HOW GABAA RECEPTORS ARE CREATED AND BENZO INDUCED CHANGES

The purpose of this thread is to explain the processes involved in the creation of GABAA receptors (GABAARs) and discuss research exploring benzo induced receptor changes.  I am hoping this will be a collective effort, so please feel free to contribute information relevant to this subject and address each other’s questions (especially since I will not be able to be here all of the time).

 

By the end of this paper I am hoping you will understand how GABAARs are formed all the way from genetic information in your DNA to their appearance on the surface of the neuronal membrane.  At the end of this paper I will present some of the latest research findings that offer insights as to how benzodiazepines may have effected these processes.

 

Unfortunately, in order to really understand any of the benzo research you have to know how GABAARs are created.  I have tried to make this topic easy to understand by breaking it down to fundamentals and including lots of pictures- and even provided links to some videos to help you grasp it all.

 

The making of a GABAAR involves a ton of steps, so I recommend that you find a comfortable place to sit where you can focus without distractions- and let’s begin!

 

We will begin with the neuron (shown below).  Inside the neuron is a nucleus.  The nucleus is enclosed in a double lipid bilayer membrane called the nuclear envelope (aka- nuclear membrane, nucleolemma or karyotheca). (30)

 

http://o.quizlet.com/gQIYAV3hswkyxSDQ5PJy8A.png

 

Inside the nucleus are Chromosomes (shown below).  Each Chromosome is made up of Chromatin.  Chromatin is made up of a long DNA (Deoxyribonucleic acid) molecule tightly coiled many times around proteins called histones that support its structure.

 

http://desybio.files.wordpress.com/2010/03/kromosom-mengandung-dna.jpg?w=390&h=312

 

Here is a video showing you how Chromosomes are put together:

 

Chromosomes are not visible in the cell’s nucleus—not even under a microscope—when the cell is not dividing.  However, the DNA that makes up Chromosomes becomes more tightly packed during cell division and is then visible under a microscope. Most of what researchers know about Chromosomes was learned by observing Chromosomes during cell division.

 

Each Chromosome has a constriction point called the centromere, which divides the Chromosome into two sections, or “arms.” The short arm of the Chromosome is labeled the “p arm.” The long arm of the Chromosome is labeled the “q arm.” The location of the centromere on each Chromosome gives the Chromosome its characteristic shape, and can be used to help describe the location of specific Genes. (1)

 

 

http://histology-world.com/photoalbum//albums/userpics/normal_chromosomes.jpg

 

Chromosomes in humans can be divided into two types: autosomes and sex chromosomes. Certain genetic traits are linked to a person's sex and are passed on through the sex chromosomes. The autosomes contain the rest of the Genetic hereditary information.  Human cells have 23 pairs of Chromosomes (22 pairs of autosomes and one pair of sex chromosomes), giving a total of 46 located in each cell.  One Chromosome from each of your 23 pairs came from each of your parents.

 

Human cells are called Diploid, which means the nucleus of each cell contains two homologous copies of each Chromosome.  Homologous means they carry the same type of information, yet they are different- this is because one is from your Mother and one is from your Father.

 

The DNA is made up of a double helix formed by double-stranded molecules carrying sequences of nucleic acids called Nucleotides.  These Nucleotide sequences are made up of combinations of four different monomers: Adenine, Guanine, Cytosine, and Thymine represented by the letters A, G, C, and T respectively (shown below). It is the Nucleotide sequences that make up our Genetic information.  The entirety of your hereditary information, known as the Genome, is encoded on your DNA through these Nucleotide sequences.

 

http://cyberbridge.mcb.harvard.edu/images/dna1_7.png

 

Each end of each strand of the DNA double helix is designated as either the 5' end (pronounced 5 prime end) or the 3' end (pronounced 3 prime end) (refer to picture above).  These are so named for the carbons on the deoxyribose (or ribose) ring- which we will not get into here.  You just need to know the names because they will be used later on while discussing orientation and direction along the DNA strands.  The relative positions of structures along a strand of DNA, including Genes and various protein binding sites, are usually noted as being either upstream (towards the 5′ end) or downstream (towards the 3′ end). The 3’ end is also known as the tail end.

 

The Nucleotide sequences along each strand in the double helix are complimentary to each other, or in other words, they run parallel to each other but are in reverse order.  Each Nucleotide is referred to as a Base.  Their order always follows a certain rule-  Adenine (A) Bases complement Thymine (T) Bases and vice versa; Guanine (G) Bases complement Cytosine © Bases and vice versa, as shown below.  These compliments are called Base Pairs.  They are held together, or to each other, by a hydrogen bond which in turn also holds the strands of the double helix in position providing structural support.

 

http://biology12qe.wikispaces.com/file/view/SK195_2_005i.jpg/33413899/195x358/SK195_2_005i.jpg

 

A Gene is a section of the DNA.  It consists of a specific sequence of Nucleotides encoded on the DNA strand.  The picture below depicts a double helix strand of DNA being unraveled from a Chromosome, and shows how a section of the DNA is designated as a Gene.  The specific location of a Gene or DNA sequence on a Chromosome is called the Locus (plural Loci).  Some Genes contain sequences known as Introns and Exons, which will be discussed later.

 

http://upload.wikimedia.org/wikipedia/commons/0/07/Gene.png

 

We all have Genes that carry the same type of information, but differences in the nucleotide sequences collectively make each one of us unique.  These differences in the nucleotide sequences within a Gene are called Alleles.  A pair of Chromosomes are shown below.  The Alleles are identified by the Gene loci (locations) on the chromosome.  As I mentioned before, humans have two copies of each Chromosome (one from the father and one from the mother) which make up the pair.  If a mutation (difference) occurs in just one copy of the Gene then that mutation is considered a heterozygous genotype.  On the other hand, if both copies of a Gene are mutated then that mutation is a called a homozygous genotype. (3)  Mutations come in different forms such as a substitution of one Nucleotide for another, deletion of single or a group of Nucleotides, insertion of one or more Nucleotides and so on. (4)

 

http://click4biology.info/c4b/4/images/4.1/homologous.gif

 

Single Nucleotide Polymorphisms, frequently called SNPs (pronounced “snips”), are the most common type of these genetic variations among people.  Each SNP represents a difference in a single Nucleotide of one allele.  For example, a SNP may replace the Nucleotide cytosine © with the Nucleotide thymine (T) in a certain stretch of DNA.

