The Chemistry of Thought

                                                                                        

                                    

Astrophysicist Sir James Jean (1937):
I incline to the idealistic theory that consciousness is fundamental, and that the material universe is derivative from consciousness, not consciousness from the material universe... In general the universe seems to me to be nearer to a great thought than to a great machine. It may well be, it seems to me, that each individual consciousness ought to be compared to a brain-cell in a universal mind.
A primary feature of the Universe is entropy, the movement of energy across a gradient, from a higher order of energy, beginning with The Big Bang, the primordial explosion that marks the genesis of the Universe, to a lower order of energy, until all energetic movement ceases, as in the cold of interstellar space.  The emergent qualities of the Universe, derived from the quantum energy of electrical reactions moving across a gradient, most closely resembles a Thought, an emergent quality arising from quantum energy transfer across a gradient located in the brain.  Life harnesses the energy gradient inherent in entropy and creates order out of decay.
                                       




            
                                               THE CHEMISTRY OF THOUGHT

            Thought is an electro-chemical event located in the time and space of the human brain.

1.         The mind consists of:
            A.  The conscious mind (where “I” live)
            B.  The subconscious mind (the emotional mind)
            C.  The unconscious mind (instinct) (“reptile mind”).

2.   Conscious memory consists of:
            A. The actual event generating electro-chemical activity in the brain.
            B. The presentation of this electro-chemical event for storage.
            C. The storage of the electro-chemical event.
            D. The retrieval and expression of the electro-chemical event (remembering).



             I believe one can divide human mental activity into "genetic thought" and "conscious thought".  In a study of permanently vegetative patients, total brain glucose metabolism was less than half that of conscious normals.  This implies that the brain stem (reptile brain) uses about half the total energy consumption of the brain for genetically determined housekeeping functions.  

During anesthesia, brain glucose consumption falls 20%.  This resembles the glucose consumption fall of 25% during sleep.  These results imply that subconscious thought consumes 25-35% of total glucose consumption.  

A fourth study found a 16% increase in global glucose consumption over baseline in test subjects awake during the performance of a voluntary, conscious sensory-motor task. Baseline was determined by test subjects sitting quietly in a room while blindfolded and hearing impaired, but not asleep.  

Taken together, these studies suggest that genetic thought, or unconscious and subconscious brain metabolism, comprises 75-85% of the total, with conscious thought consuming up to an additional 20% when needed.

    

         In a paper published in 1984, (Nature, Vol. 312, #8, p. 101) Sir Francis Crick, knighted for co-discovering the structure of DNA, speculated that memories might be coded and stored on DNA.  If so, the storage of the electro-chemical event (C, above) may occur on the DNA of neuronal memory cells.   Memory-related enzymes may modify DNA control regions with 200 different chemical signals, including methyl groups, to store the pattern of electro-chemical activity. Following the retrieval signal (D, above), the altered control regions then regulate transcription of this information through messenger RNA.  Memory proteins are then expressed.

Only 15% of the DNA of a fertilized ovum regulates the expression of “humanness”, in other words, all of the information necessary to synthesize and fold proteins into the shape of a human being is present in less than 15% of the DNA of a single fertilized ovum.   Present day understanding of DNA considers the remaining 85% of DNA as “nonsense”, in that no researcher can demonstrate a specific use for 85% of non-coding DNA.

           
            A single strand of human DNA is comprised of 3,000,000,000 base pairs.  200 different chemical signals can modify these base pairs, changing the electro-chemical information stored in the DNA.     Thus, the total number of the possible combinations of a single strand of DNA is 3,000,000,000²⁰⁰.  By way of comparison, the total number of atoms in the known Universe is about 10⁸⁰.  If all the information in a single human DNA molecule were in book form, it would fill a phone book the height of the Washington Monument.  When uncoiled, the DNA in a single cell would be about six feet long.  If the entire DNA in the human body were laid end to end, it would stretch over 6.5 billion miles.

