to the conclusion that short acting benzos have NO therapeutic value.

August 10, 2017

If I were to give information here to doctors and nurses, dm123, what in particular would I give them in order that they TRULY UNDERSTAND what goes on with having benzos in the system? And thank you again for all of your valuable research!

What would make the most impact, in other words, so finally they would GET IT!

Hi Terry,I'm not sure. The more research I do on this, the more damning the evidence is for these drugs. Pers put together a document for doctors. I don't remember the title but it's pinned above, and nice and short.

I think ADDENDUM 6 alone says a lot. There's so much more , unfortunately. There are metabotropic Glutamate receptors, and serotonin, ACh and dopamine receptors that are effected as well, and of course what is truly behind kindling.

I truly feel that we are in an era like the tobacco industry was in 50 years ago. The science is all there, everyone knows what the drugs do to our physiology, but the regulatory standards of care are simply not in place yet. We have a lot of work to do. Knowledge always is power....

Can anyone make sense of this to explain to a layperson?

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