 

SNPs occur normally throughout a person’s DNA. There are roughly 10 million SNPs in the average person’s Genome.  Most SNPs have no effect on health or development.  Some of these genetic differences, however, can be very important to human health.  Researchers have found SNPs that may help predict an individual’s response to certain drugs, and therefore may hold the key as to why some people are more susceptible to benzodiazepine tolerance and withdrawal and others aren’t. (2)

 

The process in which information from a Gene is used in the synthesis (creation) of a functional product is known as Gene Expression.  Before we continue I think it would be helpful if you watch this video animation first.  Don’t worry if you don’t understand it all now- but it will give you a visual reference to draw from in your mind as you read along.  Later, once you have read through this paper you should be able to come back, watch it again, and hopefully understand all that you are viewing.

 

 

During Gene Expression, Gene nucleotide sequences are copied from the DNA and used as a blueprint for manufacturing various things in your body.  Some are used to create mRNA strands which are then used to synthesize proteins- these are the ones we will be focusing on.

 

The DNA itself never leaves the nucleus, only copies of the Genes on the DNA are allowed to leave.  Genes that contain the Nucleotide sequence codes used to make proteins (long chains of amino acids) are composed of tri-Nucleotide units (a sequence of 3 Nucleotides) which are called Codons.  Each Codon will be used to code for a single amino acid in the protein chain.

 

The actual process of copying these Nucleotide sequences from the Gene is called Transcription.  Located at the end of each Gene on the DNA are areas called Promoters and Terminators (shown below), which are made up of their own specific sequences.  They specify where the Gene Transcription will begin and end.  In order for the Gene to be copied, an enzyme called RNA Polymerase (RNAP) and a set of DNA-binding proteins called Transcription Factors must bind to the Promoter sequence to initiate the process.

 

http://www.phschool.com/science/biology_place/biocoach/images/transcription/gene.gif

 

Proteins are produced when they are needed in a cell.  Some Genes are expressed (copied) to produce proteins all of the time for housekeeping purposes, this is known as Constitutive Transcription.  Other proteins are only produced at specific times when they are needed, this is called Regulated Transcription.  Transcription is regulated by Transcription Factors.  Transcription Factors can bind to either the enhancer or promoter regions of the DNA adjacent to the Genes that they regulate.  Depending on the Transcription Factor, the transcription of the adjacent Gene is either up- or down-regulated (turned on or off).  This is known as Transcription Regulation.

 

During Transcription, an enzyme called Helicase unwinds a short section of the DNA double helix strand near the start of the Gene to be copied like a zipper, separating it into two strands.  One is designated as the Template Strand (aka Antisense strand or noncoding strand), which serves as the blueprint for the copy, and the other is the Coding Strand (aka Sense Strand or Non-Template Strand).  The unwound section is known as the transcription bubble.

(Continued on next page- post was too big to fit on one page)

 

http://ewascienceputnam.weebly.com/uploads/5/1/1/0/5110389/237108.gif?378

 

The RNAP and transcription bubble travel along the section of the DNA to be copied from the Promoter sequence to the Terminator sequence.  The RNAP reads the sequence of the Nucleotides off the Template Strand as it goes, creating a new ever growing strand- adding one Nucleotide at a time to the new strand.  The result is a new strand is called a Precursor mRNA (or Pre-mRNA) transcript which will shortly become Messenger RNA (mRNA).  As the transcription bubble travels along the DNA double helix, the RNAP simultaneously rewinds the helix at the rear of the transcription bubble.  Like how a zipper works, it simultaneously unzips and re-zips it without going back and forth.

 

http://www.phschool.com/science/biology_place/biocoach/images/transcription/startrans.gif

 

The resulting strand of Pre-mRNA is a complimentary copy of the Template Strand (the Nucleotides are in the same but reverse order) and identical to the Coding strand with the exception that Thymine (T) Nucleotides are replaced with Uracil (U) Nucleotides, as shown above.

 

Next, Post-Transcriptional Modulation (aka- Pre-mRNA Processing, RNA Processing, or Co-transcriptional modification) occurs to turn the pre-mRNA into mature mRNA that will be ready for Translation.  This processing consists of several stages; capping, addition of a Poly-A tail, splicing, and RNA editing:

 

Capping - A methylated cap (aka 5’ cap or 7-methylguanylate cap, abbreviated m7G) is attached on the 5’ end.  This is done to protect the mRNA from degradation, promote RNA movement from the nucleus out to the cytoplasm, and to help facilitate Ribosomal binding later on during Translation. (19)

 

Addition of a Poly-A tail - Otherwise known as polyadenylation, is where a poly-adenalated tail (aka- poly-A tail) is added at the 3’ end of the mRNA.  It protects against mRNA degradation, helps promote mRNA movement from the nucleus out to the cytoplasm, and helps facilitate the mRNA attachment to the Ribosome during Translation later on. (19)

 

Splicing – Pre-mRNAs include two different types of segments, Exons and Introns.  During splicing the Introns are removed by a Spliceosome.  The mRNA contains only Exons after splicing is complete, as shown below.

 

http://www.phschool.com/science/biology_place/biocoach/images/transcription/eunotcol.gif

 

Here is a video animation of splicing which may help you better understand this process:

 

There is also another form of splicing known as Alternative Splicing, where different types of proteins, called Isoforms (which will be used later to create the GABAAR subunits), can be coded from a single Gene. (22)  In this process, particular Exons of a Gene may be included within, or excluded from (Exon Skipping) the final mRNA (shown below).  Consequently, the proteins translated from Alternatively Spliced mRNAs will contain differences in their amino acid sequence which later translates into differences in their biological functions. This process greatly increases the diversity of proteins that can be encoded from a single Gene. (5)  (Note- A large number of Isoforms are also the result of SNPs, which we discussed earlier.)

 

http://upload.wikimedia.org/wikipedia/commons/thumb/6/6e/Splicing_overview.jpg/320px-Splicing_overview.jpg

 

RNA editing – In this process discrete changes to specific Nucleotide sequences within certain pre-mRNA molecules are made, which result in mRNA transcripts with sequences that are different from the original Gene. (24)  Like Alternative splicing, RNA Editing increases the number of different proteins available without the need to increase the number of Genes in the Genome.  In other words, multiple Isoforms (forms of protein) can be generated from the same Gene.  So it can create proteins with slightly different functions to use in specialized circumstances. (25)  Editing may include the insertion, deletion, and Base substitution of Nucleotides. (24)

 

The GABAAR contains 5 subunits that are made from Isoforms (produced by either Alternative Splicing or RNA Editing). (28)  Researchers have discovered that the Pre-mRNA of at least one of the GABAAR subunits, the GABA-alpha3 receptor subunit, goes through the RNA Editing process. (26)  19 different Isoforms (α1-6, β1-3, γ1-3, δ, ε, θ, π, and ρ1-3) have been discovered that make that the GABAAR subunits, which appear in different combinations.