Extrapolating from these numbers, then the storage capacity of the totality of neuronal cells (approximately 86 billion) becomes effectively infinite over the course of a human lifetime.     A Bell Lab researcher, using a measured single nerve conduction rate of 2 bits per second, estimated that the total possible data generated over the course of a human lifetime requiring storage by the human brain approximated 125 megabytes of data. Thus, the theoretical storage capacity of neural DNA far exceeds the actual data amounts generated during a human lifetime.  This may mean that memory storage neurons comprise only a small portion of the overall brain. 

 



                                                   OUTRAGEOUS PROPOSITION:

That we can build chemical thoughts to be transmitted by virus to order to change behavior. 
This can take the form of either changing present thoughts or altering memories, to influence present or future behavior.

Theoretical possibilities include:

           

           An experimenter places a well-paid, voluntary test subject in a training scenario.  The subject is taught, through audio-visual means, to experience, and then remember, a happy occasion.  We can take a positron emission tomograph of their brain while it stores the memory, so that a 3D computer map will show the exact location of the neurons which stored the memory.  After strapping the person into a head restraint, a computer guides a minuscule laser while it burns a hole in the skull, which allows the insertion of a miniature needle directly into the location of the memory.  The surgeon then performs a memorectomy by applying suction through the needle, so that the 5 or 10 neurons involved are removed and stored.

            

         The experimenter extracts the DNA from the neurons and injects it into a DNA sequencer.  A computer program then stores the DNA code.  This allows another team member to construct a virus containing the stored code, effectively replicating the thought pattern.  The researcher then inserts the virus into a double-stranded DNA insect virus like Baculovirus, encapsulating it in a buckminsterfullerene protein coat, and creates an infectious agent to transmit the thought.  Using aerosol infectivity, spray can thoughts become possible: “thought graffiti”; Ph.D. in a pill; or doctor in a can; each becomes feasible.  We can either enhance present capabilities by encoding for additional knowledge, or change existing memories by encoding for “altered history”.  We can infect various parts of the brain with appropriate memories, or change the “state” of the parts. 

            

         Thus, encoding a virus to infect the aggressive center of the brain with highly aggressive thoughts could construct the perfect soldier.  Or, using it as a bio-weapon, you might infect a population to make them hyper-violent, so that they kill each other. 

            

            Or, encoding a virus to infect the pleasure center of the brain, (the opiate center) and increase the release of endorphins could render drug addiction meaningless.  If everyone were happy, would everyone quit work?  Or, would people only do what they liked, so that overall productivity increases?  Would war end?  Is this Soma?  It isn’t that people cannot think about happiness, it’s that they cannot hold that thought.  Create DNA to “hold it” in the happiness center.

          

         Could an alien civilization construct a virus that contains their intelligence, send it to Earth, infect the population, and take over humankind?

            

        Could unscrupulous researchers infect the world with a passivity virus, retain the antidote for themselves, and rule the world?

Scientists estimate that modern humans first emerged about 200,000 years ago.  Using 20 years as a generational cycle, then 10,000 past lives becomes possible for a human living today.  There exists sufficient excess storage capacity in a single fertilized ovum to permit the inter-generational transmission of memories.  Does this explain recall of past life experiences?  
It is also likely that “intelligence” (the difference between a chimpanzee and a man) is located on DNA.

            The neighbors’ dog comes into the yard of a genetic researcher to crap.  He and his chemist friend decide to make a DNA virus to infect the dog’s memory so it remembers to shit at home.  The virus mutates, infects the sinus emissions of the dog (Law of Unintended Consequences), and becomes highly contagious.  What happens?

            Is it God, or is it Man?   Is the viral information contained in the AIDS virus the product of GOD (Buckminster Fuller believed homosexuality to be God’s response to overcrowding), an accident (a meteorite containing the virus falls to Earth), or is it Man-made (a genetically engineered RNA that codes for the disease)? 

