« on: November 01, 2019, 08:50:00 pm »
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Important Note:  This opening post has been revised due to some of the links becoming deactivated.  The corrected copy of the original review can be found here:

Benzodiazepine (BZ) withdrawal can initiate a cascade of events within the neurons creating conditions favorable to the establishment of long term potential (LTPs) at synapses in the brain.  As we shall see, abrupt BZ discontinuation has been shown to cause a depolarization in the neuron, which in turn activates mechanisms essential to the establishment of LTPs, including the NMDA and AMPA ionotropic glutamate receptors and protein kinases.  This paper will discuss the following:

I  The difference between Tolerance, Dependence, and the Withdrawal Syndrome
II  Long Term Potentiation
III  How BZ Withdrawal Sets up LTPs
IV  Environmental Cues Trigger Withdrawal Symptoms
V  Are there any cures for LTPs?
VI  Can Withdrawal Symptoms be Prevented?
VII  Kindling

The difference between Tolerance, Dependence, and the Withdrawal Syndrome

Before discussing long term potentiation (LTP) in connection with BZ withdrawal, it is important to understand the 3 scenarios associated with long term BZ use- tolerance, dependence, and the withdrawal syndrome.

First off, let’s look at tolerance.  Tolerance happens when the neurons have made changes that counteract the effects of the drug, which causes the initial effect of the drug to disappear.  In order to get the initial effect of the drug again you would have to increase the dosage after tolerance has set up.  Here is how it happens.

BZs bind to the GABAA receptor and as a result cause the chloride channel in the center of the receptor to open more frequently allowing more chloride into the neuron. 

After long term BZ administration the neurons begin to make some changes to the receptors to counteract the effect of the drug.  Scientists have proposed different ways in which this may occur, such as:

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

   b) 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) 

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

The neuroadaptations essentially cancel out the BZ-induced chloride increase – thus the drug becomes ineffective.  So now if you wanted to feel the effects you did before, you would have to increase the dosage to raise the chloride level again.

Next, let’s look at dependence.  Dependence happens when enough of these neuroadaptations have been put into place to the point that you must continue to take the drug in order to avoid withdrawal symptoms.  After the neuroadaptations have been put in place, both the BZ and the neuroadaptations now maintain the chloride level together.  BZs caused an increase in chloride by making GABAA receptors more efficient, passing more chloride into the neuron.  The neuron made neuroadaptations to bring the chloride level back down.  Now, if you remove the drug the receptors will no longer be enhanced by the BZ, however, the neuroadaptations are still in place.  The result is that chloride level will now be too low.  This chloride deficiency will trigger a series of events leading to withdrawal symptoms.  So basically, in dependence you take the drug to avoid triggering the commencement of withdrawal symptoms.

The withdrawal syndrome begins after the drug has been discontinued (and possibly during tapering to varying degrees).  This is where the changes happen that can set up LTPs.

II  Long Term Potentiation

So what is long term potentiations (LTP)?  LTP is a long lasting increase in strength at synaptic connections between neurons, which scientists believe the brain uses as a way to store information. (12)  LTPS have been shown to have the ability to persist for long periods of time- anywhere from hours to weeks to months. (4) LTPs have the ability to occur in many regions of the brain including but not limited to the cerebral cortex, hippocampus, cerebellum, and amygdala. (13)  It is thought that LTPs are the basis for memories and that different forms of LTP may underlie conditions such as phantom pain syndrome, (6) PTSD (29), and chronic pain. (6, 16)  Studies have shown that LTPs may be a major factor in BZ withdrawal and other drugs of dependence. (7, 13, 14, 16)

There are different types of LTPs.  Determining factors include aspects such as the area of the brain in which it occurs and the signaling pathways used by a particular cell. (13)  LTPs can be further classified as Hebbian, non-Hebbian, and anti-Hebbian depending on the pre and post synaptic mechanisms involved with their induction.  A Hebbian LTP requires simultaneous pre- and postsynaptic depolarization for its induction; a non-Hebbian LTP does not require such simultaneous depolarization of pre- and postsynaptic cells; an anti-Hebbian LTP requires simultaneous presynaptic depolarization and relative postsynaptic hyperpolarization for its induction. (13)  Based on this criteria, the type of LTPs that we will look at in connection with BZ withdrawal would involve the non-Hebbian type.