 

After Post-Transcriptional Modulation is complete we now have a mature mRNA that can go on to the next phase in Gene Expression, which is called Translation.  Translation is the process through which cellular Ribosomes manufacture proteins using the instructions encoded on the mRNA.  Essentially, the mRNA carries instructions from the Genes in the DNA out of the nucleus to Ribosomes suspended in the cytosol- and then to the rough part of the Endoplasmic Reticulum (ER).  (Note- The rough part of the Endoplasmic Reticulum is so named because the Ribosomes attached to the walls give it a ‘rough’ appearance, shown below).  The Endoplasmic Reticulum is an organelle, which literally means ‘little organ.’  There are many different ‘little organs’ in a cell that all have specialized functions. (48)

 

http://images.protopage.com/view/722248/e5otxffoui7se5xlfmb6gqlak.jpg

 

The Ribosomes, with the help of Transfer RNA (tRNA) molecules, translate the information on the mRNA into an amino acid chain, also known as a polypeptide chain.

 

http://themicroscopicplant.weebly.com/uploads/9/1/8/9/9189496/1330842542.jpg

 

The processed mature mRNA strand contains a series of Codons that will dictate the sequence of the amino acids needed to make the protein.  The Ribosome (shown above) consists of two major units— a small subunit and a large subunit.  The large subunit joins amino acids to form a polypeptide chain. (14)  The Translation processes begins when a Ribosome binds to a special nucleotide sequence on the mRNA called a Start Codon.

 

Amino acids that will be used to manufacture the protein are selected, collected, and carried to the Ribosome by tRNA  molecules.  On one end of the tRNA molecule there is a 3 nucleotide sequence called and Anticodon, and on the other end is a corresponding amino acid, as shown below.

 

http://www.nature.com/scitable/content/ne0000/ne0000/ne0000/ne0000/6903578/EssGen1-5_tRNA-labeled_0.jpg

 

The tRNA molecule enters one part of the ribosome and binds to the mRNA Codon if there is a match between the Codon on the mRNA and the Anticodon on the tRNA.  In order for the tRNA to bind, the nucleotide sequence on the Anticodon must be a complementary match to the sequence of nucleotides of mRNA Codon.  This is how the mRNA indirectly dictates which amino acids will be used in the polypeptide chain.

 

http://cklabrie.wikispaces.com/file/view/mRNA_to_protein.gif/285948376/503x262/mRNA_to_protein.gif

 

If there is a complementary match, the tRNA will bind to the Codon on the mRNA.  The Ribosome then slides over to the next Codon on the mRNA where another tRNA will come along and bind to the mRNA Codon (refer to picture above).

 

 

The amino acids on the other end of the tRNA molecules will then be joined together by a peptide bond to create a polypeptide chain.  The mRNA then releases the first tRNA molecule and the Ribosome slides over to the next Codon on the strand.  Then, another tRNA molecule will come along and bind to this Codon and the process is repeated- resulting in an ever growing polypeptide chain of amino acids.  This stepwise addition of amino acids to the growing protein chain is called Elongation. (42)  The process will repeat until the polypeptide chain is complete, which happens when the Ribosome reaches the Stop Codon on the mRNA strand.

 

Here is a video animation of all the steps in Translation that I just discussed:

 

So now we have a translated polypeptide chain made out of amino acid sequences.  In order to understand the peptide bonds that holds the chain together we first must look the molecular structure of the amino acids.  The amino acids within these sequences are molecules made from amine (-NH2) and carboxylic acid (-COOH) functional groups, along with a side-chain (aka R Group) that is specific to each amino acid (shown on next page).  The amine group of one amino acid links to the carboxyl group of the next amino acid creating a peptide bond in order to form the chain.  During this process a water molecule is lost and thus this process is called Dehydration Reaction.  (continued on next page)

 

http://ranubisnis.files.wordpress.com/2012/02/amino.jpg?w=320&h=305

 

So the beginning of the chain will start with an amine group and the end of the chain will end with a carboxyl group.  The amine end is referred to as the N-terminus and the carboxyl end is called the C-terminus.  When the protein is translated from messenger RNA, it is created from N-terminus to C-terminus.

 

The portion of the amino acid remaining in the center after the peptide bonds are formed is called an amino acid residue (aka- side chain, or R-Group Variant) and is also referred to as the primary structure of the peptide chain. (21)

 

 

http://www.chemistry.ccsu.edu/crundwell/Galleries/gallery/protein.gif

 

Due to repulsions and attractions between atoms that make up the amino acids of the peptide chain the shape will begin to conform into twists and sheets, which are referred to α helices and beta sheets respectively (shown above).  This is also referred to as the secondary structure.

 

http://www.quia.com/files/quia/users/lmcgee/genetics/AP_chapter17_Proteinsynthesis/proteintoER_L.gif

 

Referring to the picture above, at the very beginning of Translation, as the polypeptide chain begins to emerge from the Ribosome, a short Signal Sequence (called a ‘signal peptide’ in the picture above) is created on the N-Terminal of the polypeptide chain.  At this point the Ribosome, along with the polypeptide chain that it is synthesizing, is referred to as a Ribosome-nascent chain complex (RNC).  While the polypeptide is still being synthesized from the Ribosome and the Signal Sequence begins to emerge, a Signal Recognition Particle (SRP) (aka Docking Protein) binds to the Signal Sequence.  This temporarily halts Translation of the polypeptide chain by the Ribosome as the SRP guides and anchors the RNC to an SRP Receptor located on the surface of the rough Endoplasmic Reticulum (ER) membrane that is associated with a Translocon. (31, 32)  The Translocon acts as a channel through the ER membrane.  The Ribosome then docks on the Translocon and the Signal Sequence inserts into the channel.  The SRP and SRP receptor then release.  Translation then proceeds once again, during which time the elongating polypeptide chain will begin to pass though the Translocon.  This is known as Vectorial Synthesis.  Once Translation is complete a Signal Peptidase will come along and cleave (cut) the polypeptide chain off from the Signal Sequence portion, leaving the polypeptide chain in the lumen (interior cavity) of the ER.  This entire process is known as Co-translational Translocation.