Profit-Driven Drug Companies:

            

         Drug companies collectively spend in excess of $10 billion per year on research.  This represents less than 10% of pretax profits, and earns the companies a dollar-for-dollar tax credit.  In 1975, one such company discovers a treatment for Retroviruses.  However, this family of viruses primarily infects animals.  When the researchers discover that infection of chimps occurs best in the mucus membranes of the colon, they realize that human homosexuals represent their ideal host population.  They clone the homosexuality gene (the fruitless gene), infect the population, the number of homosexuals increases, then they introduce the AIDS virus, a lab creation containing maximum infectivity combined with delayed lethality to insure serial transmission, and thus guarantee sales of their viral treatment. 

Teenage Sexuality:

            

         The teenage desire to procreate is genetic in origin (coded on DNA).  Once DNA is altered by events (memories cause methylation changes on DNA) caused by the genetically driven urge to reproduce (pregnancies, Sexually Transmitted Diseases, etc.) the person is left calmer, ready for “conscious driven” actions:

The Maturity of Aging.

           
           Do teenagers “fall in love” because they infect each others nervous system with genetic thoughts? “I can’t get her out of my mind”.    Does infatuation have a genetic basis?  Does swapping spit lead to love blindness?   Is a Love Virus possible?


The Black Widow Husband and the Psycho-Bitch Wife:

            

       The Black Widow male knows it will be eaten immediately following sexual transfer of DNA.  The Black Widow female knows she will eat the male.  Thus they both participate in a genetically driven murder-suicide following procreation: Fatal Attraction.

World Domination:

           

         Create a virally infected aerosol Ecstasy, which infects the aggression center, causing “other” directed violence.  Code the virus so that after x divisions, using the methyl time clock, it deactivates, making the virus self-limiting.  Then, infect a population in the middle of Olympic Village: they sink into anarchy.  The biological researchers working for Saddam Hussein tell him they can create Mr. Hyde in Israel.  Use Sarajevo, Mogadishu, and Rwanda as examples.  Or, an international weapons corporation sets up a lab to make the virus in order to boost munitions sales to third world countries.  Or, an international dam construction company pays for construction of the virus to get UN money to rebuild dams after anarchy destroys them.

Star Trek:
           
          Dr. Frankenstein constructs a thought virus to transmit his memories to the infant he clones of himself... 

                                                                          Robert Bayless
                                                                                 2015
                                                                     All Rights Reserved

                
               

Viral transmission of genetic material

http://www.google.com/patents/US5403484

Mice Epigenetics and Memory

• National Geographic
• December 1, 2013

Mice Inherit Specific Memories, Because Epigenetics?

