Author Topic: Nuclear Mechanisms of the Protracted Benzodiazepine Withdrawal Syndrome  (Read 1509 times)

[Buddie]

If you wish to discuss the below document by Perseverance, please visit the original discussion thread:

http://www.benzobuddies.org/forum/index.php?topic=106459.0


I put this together for doctors to read and thought it might help those of you who are looking for documents to present to your physicians regarding the protracted nature of BW.  I don’t expect all of the members of this support group to understand it, however I always like to encourage members to try.  I hope it will help in our efforts to spread awareness throughout the medical community.

Nuclear Mechanisms of the Protracted Benzodiazepine Withdrawal Syndrome

This review will discuss relevant theories and research regarding contributing factors to protracted symptoms associated with the benzodiazepine withdrawal syndrome.  Topics discussed will include neuroplastic homeostatic changes to GABA A receptors (GABAARs), non-homeostatic changes to the Glutamate system, and the formation and maintenance of Long Term Potential (LTP) like occurrences in the hippocampus.  The lack of research with regards to protracted symptoms associated with benzodiazepine (benzo, BZ) discontinuation after long term use will also be addressed.

Professor Heather Ashton, a world-wide renowned expert in benzodiazepine addiction and withdrawal, made the following observation in the 2004 edition of the “Comprehensive Handbook of Drug & Alcohol Addiction”:

“For some chronic benzodiazepine users, withdrawal can be a long, drawn-out process. A sizeable minority, perhaps 10 to 15% develop a "post-withdrawal syndrome", which may linger for months or even years. (16)

In the 2011 supplement to the Ashton Manual she wrote about changes to the neurons that may be involved:


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.
(1)


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:


“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.” (6)


In the quote from the 2011 supplement above, 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.”  There are 2 ways in which GABA receptors can be delivered to the cell surface:

GABAARs can be delivered to the cell surface either as newly assembled channel complexes via a de novo secretory pathway or reinserted following internalization. (6)

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

Endocytosed GABAARs that fail to be recycled are targeted for lysosomal degradation… (7)

Researchers have observed changes in subunit configuration after chronic benzodiazepine use in recombinant GABAARs:

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.” (8 )


In other words, the GABAARs that had high sensitivity to benzodiazepines were replaced with GABAARs with low sensitivity.  This would be an example of a homeostatic response to counteract the effect of the drug.  Other benzo induced changes which could lead to receptor uncoupling have been suggested.  These changes include changes to phosphorylation, conformal changes, changes in domains, and changes to receptor subunit configuration:


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. (10)

The mechanism of GABA-A receptor uncoupling by benzodiazepines in vitro may be a permanent conformational change in existing receptors, production of an endogenous regulatory GABA-A receptor ligand, a change in phosphorylation, or a displacement of GABA-A receptor subunits. The favored mechanism is a lasting conformational change in existing receptors.” (2)

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. (9)


It stands to reason that any homeostatic 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 these types of neuroadaptations were functional or plastic in nature and were made in response to the presence of the drug:


“Benzodiazepine tolerance is considered to constitute an adaptive mechanism following chronic treatment, and it may thus be regarded as an example of neuronal plasticity. (10)

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. (15)

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. (10)

Benzos have also been shown to cause changes to the Glutamate system:


“According to glutamate hypothesis of BZ tolerance and dependence, excitatory mechanisms become up-regulated to compensate for BZ-induced enhancement of inhibition (Stephens, 1995). Expression of N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors has been shown to be regulated after long-term BZ treatment.” (9)

Benzo discontinuation after tolerance has set up can lead to a depolarizing event resulting in the activation of NMDA receptors (via a voltage triggered release of the magnesium ion channel block) and/or activation of high-voltage activated (HVA) L-type Ca2+ channels:

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.” (13)

"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." (14)

The resulting influx of calcium into the neurons can activate protein kinase CaMKII, which in turn can cause 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. (11):

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…

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…


In models of activity-dependent plasticity, such as long-term potentiation (LTP), CaMKII is primarily activated by Ca2+ influx through N-methyl--aspartate receptors (NMDARs) (Collingridge et al, 2004) and potentiates synaptic efficacy by inducing synaptic insertion of AMPARs, as well as increasing AMPAR single-channel conductance through CaMKII-mediated Ser831 GluA1 subunit phosphorylation (Lisman et al, 2002). AMPAR conductance in CA1 neurons from 2-day FZP-withdrawn rats nearly doubled from 8.5 to 14.7 pS (Shen et al, 2009), an observation that resembled CaMKII-mediated phosphorylation of GluA1 subunits in LTP (Barria et al, 1997b) and modulation of AMPAR conductance in recombinant GluA1 homomeric AMPARs (Derkach et al, 1999)...”
(14)