 

Here is a video animation you can watch showing what I have just described:

http://www.youtube.com/watch?v=SC0TNIrT_lg’

 

http://faculty.weber.edu/nokazaki/Human_Biology/Chp%202-Chemistry_files/image012.jpg

 

 

In the ER the polypeptide chain folds to become a functional protein that becomes one of the subunits for the final GABAA receptor.  Folding into this tertiary structure (3 dimensional shape) is facilitated through N-Linked Glycosylation (addition of sugar residues) and proteins present in the lumen of the ER called molecular Chaperones (e.g. calnexin and binding immunoglobulin protein (BiP)), which protect it from the high concentration of other proteins in the ER, giving it time to fold correctly. (28, 33, 36, 41)  The folded protein is now said to be in a native state. (44)  Then the various folded protein isoforms that will comprise the 5 subunits within the GABAAR (that will become the pentameric GABAA transmembrane receptor) Oligomerize (bind to each other) in the ER forming a quaternary structure. (34)  Ultimately it is the shape of the proteins which make up the subunits that determines their function.  So the receptor is made from the primary structure (sequence of amino acid residues) plus the secondary structure (conformation of polypeptide chains into α helices and beta sheets) plus the tertiary structure (protein folding to make subunits) plus the quaternary structure (combination of subunits to form the receptor) (shown above).

 

 

This assembly process plays a critical role in determining the diversity of receptor subtypes expressed on the neuronal plasma membrane. (37)  Many different subunit combinations are theoretically possible, however only a limited number of these combinations can actually exit the ER to later be inserted into the neuronal cell surface.  Most GABAARs are composed of 2α, 2β, and 1γ subunit, the γ subunit can be replaced by δ, ε or π depending on the neuron type receptor location on the neuron cell membrane. (53)  The most abundant type of GABARs the body makes have the α1β2y2 subunit configuration. (36)

 

Within the ER, unassembled or improperly folded receptor subunits are subjected to Polyubiquitination that targets them for proteasomal degradation, a phenomenon that is dependent on the level of neuronal activity. (37, 46, 53)  In Polyubiquitination, Ubiquitin molecules are signaled to bind to the target protein.  These Ubiquitin molecules are known as ‘the kiss of death’ to a protein.  Ubiquitin molecules tag which proteins will be ferried by protein-transport machinery to the Proteasome for degradation.  A Proteasome is a protein degradation "machine" within the cell that can break down proteins into short polypeptides and amino acids.  However, the ubiquitin-like protein PLIC can bind directly with receptor α and β subunits to block targeting for proteasomal degradation, allowing the subunits to continue on their journey. (36, 37, 45, 46)  This one way the cell controls how many receptors will be made to populate the cell membrane. (37)

 

(Note- the terms proteins, receptors, and subunits will be used interchangeably from here on out- but all refer to the receptor complex of subunits assembled in the ER)

 

Properly folded and assembled subunits are exported from the ER at ER exit sites (ERES) on its membrane where COPII proteins are present.  COPII proteins direct a portion of the ER to encapsulate the subunits and bud off to become a transport vesicle used to shuttle subunits to the Golgi apparatus. (39)

 

The Golgi apparatus (aka Golgi complex) is an organelle that functions as a factory in which proteins received from the ER are further processed and sorted for transport to their eventual destinations, which could be to lysosomes inside the cell, the plasma membrane, or secretion outside of the cell. (43)  Our subunits will be destined for the plasma membrane.

 

The Golgi apparatus is composed of stacks of membrane-bound structures known as cisternae (singular: cisterna).  The cisternae stack has four functional regions: the cis-region, medial-region, endo-region, and trans-region (shown below). (37, 38)

 

http://organelles2011.wikispaces.com/file/view/Z_A_3_-_D_Visualisation_of_the_Golgi_Apparatus_.gif/263686496/Z_A_3_-_D_Visualisation_of_the_Golgi_Apparatus_.gif

 

The transport vesicles travel to the cis-cisterna (side closest to the ER) of the Golgi apparatus where the membrane of the transport vesicle fuses with the membrane of the Golgi apparatus, merging their contents. (33)  The protein is processed as it travels through the Golgi apparatus, from the cis-cisterna to the trans-cisterna (side furthest away from the ER). The Golgi apparatus sorts and packages proteins into vesicles that determine their ultimate destination. The Golgi is known as the ‘Post Office’ of the cell because it attaches routing directions to the proteins so that they are delivered to their proper destinations. (35)  This type of controlled movement of the protein from place to place in the cell is known as Trafficking.

 

Therefore the modifications which happen to the receptor proteins within the Golgi apparatus will determine its final destination.  For example, the GABAAR-associated protein (GABARAP) associates with the y2 subunit of GABAARs and aids in the trafficking of GABAARs from the Golgi network to the plasma membrane.  N-ethylmaleimide-sensitive factor (NSF) and brefeldin-A-inhibited GDP/GTP exchange factor 2 (BIG2) bind to the β subunits and modulate GABAA receptor trafficking.  Golgi-specific DHHC zinc-finger-domain protein (GODZ) is a Golgi resident palmitoyltransferase that regulates Palmitoylation of γ-subunits and is a critical step in the delivery of GABAARs to the plasma membrane (Note: Palmitoylation is the attachment of fatty acids).  GABAAR-interacting factor proteins (GRIFs) have a role in the trafficking of GABAARs to the membrane. (47)  Glycan trimming (modification of sugar chains by enzymes) and Phospholipase-C-related catalytically inactive proteins (PRIP) are also involved and play essential roles in the trafficking of receptors and in modulating their Phosphorylation state. (28, 37, 40, 47)  The journey of the protein from the ER, to the Golgi apparatus, to the cell membrane and the various steps in between is called the Secretory Pathway (shown below). (44)

 

http://ars.els-cdn.com/content/image/1-s2.0-S089662731100300X-gr2.jpg

 

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’).

 

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.