by Virginia Hughes

Two weeks ago I wrote about some tantalizing research coming out of the Society for Neuroscience meeting in San Diego. Brian Dias, a postdoctoral fellow in Kerry Ressler’s lab at Emory University, had reported that mice inherit specific smell memories from their fathers — even when the offspring have never experienced that smell before, and even when they’ve never met their father. What’s more, their children are born with the same specific memory.
This was a big, surprising claim, causing many genetics experts to do a double-take, as I discovered from a subsequent flurry of Tweets. “Crazy Lamarkian shit,” quipped Laura Hercher (@laurahercher), referring to Lamarckian inheritance, the largely discredited theory that says an organism can pass down learned behaviors or traits to its offspring. “My instinct is deep skepticism, but will have to wait for paper to come out,” wrote Kevin Mitchell (@WiringTheBrain). “If true, would be revolutionary.”
The paper is out today in Nature Neuroscience, showing what I reported before as well as the beginnings of an epigenetic explanation. (Epigenetics usually refers to chemical changes that affect gene expression without altering the DNA code).
Having the data in hand allowed me to fill in the backstory of the research, as well as gather more informed reactions from experts in neuroscience and in genetics. I’ve gone into a lot of detail below, but here’s the bottom line: The behavioral results are surprising, solid, and will certainly inspire further studies by many other research groups. The epigenetic data seems gauzy by comparison, with some experts saying it’s thin-but-useful and others finding it full of holes.
So what is the surprising part, again? 
If you’ve followed science news over the past decade then you’ve probably heard about epigenetics, a field that’s caught fire in the minds of scientists and the public, and understandably so. Epigenetic studies have shown that changes in an organism’s external environment — its life experiences and even its choices, if you want to get hyperbolic — can influence the expression of its otherwise inflexible DNA code. Epigenetics, in other words, is enticing because it offers a resolution to the tedious, perennial debates of nurture versus nature.
But some scientists dispute the notion that epigenetic changes have much influence on behavior (see this Nature feature for a great overview of the debate). Even more controversial is the idea that epigenetic changes can be passed down from one generation to the next, effectively giving parents a way to prime their children for a specific environment. The key question isn’t whether this so-called ‘transgenerational epigenetic inheritance’ happens — it does — but rather how it happens (and how frequently, and in what contexts and species).
That’s what Dias and Ressler wanted to investigate. Trouble is, environmental influences such as stress are notoriously difficult to measure. So the researchers focused on the mouse olfactory system, the oft-studied and well-mapped brain circuits that process smell. “We thought it would give us a molecular foothold into how transgenerational inheritance might occur,” Dias says.
The researchers made mice afraid of a fruity odor, called acetophenone, by pairing it with a mild shock to the foot. In a study published a few years ago, Ressler had shown that this type of fear learning is specific: Mice trained to fear one particular smell show an increased startle to that odor but not others. What’s more, this fear learning changes the organization of neurons in the animal’s nose, leading to more cells that are sensitive to that particular smell.
Ten days after this fear training, Dias allowed the animals to mate. And that’s where the crazy begins. The offspring (known as the F1 generation) show an increased startle to the fruity smell even when they have never encountered the smell before, and thus have no obvious reason to be sensitive to it. And their reaction is specific: They do not startle to another odor called propanol. Craziest of all, their offspring (the F2 generation) show the same increased sensitivity to acetophenone.
The scientists then looked at the F1 and F2 animals’ brains. When the grandparent generation is trained to fear acetophenone, the F1 and F2 generations’ noses end up with more “M71 neurons,” which contain a receptor that detects acetophenone. Their brains also have larger “M71 glomeruli,” a region of the olfactory bulb that responds to this smell.
“When Brian came in with the first set of data, we both just couldn’t believe it,” Ressler recalls. “I was like, ‘Well, it must just be random, let’s do it again.’ And then it just kept working. We do a lot of behavior [experiments], but being able to see structural change that correlates with behavior is really pretty astounding.”
Still, those experiments couldn’t rule out some kind of social, rather than biological transmission. Perhaps fathers exposed to the fear training treated their children differently. Or maybe mothers, sensing something odd in their mate’s behavior, treated their children differently.
To control for these possibilities, the researchers performed an in vitro fertilization (IVF) experiment in which they trained male animals to fear acetophenone and then 10 days later harvested the animals’ sperm. They sent the sperm to another lab across campus where it was used to artificially inseminate female mice. Then the researchers looked at the brains of the offspring. They had larger M71 glomeruli, just as before. (The researchers couldn’t perform behavioral tests on these animals because of laboratory regulations about animal quarantine.)
“For me it clicked when we did the IVF
Other researchers also seem convinced. “It is high time public health researchers took human transgenerational responses seriously,” says Marcus Pembrey, emeritus professor of paediatric genetics at University College London, who has been championing the idea of epigenetic inheritance for over a decade. “I suspect we will not understand the rise in neuropsychiatric disorders or obesity, diabetes and metabolic disruptions generally without taking a multigenerational approach,” he says.
In an interesting historical aside, Pembrey also notes that the new study echoes an experiment that Ivan Pavlov did* 90 years ago, in which he trained mice to associate food with the sound of a bell. Pavlov “reported that successive generations took fewer and fewer training sessions before they would search for food on hearing a bell even when food was absent,” Pembrey says. Nevertheless, the idea that experience could be biologically inherited fell out of favor in the 20th century. “If alive today, Pavlov would have been delighted by the Dias and Ressler paper, first as a vindication of his own experiment and results, and second by the amazing experimental tools available to the modern scientist.”
Neuroscientists, too, are enthusiastic about what these results might mean for understanding the brain.
“To my knowledge this is the first example, in any animal, of epigenetic transmission of a simple memory for a specific perceptual stimulus,” says Tomás Ryan, a research fellow at MIT who studies how memories form in the brain. “The broader implications for the neuroscience of memory and to evolutionary biology in general could be paradigm shifting and unprecedented.”
There are still some unanswered questions, Ryan notes. For example, the researchers didn’t do a control experiment where the F0 animals are exposed to the fruity odor without the shock. So it’s unclear whether the “memory” they’re transmitting to their offspring is a fear memory, per se, or rather an increased sensitivity to an odor. This is an important distinction, because the brain uses many brain circuits outside of the olfactory bulb to encode fear memories. It’s difficult to imagine how that kind of complicated brain imprint might get passed down to the next generation.
Ressler and Dias agree, and for that reason were careful not to refer to the transmitted information as a fear memory. “I don’t know if it’s a memory,” Dias says. “It’s a sensitivity, for now.”
What’s that got to do with epigenetics?
So let’s call it a sensitivity. How could a smell sensitivity, formed in an adult animal’s olfactory bulb in its brain, possibly be transmitted to its gonads and passed on to future generations?
The researchers are nowhere near being able to answer that question, but they have some data that points to epigenetics.
There are several types of epigenetic modifications. One of the best understood is DNA methylation. There are millions of spots along the mouse genome (and the human genome), called CpG sites, where methyl groups can attach and affect the expression of nearby genes. Typically, methylation dials down gene expression.
Dias and Ressler sent sperm samples of mice that had been fear-conditioned to either acetophenone or propanol to a private company, called Active Motif, which specializes in methylation analyses. The company’s researchers (who were blinded to which samples were which) mapped out the sperm methylation patterns near two olfactory genes: Olfr151, which codes for the M71 receptor that’s sensitive to acetophenone, and Olfr6, which codes for another odor receptor that is not sensitive to either odor.
It turns out that Olfr151, but not the other gene, is significantly less methylated in sperm from animals trained to fear acetophenone than in sperm from those trained to fear propanol. Because less methylation usually means a boost in gene expression, this could plausibly explain why these animals have more M71 receptors in their brains, the researchers say.