In order for an LTP to occur a series of events must take place in the neuron.  It begins with a depolarizing event, in which there is a rise in the neurons membrane voltage potential.  This triggers voltage gated calcium channels to open- which usually involves the NMDA Glutamate receptor, but may also involve other modes of entry which will be discussed later.

Under conditions of rest or low levels of input activity, the channel of the NMDA receptor is normally blocked by positively charged extra-cellular magnesium (Mg2+) ions. (4) This prevents positively charged Calcium (Ca2+) from entering the neuron.  A depolarizing event which causes rise in the membrane voltage potential will cause the Mg2+ block to disassociate from the NMDA receptor.  Without the Mg2+ block, Ca2+ can now pass through the receptor when agonists are present.  The NMDA receptor is unique in that it is both ligand and voltage gated, and therefore only allows Ca2+ to enter if both the pre and post synaptic neurons are depolarized.

A rise in membrane potential can also trigger high-voltage activated (HVA) L-type Ca2+ channels, which would also permit Ca2+ to enter the neuron.

When Ca2+ enters a neuron, a multitude of signaling molecules can be activated. (17)  The amount of Ca2+ that enters the neuron determines the sequence of events that will take place.  A large increase in Ca2+ will activate protein kinases which in turn initiate a cascade of events that result in an LTP. (14)  One of these kinases, known as Ca2+/calmodulin-dependent protein kinase (CaMKII), enhances the single channel conductance of the AMPA glutamate receptors at the synapse through phosphorylation.  This means the current flow of positively charged sodium ions through the AMPA receptor ion channel is increased. (15)  CaMKII also promotes movement of AMPA receptors from intracellular stores into the synapse, thereby increasing their number. (10)  The combination of these two events significantly increases the synaptic strength.  This all happens as part of the first phase of LTP, which is also known as early phase LTP, or E-LTP. (13, 16)

The influx of Ca2+ also activates enzymes which in turn activate transcription pathways that alter gene expression.  This leads to synthesis of new AMPA receptors and, perhaps, a change in translation which leads to synthesis of an LTP maintenance kinase, known as Protein Kinase M Zeta (PKMζ). (13, 22, 24)  (Note: There is at least one conflicting report debating whether PKMζ is a true factor in LTP maintenance or not, outside of this report PKMζ is generally accepted as the maintenance molecule). (20)  Initiation of gene expression and protein synthesis are part of the second phase of LTP, which is known as late phase LTP or L-LTP. (13, 16)

III  How BZ Withdrawal Sets up LTPs

Researchers demonstrated that onset of anxiety-like behavior during BZ withdrawal coincided with the beginning of LTP like activity in hippocampal neurons.  They found that a GABA receptor mediated depolarizing potential, which was present on the second day of withdrawal in pyramidal neurons from the CA1 region of the hippocampus, activated NMDA receptors.

“A GABAR-mediated depolarizing potential, which is present in 2-day FZP-withdrawn CA1 neurons (Zeng et al, 1995), has been shown to activate NMDARs (Staley et al, 1995) and may contribute to increased postsynaptic Ca2+-mediated signal transduction.” (9)

This matched criteria for initiation of an LTP.  Researchers later realized that in addition, depolarization activation of high-voltage activated (HVA) L-type Ca2+ channels may not only contribute to the Ca2+ influx, but may actually be the primary source of Ca2+ influx:

"Distinct from NMDAR-dependent LTP, in which Ca2+ influx primarily through NMDAR initiates CaMKII activation and AMPAR potentiation, Ca2+ entry during benzodiazepine withdrawal may primarily occur through an increase in high voltage-activated Ca2+-channel current; and perhaps subsequently, through the increased density of Ca2+-permeable, GluA1 homomeric AMPARs." (7)

CaMKII activation, as noted above, causes AMPA receptor (AMPAR) potentiation in two ways, first- by facilitating the insertion of new receptors into the synapse through lateral diffusion from an extra-synaptic pool, and second- through phosphorylation of the AMPA receptors which enhances single-channel conductance (4):

“AMPAR-mediated miniature excitatory postsynaptic current (mEPSC) amplitude increased in CA1 neurons from 1- and 2-day FZP-withdrawn rats, along with increased single-channel conductance in neurons from 2-day rats.” (7)

These observations fulfilled the requirements necessary for the E-LTP phase.