An increase in calcium permeable GluA1 (aka GluR1) homomeric AMPA receptor mRNA gene expression was also observed:


"...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.” (12)

“In rats withdrawn from flurazepam, amplitudes of AMPA receptor-mediated miniature excitatory postsynaptic currents were increased in hippocampal CA1 neurons (Van Sickle et al., 2004; Xiang and Tietz, 2007). The 50% enhancement in AMPA receptor function was attributed to an increase in GluA1 polypeptide trafficking from the endoplasmic reticulum and its subsequent incorporation into membranes…” (9)

The resulting increase in AMPA receptor single channel conductance and synaptic receptor density can result in the formation of Long Term Potentiation (LTP) like occurrences, which can persist for extended periods of time.

Up-regulation of GluA1 mRNA may be significant in protracted symptoms for two reasons: 1) CaMKII enhances the gating of individual subunits in GluA1-containing AMPA receptors which increases the single channel conductance (4); 2) AMPA receptors containing GluA1 subunits are the only type permeable to Ca2+.  Therefore, the increased density along with the enhanced gating would facilitate an increase in Ca2+ influx, which could bolster the maintenance of the LTP like occurrences through perpetuation of gene transcription and construction of reinforcing proteins, such as PKMζ. (14, 5)

These LTP like occurrences may add to the effects of acute stress, which has been shown to cause an increase in NMDA and AMPA receptor activity in the hippocampus:

“Electrophysiological recording studies showed that acute stress mimicked by acute corticosterone (a glucocorticoid) injection increases synaptic NMDAR and AMPAR activity in both the prefrontal cortex4 and the hippocampus. [11] The facilitation of synaptic AMPAR and NMDAR activity is suggested to be mediated by a serum-and-glucocorticoid inducible kinase (SGK) dependent Rab4 (GTPase) mediated mechanism. [4] Acute exposure to behavioral stressors or the administration of corticosterone injection increases SGK activity, allowing it activates Rab4 downstream. [4] When activated, Rab4 is thought to simultaneously promote the synaptic trafficking of AMPARs and NMDARs. [4] Consequently, the activity of the AMPAR and NMDAR increases because more of these receptors are present to respond to incoming stimuli. (3)

While changes to GABAARs appear to be homeostatic changes, the LTP like occurrences in the glutamate system are not and therefore would probably have to fade over time in order to see improvements with the associated anxiety symptoms.  Further research is needed to determine if these occurrences can happen in neurons in other parts of the brain where benzos bind.

Success stories offer anecdotal evidence that reversal of 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 typically examined anywhere from 6 hours to 7 days after withdrawal has been induced. (9)  To my knowledge there have not been any long term studies examining reversals of neuroadaptations after withdrawal associated withlong term benzo usage.  Some researchers have made assumptions as to the reversibility of these changes based on short term studies.  Without long term studies, any claims regarding reversibility of long term changes in gene expression or LTP like occurrences would be unsubstantiated, and perhaps, reckless.  In a 2013 review of a paper compiled by representatives from the Royal College of Psychiatrists and the British Association for Psychopharmacology Ashton stated the following:

The effects on the brain of long-term benzodiazepine use have never been adequately researched despite the reports of many patients of enduring, perhaps irreversible, adverse effects. (17)




References

1)   “The Ashton Manual Supplement” Professor C Heather Ashton, DM, FRCP
A Supplement to Benzodiazepines: How They Work & How to Withdraw (2002) Published 7 April 2007 Additions 2012 and 2013
http://www.benzo.org.uk/ashsupp11.htm

2)   “Benzodiazepine Receptor Deficiency and Tinnitus” Shulman A, Strashun AM, Seibyl JP, Daftary A, Goldstein B. Benzodiazepine Receptor Deficiency and Tinnitus. Int Tinnitus J. 2000;6(2):98-111
http://www.tinnitusjournal.com/detalhe_artigo.asp?id=231

3)   “Stress Induced Plasticity in the Glumatatergic System
Neurowiki 2013 » Stress » 02 Stress Induced Plasticity in the Glumatatergic System
http://neurowiki2013.wikidot.com/individual:stress-induced-plasticity-in-the-glumatergic-syst

4)   “Mechanism of CaMKII Regulation of AMPA Receptor Gating” Anders S. Kristensen, Meagan A. Jenkins, Tue G. Banke, Arne Schousboe, Yuichi Makino, Richard C. Johnson, Richard Huganir, Stephen F. Traynelis
Nat Neurosci. Author manuscript; available in PMC Dec 1, 2011. Published in final edited form as: Nat Neurosci. Jun 2011; 14(6): 727–735. Published online Apr 24, 2011. doi:  10.1038/nn.2804
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3102786/