 

http://totalpict.com/images/92/922615272503c6da9773a7.png

 

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)

 

http://1.bp.blogspot.com/_a6500SnWswg/SnLrhO48l1I/AAAAAAAACn0/0Uff4oDMyPo/s400/receptors.bmp

 

The receptors located in the postsynaptic terminal are activated by GABA neurotransmitters released by the presynaptic terminal of the previous neuron, which transiently (lasting for only a short time) activate the receptor and cause inhibitory postsynaptic currents (IPSCs)- which makes the neuron less likely to fire.  This type of modulation is called is called Phasic inhibition. (28) The receptors located in peri or extra synaptic positions are persistently activated by ambient concentrations of the neurotransmitter GABA.  This type of modulation is called Tonic inhibition. (28)

 

The final GABAARs are classified as transmembrane proteins (TPs) (aka- transmembrane receptors) because they penetrate the cell membrane and provide a gateway that for a substance (in this case chloride) be transported into the cell.  They are also considered to be part of the Cys-loop ligand-gated ion channel superfamily (shown in the picture below on the left) because the subunits of all these receptors share a common ancestral structural elements that include- five subunits that form a pentameric arrangement around a central pore, an extracellular N-terminal domain, a characteristic loop formed by a disulfide bond, four transmembrane domains (TM1–4), and an extended cytoplasmic loop region between TM3 and TM4. (36, 49)

 

http://ars.els-cdn.com/content/image/1-s2.0-S0028390808002293-gr1.jpg

 

Referring to the schematic below, on the left- four transmembrane α-helices (1-4) of a subunit are depicted as cylinders. A single transmembrane alpha helix is called a transmembrane domain.  On the right- five subunits are symmetrically arranged around the central chloride conduction pore (aka- ion channel).

 

http://upload.wikimedia.org/wikipedia/commons/thumb/0/06/GABAA_receptor_schematic.png/320px-GABAA_receptor_schematic.png

 

The ion channel part of the receptor is formed by transmembrane domains. Five subunits, each with four transmembrane domains (M1-M4), form a pentameric structure with the ion channel pore in the center. The second transmembrane domain (M2) lines the pore. The M2 domains are slightly kinked in the middle and form the ion channel gate to control opening and closing of the channel. (50)

 

This completes the explanation of how a GABAAR is made.  If you want to learn how the receptor functions, you can read a previous paper I posted entitled “A LAYMAN’S EXPLANATION OF NEURONS, BENZOS, AND RECOVERY” at this link:

http://www.benzobuddies.org/forum/index.php?topic=77803.msg1026651#msg1026651

 

I would include it here, but this paper is just becoming too long.

 

Now that you know more about Gene Expression and how a GABAA receptor is made, let’s examine some of the research regarding receptor changes.

 

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.

 

Well, that’s it.  I will try to stick around to answer questions pertaining to what I have discussed here for a while, but then I must get back to work breaking down the next topic on the menu- The Glutamate Hypothesis.  This one will be important as it may explain why reinstatement does not always take away all of the withdrawal symptoms, why it must be done within a short window after the drug was initially discontinued, and may be what is behind the kindling phenomenon.

 

I hope that this will thread will become an interactive dialogue regarding receptor changes, a place where members here doing research can bounce ideas off each other, and also help to educate our members about the molecular components involved.

 

 

References

1) http://ghr.nlm.nih.gov/handbook/basics/Chromosome

2) http://ghr.nlm.nih.gov/handbook/genomicresearch/snp

3) http://www.makGene.com/index.cfm?fa=content.display&content_id=39

4) http://www.makGene.com/index.cfm?fa=content.display&content_id=26

5) http://en.wikipedia.org/wiki/Alternative_splicing

6) http://www.benzo.org.uk/ashsupp11.htm

7) http://bjp.rcpsych.org/content/179/5/390.full

8 ) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3375401/

9) http://ghr.nlm.nih.gov/Gene/GABRA1

10) http://www.Genenames.org/Genefamilies/LGIC

11) http://en.wikipedia.org/wiki/Regulation_of_Gene_expression#Up-regulation_and_down-regulation

12) http://pharmrev.aspetjournals.org/content/62/1/97.long#title29

13) http://en.wikipedia.org/wiki/Down-regulation#Mechanism:_The_Insulin_Receptor

14) http://en.wikipedia.org/wiki/Ribosome

15) http://en.wikipedia.org/wiki/AntiCodon#AntiCodon

16) http://en.wikipedia.org/wiki/Protein_folding

17) http://www.jneurosci.org/content/21/1/330.long#sec-10

18) http://pharmrev.aspetjournals.org/content/62/1/97.full#title2

19) https://www.boundless.com/physiology/cellular-structure-and-function-1/rna-processing/adding-5-cap-and-poly-tail-to-rna/

20) http://www.ncbi.nlm.nih.gov/pubmed/10968650/

21) http://www.healthknot.com/body_protein.html

22) http://link.springer.com/chapter/10.1007%2F978-1-59745-465-0_8#page-1

23) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2651003/

24) http://en.wikipedia.org/wiki/RNA_editing

25) http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RNA_Editing.html

26) http://en.wikipedia.org/wiki/Post-transcriptional_regulation

27) http://www.jbc.org/content/271/21/12221.long

28) http://vrn.vanderbilt.edu/2010/PDFs/VRN2010%2025-32%20(Gurba).pdf

29) http://en.wikipedia.org/wiki/Posttranslational_modification

30) http://en.wikipedia.org/wiki/Nuclear_membrane

31) http://en.wikipedia.org/wiki/Protein_targeting#Co-translational_translocation

32) http://en.wikipedia.org/wiki/Translocon#The_ER-translocon

33) http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/ProteinKinesis.html

34) http://etd.library.vanderbilt.edu/available/etd-11242008-214043/unrestricted/Wenyi_Lo_Dissertation.pdf

35) http://droualb.faculty.mjc.edu/Course%20Materials/Physiology%20101/Chapter%20Notes/Fall%202011/chapter_2%20Fall%202011.htm

36) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3093971/

37) http://physrev.physiology.org/content/91/3/1009.full

38) http://en.wikipedia.org/wiki/Golgi_apparatus

39) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2228377/

40) http://gallus.reactome.org/cgi-bin/eventbrowser?DB=test_gallus_reactome_release_2_myisam&FOCUS_SPECIES=Homo sapiens&ID=446203&

41) http://en.wikipedia.org/wiki/Protein_translocation#Protein_translocation

42) http://www.nobelprize.org/educational/medicine/dna/a/translation/elongation.html