Next researchers observed an increase in mRNA and density of GluA1 (aka GluR1) homomeric AMPARs, which matched L-LTP criteria for gene expression and protein synthesis:

"...the levels of GluR1 mRNA were significantly increased in frontal cortex (48%), occipital cortex (38%), and hippocampus (56%), but failed to change in the cerebellum of 96-hr diazepam-withdrawn rats when compared with 96-hr vehicle-withdrawn rats. Notably, the increase in GluR1 mRNA expression detected in the frontal cortex and hippocampus of 96-hr diazepam-withdrawn rats was either not detectable or not statistically significant after 12 or 48 h of diazepam withdrawal.” (1)

“Benzodiazepine withdrawal anxiety is associated with potentiation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) currents in hippocampal CA1 pyramidal neurons attributable to increased synaptic incorporation of GluA1-containing AMPARs.”  (7)

Up-regulation of GluA1 mRNA is significant for two reasons: 1) CaMKII enhances gating of individual subunits in GluA1-containing AMPA receptors which increases the single channel conductance (21); 2) AMPA receptors containing GluA1 subunits are the only ones permeable to Ca2+.  Therefore, the increased density along with the enhanced gating would facilitate an increase in Ca2+ entry into the neuron during the L-LTP phase.  This could could be a factor in maintenance of the LTP by perpetuating gene transcription and construction of reinforcing proteins, such as PKMζ. (7, 22)

Up-regulation of AMPA receptors via protein synthesis fulfills criteria necessary for the L-LTP phase.

IV  Environmental Cues Trigger Withdrawal Symptoms

After discovering PKMζ researchers developed a zeta inhibitory peptide (ZIP) which was thought to inhibit PKMζ and was actually shown to eliminate LTPs. (23, 24)  One research team hypothesized LTP, and therefore PKMζ, might be involved with contextual memory cues triggering a re-emergence of withdrawal symptoms.

In this study, rats were administered diazepam (DZ) for 18 days then abruptly taken off the drug.  The rats exhibited symptoms of withdrawal anxiety for 4 days after the drug was withdrawn.  On the 15th day the rats were re-exposed to the same environment (a plus maze, or PM) where they had originally experienced the withdrawal symptoms.  The contextual cues in the environment of the PM triggered a recurrence of the withdrawal symptoms. If the contextual cues were changed in the environment of the PM, the rats did not experience a recurrence of the withdrawal symptoms on re-exposure. 

“In the present investigation we demonstrated that changes in the environmental cues associated with DZ withdrawal prevented the expression of the anxiety-like behavior observed during PM test re-exposure.” (19)

The researchers attributed the recurrence of symptoms in conjunction with contextual cues to a lowered threshold to generate LTP.  Next they repeated the experiment, but this time they administered the PKMζ inhibitor ZIP two hours prior to re-exposure and observed the following:

“In the present study we found that inhibition of PKMζ prior to re-exposure to the withdrawal environment with preserved contextual cues not only prevented the anxiety expression but also reversed the facilitated LTP in all DZ-dependent animals, suggesting that plasticity in the hippocampus has a crucial role in maintenance of these memories that were vulnerable to the amnesic effects of ZIP.” (19)

It was interesting that this study showed environmental cues could trigger withdrawal symptoms in conjunction with LTPs, which may demonstrate an overlap in functional characteristics between memory LTPs and BZ withdrawal LTPs.  This poses a question as to whether LTPs which occur during withdrawal could share other commonalities with memories.  Other LTPs, such as LTPs involved in chronic pain conditions, have been shown to share characteristics with memory LTPs:

“…these findings point to a spinally encoded mechanism for the persistence of nociceptive sensitization with molecular underpinnings that closely resemble those involved in L-LTP and the maintenance of long-term memory traces.” (24)