5)   “Long Term Memory
From Wikipedia, the free encyclopedia
http://en.wikipedia.org/wiki/Long-term_memory

6)   “The Dynamic Modulation of GABAA Receptor Trafficking and Its Role in Regulating the Plasticity of Inhibitory Synapses” Mansi Vithlani , Miho Terunuma , Stephen J. Moss
Physiological Reviews Published 1 July 2011Vol. 91no. 1009-1022DOI: 10.1152/physrev.00015.2010
http://physrev.physiology.org/content/91/3/1009.full

7)   “GABAAR Trafficking-mediated Plasticity of Inhibitory Synapses” Bernhard Luscher, Thomas Fuchs, Casey L. Kilpatrick Published in final edited form as: Neuron. May 12, 2011; 70(3): 385–409. doi:  10.1016/j.neuron.2011.03.024
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3093971/

8 )   “New Insights into the Role of the GABAA—Benzodiazepine Receptor in Psychiatric Disorder
David J. Nutt, FRCPsych, Andrea L. Malizia, MRCPsych
The British Journal of Psychiatry (2001) 179: 390-396 doi: 10.1192/bjp.179.5.390
http://bjp.rcpsych.org/content/179/5/390.full

9)   “Regulation of GABAA Receptor Subunit Expression by Pharmacological Agents” Mikko Uusi-Oukari, Esa R. Korpi
Published online before print February 1, 2010, doi: 10.1124/pr.109.002063 Pharmacological Reviews March 2010 vol. 62 no. 1 97-135
http://pharmrev.aspetjournals.org/content/62/1/97.full#title2

10)   “Mechanisms Underlying Tolerance after Long-Term Benzodiazepine Use: A Future for Subtype-Selective GABAA Receptor Modulators?” Christiaan H. Vinkers, Berend Oliver
Advances in Pharmacological Sciences Volume 2012 (2012), Article ID 416864, 19 pages http:dx.doi.org/10.1155/2012/416864
http://www.hindawi.com/journals/aps/2012/416864/

11)   “Plasticity in the human central nervous system” S.F.Cooke, T.V.P. Bliss
Brain (2006) 129 (7): 1659-1673. doi: 10.1093/brain/awl082 First published online: May 3, 2006
http://brain.oxfordjournals.org/content/129/7/1659.long

12)   “Glutamic acid decarboxylase and glutamate receptor changes during tolerance and dependence to benzodiazepines” Emanuela Izzo, James Auta, Francesco Impagnatiello, Christine Pesold, Alessandro Guidotti, Erminio Costa PNAS March 13, 2001 vol. 98 no. 6 3483–3488, doi: 10.1073/pnas.051628698
http://www.pnas.org/content/98/6/3483.full

13)   “Transient Plasticity of Hippocampal CA1 Neuron Glutamate Receptors Contributes to Benzodiazepine Withdrawal-Anxiety” Bradley J Van Sickle, Kun Xiang, Elizabeth I Tietz
Neuropsychopharmacology (2004) 29, 1994–2006, advance online publication, 21 July 2004; doi:10.1038/sj.npp.1300531
http://www.nature.com/npp/journal/v29/n11/full/1300531a.html

14)   “Calcium/Calmodulin-Dependent Protein Kinase II Mediates Hippocampal Glutamatergic Plasticity During Benzodiazepine Withdrawal” Guofu Shen, Bradley J Van Sickle, Elizabeth I Tietz
Neuropsychopharmacology. Aug 2010; 35(9): 1897–1909. Published online May 5, 2010. doi:  10.1038/npp.2010.61
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2904841/

15)   “Mechanisms of GABAA and GABAB Receptor Gene Regulation and Cell Surface Expression
David H. Farb PhD, Janine L. Steiger PhD, Stella C. Martin PhD, Maria C. Gravielle PhD, Terrell T. Gibbs PhD, Shelley J. Russek PhD The GABA Receptors. The Receptors 2007, pp 169-238
http://link.springer.com/chapter/10.1007%2F978-1-59745-465-0_8#page-1

16)   “Protracted Withdrawal Symptoms from Benzodiazepines” Professor C Heather Ashton, DM, FRCP
Comprehensive Handbook of Drug & Alcohol Addiction 2004
http://www.benzo.org.uk/pws04.htm

17)   “Benzodiazepines; Risks and Benefits. a Reconsideration- a Review” by Professor C Heather Ashton, DM, FRCP November 2013
http://www.benzo.org.uk/amisc/ashreview11-13.pdf

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