43) http://www.ncbi.nlm.nih.gov/books/NBK9838/

44) http://www.cureffi.org/2013/02/24/cell-biology-04-the-secretory-pathway/

45) http://www.hindawi.com/journals/aps/2012/416864/

46) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3093971/figure/F2/

47) http://www.nature.com/nrn/journal/v9/n5/fig_tab/nrn2370_F2.html

48) http://sunburst.ud.edu/~bgoodman/ReviewFrames.htm

49) http://en.wikipedia.org/wiki/Cys-loop_receptors

50) http://www.thebarrow.org/Education_And_Resources/Barrow_Quarterly/204387

51) http://f1000research.com/articles/1-69/v1

52) http://www.ncbi.nlm.nih.gov/pubmed/22531784

53) http://www.ncbi.nlm.nih.gov/pmc/articles/ PMC2709246/[/size]

 

Hi Perseverance

Another outstanding post! I hope you decide to go into molecular biology as a career. In your post you mentioned that GABA receptors come back in a slightly altered form less sensitive to benzodiazepines following withdrawal.  Ashton supports this as do experimental animal models. I note that most authorities including Ashton believe that post acute withdrawal syndrome (PAWS) is more likely to develop in people who have cold turkeyed or withdrawn rapidly. There is no experimental evidence for this, but based on my knowledge of other cellular processes I reasoned that what may be happening here is that when the body is suddenly denied benzodiazepines it "senses" the need to produce more GABA receptors as well as re-regulate other pathway expression as rapidly as possible. This could lead to "shortcuts" at the gene expression level resulting in slightly different variants of the GABA receptor less sensitive to GABA. Again, there is no experimental evidence for this and it is only my working hypothesis, but it is precisely the reason I have chosen to taper so slowly. I believe that a gradual taper may result in more normal functioning GABA receptors in the long run. We'll see.

Bart

 

Hi Bart,

 

What do you think the 'short cuts' might be/which stage of gene expression?

Hi Pers

I sent you a PM but it was blocked. "Short cuts" can happen at multiple locations, but post transcriptional editing is common as well as various ligand effects on the promoter region.

 

Bart

Sorry about that Bart, I had to close my box due to restrictions on my time.

 

I thought you might mention the Post Translational editing- however, could you elaborate on the ligand effects on the promoter region?

Perseverance,

 

Thanks so much for this post. I hope to possibly participate in the discussion, but need to spend more time working with the material in order to really grasp some of the science involved. I didn't want to miss the chance to thank you now though for your diligence in researching and then relaying detailed information on the many processes underlying benzodiazepine related changes and adaptations in the body and brain, in this case the GABBAA receptors. Like bart, I hope you consider pursuing molecular biology and take your interest and passion for this subject into the professional arena. I've always thought it will most like be someone who has been through BZD w/d and healed who can really delve into the science of it and make discoveries that might finally improve understanding and lead to change as well.

 

Thank you.

Hi Pers

If you're interested in more details, here is a link to a fairly inexpensive but classic  introduction to all this.

http://www.amazon.com/Recombinant-DNA-Genomes-Course-Watson/dp/0716728664/ref=sr_1_24?s=books&ie=UTF8&qid=1369264427&sr=1-24

Or you could just Wiki  "Regulation of Gene Expression" for a thumbnail picture.

http://en.wikipedia.org/wiki/Regulation_of_gene_expression

The take home message is our cells, including neurons, are wonderfully adaptive to whatever environment we put them in. These processes are the mechanism for what we call neuroplasticity. When we suddenly start or stop taking benzodiazepines, our neurons adapt as best they can. Gradual change tends to cause less disruption for adaptive changes as a general rule. This may or may not be true for benzodiazepine induced alterations. Nobody really knows for sure. A lay person analogy might be if your house was destroyed in a hurricane and you rebuilt it slowly it would likely turn out better than if you threw up your new house in a very short period of time.

Bart

I don't understand the science, but this is an upsetting theory for those of use who have rapid tapered or cold turkeyed. It makes me wonder if reinstatement and doing a slow taper would make sense.

 

This is clearly a 'benzo thought', but nonetheless, it's scary to read stuff like this after you have CT or rapid tapered and still symptomatic.

Perseverance,

 

Thanks so much for this post. I hope to possibly participate in the discussion, but need to spend more time working with the material in order to really grasp some of the science involved. I didn't want to miss the chance to thank you now though for your diligence in researching and then relaying detailed information on the many processes underlying benzodiazepine related changes and adaptations in the body and brain, in this case the GABBAA receptors. Like bart, I hope you consider pursuing molecular biology and take your interest and passion for this subject into the professional arena. I've always thought it will most like be someone who has been through BZD w/d and healed who can really delve into the science of it and make discoveries that might finally improve understanding and lead to change as well.

 

Thank you.

 

Hi Perseus,

 

It may be a little bit too late in life for me to start looking at delving into a new career that would require an enormous amount of education, lol...but I am flattered nonetheless by your remark.

 

My present goal is to learn about how all this works in order to understand the current research and also to present reasons that carry some sort of weight why people should not reinstate after being off the drug for an extended period, why not to try to treat their sxs erroneously with supplements that might have benzo like characteristics, to educate people so they can advocate for themselves, and to show people that all of these changes fall under the umbrella of neuroplasticity--meaning they are adaptive changes, where the body uses its own set of tools to adjust to changing conditions.  Everyone's bodies make these types of changes all of the time to adjust to everything from changing hormone levels to adjusting to stress and anxiety.  Of course the changes in our case were more profound, but I am trying to show people that the body has the capability to reverse these changes.

 

This is my driving force- but as I get more and more into my regular life I find that I have less and less time to do this type of work.

 

I do not know who will pick up where Ashton left off and carry the baton...I should think that it would be someone like Bart if it were going to be a benzo victim- I am in the process of catching up to his level of knowledge and I obviously have a ways to go.

 

I think it is important that we all try to educate ourselves on this subject so that as we discuss our dilemma with our doctors we can enlighten them and through this spread awareness in the medical community.  This will not only help our own situations, but perhaps help to curve the current trends in prescribing these types of drugs saving future patients from the same fate.

 

This gets more difficult as pharmaceutical companies continue to research and attempt to develop newer versions of benzos that have slightly different binding chacteristics, which they then market as a different class of drugs, which is obviously creating a bigger problem with regards to tolerance and withdrawal.  The current example would be the Z Drugs (ZOPICLONE (LUNESTSA), ZAPLEPLON (SONATA), AND ZOLPIDEM (AMBIEN)).