Functional similarities between withdrawal LTPs and memory LTPs might include such things as distraction and time, which are two concepts discussed frequently with regards to benzo withdrawal.  Distraction appears help reduce withdrawal symptoms, and ironically is also a factor in both avoidance and fading of memories.  Time has been suggested to be the only known cure for withdrawal, similarly it is also the cure for painful memories as ‘time heals all wounds.’  Just as withdrawal symptoms can seemingly pop up again out of the blue, so can memories.  As demonstrated in the previous study, this may be due to contextual cues in the environment, either consciously or subconsciously, triggering the ‘biological memory’ stored in the Central Nervous system (CNS).  Contextual cues may be varied and might result from single or multiple environmental perceptions involving visual, tactile, olfactory, or gustatory senses.  Formation of LTPs may be the reason why withdrawal symptoms can persist for such a long time.

Are there any cures for LTPs?

Generally, LTPs tend to spontaneously decay over time. (17, 22, 25)  In fact, a large portion of LTP research is dedicated to figuring out how to perpetuate LTPs to combat memory loss due to aging and neurodegenerative conditions. (25, 26)  Many mechanisms have been speculated to be responsible for the fading of LTPs, including changes in phosphorylation, insertion or recycling of AMPARs, or internalization mediated by Glutamate binding.  Other factors involved include the way in which the LTP was induced and patterns of synaptic activation. (27)  As mentioned earlier, there are many variables between different types of LTPs.

Research investigating ZIP to erase LTPs, while fascinating, carries inherent draw backs.  In order to be safe and effective the exact area of the brain where the undesired LTP(s) were located would have to be identified and the treatment would have to be precisely targeted in order to avoid unwanted amnesiac effects.  Extreme care would have to be taken as not to inadvertently erase desirable LTPs.  This makes the use of this medication impracticable at this point in time.

VI  Can Withdrawal Symptoms be Prevented?

Research has shown that it may be possible to prevent BZ withdrawal LTPs from setting up in the first place.

The calcium channel blocker nimodipine was shown in one study to prevent the increase in AMPA currents, and therefore the symptoms associated with withdrawal anxiety:

“injection of an L-type voltage-gated calcium channel antagonist, nimodipine (10 mg/kg, intraperitoneally) averted AMPAR current enhancement and anxiety-like behavior, suggesting that these manifestations may be initiated by a voltage-gated calcium channel-dependent signal transduction pathway.” (5)

A study in 1993 researching Glutamate receptor involvement in BZ withdrawal divided the withdrawal into two phases:

“Two major phases can be recognized in the occurrence of signs of dependence during diazepam withdrawal in mice. The initial phase lasts for ≈3 days and is symptom-poor (silent phase); the second phase is symptom-rich (active phase) and is characterized by distinct dynamics. The onset of the signs of dependence is rapid, the symptoms being most pronounced during the next 3-7 days and abating slowly up to WD 21.” ( 8 )

The period between days 1 and 3 after treatment discontinuation was symptom-free- which they called the "silent phase.”  The period between days 4 and 21 after treatment discontinuation showed a time related evolution of anxiety, muscle rigidity, and seizures- which they called the “active phase.”  These phases may perhaps be related to the E-LTP and L-LTP phases in long term potentiation.  Tests ruled out the slow elimination of the drug as being a contributing factor to the symptom free period of the silent phase.  They suspected that the active phase was somehow mediated by the NMDA receptors.

They found that the AMPA and NMDA receptor antagonists, GYKI-52466 and CPP respectively, could reduce or prevent certain signs of withdrawal:

“Treatment of mice with a-amino- 3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) antagonists 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H- 2,3-benzodiazepine (GYKI 52466) or 2,3-dihydroxy-6-nitro- 7-sulfamoylbenzo(f) quinoxaline but not with the NMDA antagonist 3-[(+)-2-carboxypiperazin-4-yl]-propyl-1-phosphonate (CPP) during the silent phase prevented signs of dependence. In contrast, treatment with CPP but not with GYKI 52466 during the active phase prevented the symptoms. The development of tolerance to and dependence on diazepam was prevented by concurrent treatment of mice with CPP but was not prevented by GYKI 52466.” ( 8 )

In summary, their findings showed:

   1) Administration of the AMPA receptor antagonists GYKI 52466 or NBQX during the initial silent phase prevented or reduced withdrawal symptoms, but it did not work during the active phase.