What’s more, the same under-methylation shows up in the sperm of F1 animals whose fathers had been trained to fear acetophenone.
“It’s a very precise signal,” Ressler says. “The convergence of this data, we think, shows that this is a really profound and robust phenomenon.”
Others, though, find a number of flaws in this epigenetic explanation.
Timothy Bestor, professor of genetics and development at Columbia University, points out that methylated CpG sites only affect gene expression when they are located in the so-called gene promoter, a region about 500 bases upstream of the gene. But the Olfr151 gene doesn’t have any CpG sites in its promoter.
That means the differences in methylation reported in the paper must have occurred within the body of the gene itself. “And methylation in the gene body is common to all genes whether they’re expressed or not,” Bestor says. “I don’t see any way by which that gene could be directly regulated by methylation.”
But what would explain the methylation differences between the trained animals and controls? They’re pretty subtle, he says, and “could easily be a statistical fluke.”
Bestor was skeptical from the outset, based on the mechanics of the reproductive system alone. “There’s a real problem in how the signal could reach the germ cells,” he says.
For one thing, the seminiferous tubules, where sperm is made inside of the testes, don’t have any nerves. “So there’s no way the central nervous system could affect germ cell development.” What’s more, he says it’s not likely that acetophenone would be able to cross the blood-testis barrier, the sheet of cells that separates the seminiferous tubules from the blood.
By this point in my conversation with Bestor, I was starting to feel a bit defensive on behalf of epigenetics and all of its wonder. “Are you saying you think epigenetic inheritance is a bunch of bologna?” I asked helplessly.
“No,” he said, laughing. “It’s just not as dynamic as people think.”
What’s next?
A good next step in resolving these pesky mechanistic questions would be to use chromatography to see whether odorant molecules like acetophenone actually get into the animals’ bloodstream, Dias says. “The technology is surely there, and I think we are going to go down those routes.”
First, though, Dias and Ressler are working on another behavioral experiment. They want to know: If the F0 mice un-learn the fear of acetophenone (which can be done by repeated exposures to the smell without a shock) and then reproduce, will their children still have an increased sensitivity to it?
“We have no idea yet,” says Ressler, a practicing psychiatrist who has long been interested in the effects of post-traumatic stress disorder (PTSD). “But we think this would have tremendous implications for the treatment of adults [with PTSD] before they have children.”
It will take a lot more work before scientists come close to understanding how these data relate to human anxiety disorders. So what, after all of these words, should we take away from this study now?
Hell if I know. Here’s the most rational and conservative appraisal I can muster: Our bodies are constantly adapting to a changing world. We have many ways of helping our children make that unpredictable world slightly more predictable, and some of those ways seem to be hidden in our genome.
Anne Ferguson-Smith, a geneticist at the University of Cambridge, put it more succinctly. The study, she says, “potentially adds to the growing list of compelling models telling us that something is going on that facilitates transmission of environmentally induced traits.”
Scientists, I have to assume, will be furiously working on what that something is for many decades to come. And I’ll be following along, or trying to, with awe.