 

Due to this trend in drug development and I believe that genetics testing may be the only viable answer to protect people as it may alert a doctor to a patients particular vulnerability to certain types of drugs.  This has the possibility to be fairly successful by means of taking human subjectivity out of the equation.

 

However, this type of testing becoming widely available plus the continuing research required pushes this possibility well off into the future.  So in the meantime creating awareness is our best weapon and is something we can all participate in.

 

Hi Pers

If you're interested in more details, here is a link to a fairly inexpensive but classic  introduction to all this.

http://www.amazon.com/Recombinant-DNA-Genomes-Course-Watson/dp/0716728664/ref=sr_1_24?s=books&ie=UTF8&qid=1369264427&sr=1-24

Or you could just Wiki  "Regulation of Gene Expression" for a thumbnail picture.

http://en.wikipedia.org/wiki/Regulation_of_gene_expression

 

Thanks for the information about the book.  I already knew about Wiki- infact I use that stuff all of the time.

 

The take home message is our cells, including neurons, are wonderfully adaptive to whatever environment we put them in. These processes are the mechanism for what we call neuroplasticity.

 

I have been saying this too.  I have been telling people for a long time that these are functional changes.  In my last thread, in one of my posts, I remember specifically calling them plastic.  I even quoted research in this thread that explained why they are considered neuroplastic changes.  I don’t think people understand that this is entirely different than structural damage… entirely.  These types of changes have the capability to turn around, albeit that may take a long time.  It is not like a disease process initiated.

 

 

When we suddenly start or stop taking benzodiazepines, our neurons adapt as best they can. Gradual change tends to cause less disruption for adaptive changes as a general rule. This may or may not be true for benzodiazepine induced alterations. Nobody really knows for sure. A lay person analogy might be if your house was destroyed in a hurricane and you rebuilt it slowly it would likely turn out better than if you threw up your new house in a very short period of time.

Bart

 

I totally got it the first time you brought this up- however I am glad that you explained it and gave an analogy so that people following this thread will all understand it too.

 

My question was about what you meant by the ligand effects on the promoter region.  Were you referring to increased activity in perhaps any of the excitatory ligands such as glutamate, acetycholine, serotin, etc. and how second messengers and other mechanisms can be triggered, going back to the nucleus to change how things come together at the promoter region?  I asked you to elaborate because I am just now beginning to learn about these regulation mechanisms.

 

I don't understand the science, but this is an upsetting theory for those of use who have rapid tapered or cold turkeyed. It makes me wonder if reinstatement and doing a slow taper would make sense.

 

This is clearly a 'benzo thought', but nonetheless, it's scary to read stuff like this after you have CT or rapid tapered and still symptomatic.

 

Hi gettingthere,

 

I understand how you feel because I am in the same boat.  However, this is really nothing new as most of us became aware after the fact that a CT or rapid detox has been connected to protracted withdrawal syndrome.  Bart’s suggestion simply provides a reasonable explanation as why it happened.

 

His suggestion should actually do the opposite and bring you a greater peace of mind about it all because as I just told Perseus in my post above, these changes are a form of neuroplasticity and-

 

“they are adaptive changes, where the body uses its own set of tools to adjust to changing conditions. Everyone's bodies make these types of changes all of the time to adjust to everything from changing hormone levels to adjusting to stress and anxiety.  Of course the changes in our case were more profound, but I am trying to show people that the body has the capability to reverse these changes.”

 

Your brain up and down regulates things all of the time, and receptors are absorbed and reinstated as a part of normal plastic processes.  The ‘short cuts’ may have made some slight alterations in nucleotide and thus amino acid changes- but as time wears on I should think that there would be a turnover of the receptors which should eventually bring it all back to normal.

 

The way I look at what Bart said is that- ok, we might have receptors that were made slightly different because of short cuts- but eventually they should all turn over as the neuron brings things back to status quo.  It is just going to take a long time.

 

I don’t think you will get the results you are looking for by reinstating if researchers are right about the Glutamate Hypothesis.  I am currently delving into this new topic and plan to post a separate thread on it in the future.

 

What I have ascertained so far from the little I have read, is that after you stop taking benzos there is a silent and then an active phase.  Once the active phase begins, something changes with the Glutamate side of things.  After that sets into motion taking more benzos will not completely relieve the sxs- like after it starts it sets the point of no return for reinstatement.  Researchers noted that the actual WD sxs did not commence until the active phase was set into motion.

 

This may explain why some people’s sxs don’t show up immediately after they discontinue the drug—in fact there have people who claimed it took weeks to a month or more after stopping their benzo before sxs began to appear- well beyond the half-life of the particular benzo they were taking.  That might have been due to the silent phase.

 

It also may explain why there appears to be a window of time where you can somewhat successfully reinstate (during the silent phase) and why that window of time expires (when the active phase begins).

 

Another thing it may also explain is kindling- as each attempt may set more Glutamate receptors into the active phase.

 

That is my take on it from the little I have read.  But as you can see- reinstatement could likely cause more problems for you- by changing more GABAARs and perhaps sending more Glutamate receptors into active phase.  The anecdotal evidence supports this.  Time and time again I have seen people reinstate only to be completely surprised that they were still experiencing WD sxs and then devastated realizing that they had to start all over again.

 

Pers - I read the following in the post (didn't have time to read the whole post):

 

"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)

 

Do you think it's more likely that the the Gene expression will be reversed, even if slowly, rather than not at all? This quote made me feel like i may have permanent CNS damage that will last forever, something i can't swallow right now.

 

Niko

 

 

Hi Niko,

 

I am not sure why Ashton said this since the changes are plastic changes.  She did not give references so I do not know who the Molecular biologists were that she spoke of.  However, I actually did address this concern in my post- here is what I said:

 

"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."

 

Hello Perseverance.  Bravo for a wonderful piece! 

 

I will need to set aside some time to digest it properly when I get a genuinely lucid interval.

 

Glancing through your article, I am intrigued and disheartened by the possibility of neuroadaptations failing to ever fully restore the former status quo.

(((Perse)))

 

Thank you so much for this and all you have done to help us understand the effect benzos have on the nervous system and the mechanisms of withdrawal.  We are so lucky to have you as our resident researcher. The more knowledge we have, the more empowered we can be when dealing with doctors or other medical personnel.