   2) Blockade of NMDA receptors by administering the NMDA antagonist CPP during the active phase eliminated withdrawal symptoms, but it did not work during the silent phase.

   3) They could prevent tolerance and dependence from developing by concomitant administration of diazepam and the NMDA antagonist CPP.

Their final conclusion was as follows:

“Our data suggest that the AMPA antagonists are of value for preventing the signs of dependence during the initial phase after withdrawal. NMDA antagonists may be indicated in the course of chronic treatment with BDZs or during the active phase after withdrawal.” ( 8 )

Another study on 2007 noted that NMDA antagonists in general prevent LTPs from setting up:

“MK-801 and other antagonists of the N-methyl-D-aspartate (NMDA) glutamate receptor subtype impair induction of LTP.” (28)

In 2004 the findings of a research team in Toledo Ohio on BZ tolerance and dependence supported the findings in the earlier study regarding AMPA antagonists. (9)  In this study rats were administered a BZ called flurazepam (FZP), then the drug was abruptly withdrawn. They observed that AMPAR Miniature Excitatory Postsynaptic Current (mEPSC) was increased in the CA1 hippocampal pyramidal neurons from 1 day withdrawn rats.  They found that if they administered the AMPA antagonist GYKI 52466 at the end of the drug treatment period (which would be during the ‘silent phase’ of the previous study) it would reverse the increase in AMPAR conductance and prevent anxiety-like behavior:

“Systemic GYKI-52466 prevented both increased AMPAR-mediated excitation in CA1 neurons and withdrawal-anxiety when injected at the onset of FZP withdrawal.” (9)

LTP like changes that were observed with BZ withdrawal in the previous studies happened under conditions where the drug was abruptly discontinued.  However, it has not been ruled out that these same type of changes may happen to varying degrees after different types of tapering methods.  Researchers have only investigated abrupt discontinuation thus far.  Abrupt discontinuation might initiate the LTP by causing a rapid decease in the influx of chloride thereby depolarizing the neurons.

Perhaps if LTPs happen during or after a taper they may not be as numerous, especially if the taper is slow- as tapering allows neurons time to make reversals to neuroadaptations and would therefore ‘soften the blow.’  Thus the decrease in chloride levels entering the neuron would not be as drastic after a taper as it will be with a rapid or abrupt discontinuation.  This may be the underlying reason why withdrawal symptoms after tapering may be fewer or less intense that Cold Turkey (CT) and rapid detox methods, and why CTs and rapid detox methods are more closely associated with protracted symptoms.  This is not to imply that symptoms cannot be numerous or intense after a taper, rather it simply offers a plausible explanation for the differences between the two scenarios.

VII  Kindling

The involvement of Glutamate receptors has also been thought to underlie the kindling phenomenon:

“Adaptational changes at the GABAA BZ receptor complex do not fully explain tolerance, dependence and withdrawal from BZs. Other receptor complexes are believed to be involved, in particular the excitatory glutamate system. The involvement of glutamate in BZ dependence explains long-term potentiation as well as neuro kindling phenomena.” (30)

Interestingly, administration of an NMDA antagonist during the in-between periods of BZ use prevented seizure sensitivity:

“Mice were treated either with diazepam (15 mg/kg s.c. in oil), for 21 days, or for 3x7-day periods interspersed with two 72-h drug-free periods. Convulsant thresholds to pentylentetrazole infused into the tail vein 72 h following the final chronic treatment were lower in multiple-withdrawal mice than in mice which had experienced the same drug load, but only a single withdrawal, consistent with sensitisation of withdrawal events following previous withdrawal experience. The increase in seizure sensitivity of repeatedly withdrawn mice was prevented by treatment with the NMDA receptor antagonist CGP 39551 (20 mg/kg, i.p.) given once daily during the 3-day breaks in diazepam treatment, suggesting a role of glutamatergic transmission in the sensitisation process.” (31)

This suggests that the sensitization experienced with kindling may be related.

   8 )

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