–
*Update, 12/1/13, 2:35pm: It seems that that Pavlov experiment may have been retracted in 1927, though I don’t know anything about that beyond what is stated here.
Style note: A few paragraphs of this post were adapted from my earlier post on this research, published November 15.

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DNA Methylation and Memory

DNA methylation-mediated control of learning and memory

Nam-Kyung Yu¹, Sung Hee Baek² and Bong-Kiun Kaang^13*

• * Corresponding author: Bong-Kiun Kaang kaang@snu.ac.kr

Author Affiliations

¹ National Creative Research Initiative Center for Memory, Department of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 151-747, Korea
² Creative Research Initiative Center for Chromatin Dynamics, Department of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 151-747, Korea
³ Department of Brain and Cognitive Sciences, College of Natural Sciences, Seoul National University, Seoul 151-747, Korea

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Molecular Brain 2011, 4:5  doi:10.1186/1756-6606-4-5

The electronic version of this article is the complete one and can be found online at: http://www.molecularbrain.com/content/4/1/5

Received:	7 December 2010
Accepted:	19 January 2011
Published:	19 January 2011

© 2011 Yu et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Animals constantly receive and respond to external or internal stimuli, and these experiences are learned and memorized in their brains. In animals, this is a crucial feature for survival, by making it possible for them to adapt their behavioral patterns to the ever-changing environment. For this learning and memory process, nerve cells in the brain undergo enormous molecular and cellular changes, not only in the input-output-related local subcellular compartments but also in the central nucleus. Interestingly, the DNA methylation pattern, which is normally stable in a terminally differentiated cell and defines the cell type identity, is emerging as an important regulatory mechanism of behavioral plasticity. The elucidation of how this covalent modification of DNA, which is known to be the most stable epigenetic mark, contributes to the complex orchestration of animal behavior is a fascinating new research area. We will overview the current understanding of the mechanism of modifying the methyl code on DNA and its impact on learning and memory.