 

PG 

I just seem to keep getting worse. I wonder if its permanent too. Sigh. I don't think I can get over this.

What I was concerned about early on when I got off these benzo's was whether these drugs cause "structural" damage to our DNA, the double helix, and therefore would alter our natural transcription process for healing repair.  Was Dr. Ashton referring to DNA damage when she said there is no permanent damage?  It does make clear that there are functional alterations in gene expression.  Why can one person take these long-term and come off with no problem and another be stuck in benzo w/d s/x's?  Our genetics.  But, how can the same person take the same drug decades earlier long term and get off them with no problem, like I did, then have benzo w/d s/x's at a later time with long-term usage?  Should I truly be looking at my psychological state and what has contributed to my GABA receptors not fixing themselves in a timely manner like they should have?  People take these drugs for a reason:  stress.       

Hello Perseverance.  Bravo for a wonderful piece! 

 

I will need to set aside some time to digest it properly when I get a genuinely lucid interval.

 

Glancing through your article, I am intrigued and disheartened by the possibility of neuroadaptations failing to ever fully restore the former status quo.

 

Hi Braban,

 

I have not seen anything that makes me think this that this is true...I think that there are some changes that happen in the signal transduction pathways- which I am looking into now.  But there is no change to our DNA, just how it is being copied and how things are phosphorylated--so while these changes may have been set into motion, I don't see why they would not eventually come back to status quo as the cell moves back towards homeostasis.  I think that because there are so many ligands that were thrown off kilter, this may take a long time to come back around...but again, these are all functional changes that were initiated to counteract the effects of the drug--and now that the drug is out of the picture, it would not make sense for the receptors to eventually revert back to their original states.

(((Perse)))

 

Thank you so much for this and all you have done to help us understand the effect benzos have on the nervous system and the mechanisms of withdrawal.  We are so lucky to have you as our resident researcher. The more knowledge we have, the more empowered we can be when dealing with doctors or other medical personnel.

 

PG 

 

Hi PG!

 

That's one of the main goals-- empowering the people to create a ripple effect.  If the patients can relay credible knowledge to their doctors, then their benzo claims will be taken seriously-  which will then create awareness in the medical community.  The study Catherine Pittman did was awesome--and will be another avenue to get the word out.  Hopefully if we hit it from enough angles this problem will gain more wide spread legitimacy.

 

I know you did your part with the doctors you were seeing--and I know how difficult it is to advocate for yourself when your brain is in disarray.  But hopefully if enough of us try, the word will spread that much faster.

 

Having knowledge can also protect people in tolerance and WD from misdiagnoses and poly-drugging to which muddy up the water.  So important for people to understand this stuff- but difficult to relay it to them in a state of WD.  We just have to keep chipping away at it.

I just seem to keep getting worse. I wonder if its permanent too. Sigh. I don't think I can get over this.

 

Don't lose hope Maymay, as I just said to Braban, I have not seen anything to make me think these changes are irreversible.  However, the changes mentioned in the research I have read are ones that can take a long time to turn around.  I really believe that patience is key.  I understand the battle fatigue, but if you look at Recovery-road.org you will see that while some people took 3-5 years- but in the end, they did recover.  While that is well outside the average recovery time, it shows that reversals continue to happen.

What I was concerned about early on when I got off these benzo's was whether these drugs cause "structural" damage to our DNA, the double helix, and therefore would alter our natural transcription process for healing repair.  Was Dr. Ashton referring to DNA damage when she said there is no permanent damage?  It does make clear that there are functional alterations in gene expression.  Why can one person take these long-term and come off with no problem and another be stuck in benzo w/d s/x's?  Our genetics.  But, how can the same person take the same drug decades earlier long term and get off them with no problem, like I did, then have benzo w/d s/x's at a later time with long-term usage?  Should I truly be looking at my psychological state and what has contributed to my GABA receptors not fixing themselves in a timely manner like they should have?  People take these drugs for a reason:  stress.     

 

Hi Becksblue,

 

I saw in your signature that you were also prescribed Ambien?  Were you taking both Ambien and a benzo the first time around?  That may have tipped the scales and made a difference.  The other thing is I have noticed some people do seem to skate by the first time around but get hit the second time they try the drug...so there may be some sort of sensitization happening.

 

Looking at your signature it looks like you are around 18 months off?  You may be getting close, I have seen many people recover around the 22 month mark, although each persons recovery is unique.  I know it is hard, believe me.  Try to hang in there, because really you never know, it could be just around the corner for you.  You have a lot of time under your belt.

Thank you perseverance.

Hi P,

 

Wow, you are just amazing! Can't wait to share this with my husband who IS a molecular biologist and has watched all my suffering while doctors didn't know what to do with me. I will be curious too to hear his thoughts about prospects for potential testing for people susceptible to benzo issues as he is working on related technology for diagnostics in general. (I'll share anything I learn.)

 

I also appreciate the info on the problems with reinstatement after the silent phase. I did reinstate successfully once long ago after 30 days off due to unbearable migraines and iatrogenic depression. Having tried it every other way, I am finding that a slow daily microtaper works much better for me.

 

I'm interested in your thoughts in terms of a few practical applications for what you have shared here:

 

Any theories why some people seem to be able to tolerate alcohol post-benzo and others not? Is there a consensus that it should definitely be always avoided due to more sensitized/altered gaba receptors? (Having a hard time coming to terms with the fact that I may never be able to have a glass of wine again.) And IF I did the wine experiment at some point after feeling totally healed and fell apart, then it would seem that reinstatement and re-tapering would not be a good idea at all.

 

Also along practical lines, I recently read this article on how meditation promotes genes for good health which was new to me. Perhaps that is one thing we can do in w/d that helps in ways more profoundly than just stopping obsessive worrying (which is also important):

http://www.newscientist.com/article/dn23480-meditation-boosts-genes-that-promote-good-health.html

 

And last, in reading your goals here for why you are sharing your research (i.e. education), I'm wondering what you think about supplements post-benzo to support return to function. It would seem that doctors who specialize in functional medicine (an example: www.drhyman.com) would be a good fit for those post-withdrawal as it would seem we have a functional vs. structural disorder as you pointed out. I have seen several but they have not been helpful DURING withdrawal. But what about, say, adrenal support supplements AFTER withdrawal to speed things along? Or do you think we should just let our bodies recover on their own?