Cytosine methylation in mammals

In mammals, methylation at symmetric CpG dinucleotides in genomic DNA is important for heritable gene silencing and regulation of gene expression [1]. It is one of the primary epigenetic mechanisms for the regulation of gene transcription along with the various histone modifications such as methylation, acetylation, SUMOylation, ubiquitination, and phosphorylation. DNA methylation has been shown to play essential roles in genomic imprinting, X chromosome inactivation, and maintenance of genome stability [1,2].

The addition of a methyl group from SAM (S-adenosyl-L-methionine) substrates to the cytosine is catalyzed by DNA (cytosine-5)-methyltransferases. Three DNA methyltransferases, DNMT1, DNMT3a, and DNMT3b, have functional enzymatic activity in mammals (Figure 1) [3,4]. DNMT1 has been called a "maintenance methyltransferase" as it has a substrate preference for hemimethylated DNA over unmethylated DNA. DNMT1 interacts with the DNA replication machinery during the S phase of dividing cells so that the methylation pattern is reliably copied to the daughter strand [5]. DNMT3a and DNMT3b are regarded as "de novo methyltransferases" since they can methylate the cytosine of CpG dinucleotides previously unmethylated on both strands, altering the epigenetic information content. They play crucial roles in establishing genomic methylation patterns during cell differentiation [6]. However, this functional distinction is not always obvious since DNMT1 also displays de novo methyltransferase activity, and its substrate preference is limited in a cellular context-dependent manner [3].

Figure 1. Overview of mammalian cytosine methylation. In mammals, cytosine methylation is necessary for regulation of DNA sequences and the following gene expression patterns. DNMT1 is active on hemimethylated DNA, which assists the maintenance of genomic methylation. The recruitment of DNMT1 to hemimethylated DNA is mediated through its interaction with UHRF1. DNMT3A and DNMT3B function as de novo methyltransferases, and they methylate the cytosine of previously unmethylated CpG dinucleotides on both strands.

The three DNMT genes display differential expression profiles in the central nervous system. DNMT1 is highly expressed in neurons from embryogenesis through adulthood [7,8]. DNMT3b expression is observed in neural progenitor tissue only during early embryogenesis. DNMT3a is expressed from late embryogenesis to adulthood with a peak during the early postnatal period, then its level declines but remains detectable in the post-mitotic neurons of the adult brain [9,10]. Aberrant regulation of DNMT expression has been shown to be related to drug abuse [11,12], suicide [13], and psychiatric disorders such as schizophrenia and bipolar disorder [14,15].

Given that the DNMTs exhibit little or no innate sequence specificity beyond the CpG dinucleotide [3], the manner in which these enzymes find a specific target DNA sequence in mammals could be a very fundamental question [4]. One of the candidate targeting molecules is UHRF1 (ubiquitin-like, containing PHD and RING finger domain 1), which has been shown to interact with hemimethylated DNA through its SET and RING-associated (SRA) domain [16]. UHRF1 is crucial for targeting DNMT1 to hemimethylated DNA, and permits the faithful transmission of genomic methylation patterns [16]. Notably, recent report observed an altered expression of this protein in the central nervous system by long-term memory formation[17]. Another reported mechanism is the use of other specific epigenetic marks such as histone methylation marks for binding to hemimethylated DNA. It has been shown that DNA methylation is dependent on previously shaped histone methylation marks. In addition, Polycomb group proteins link histone methylation and DNA methylation [18-20]. Small non-coding RNA-mediated guidance of DNMT is also a fascinating candidate mechanism, which has yet to be well described in mammals [4].

Whereas the enzymes for cytosine methylation are well characterized, the existence of enzymes for active demethylation is controversial [21]. Passive demethylation, which can occur without DNMT activity during cell proliferation and does not require enzyme activity, is widely accepted. In contrast, it has been doubted whether active removal of methyl residues from 5-methylcytosine would occur after cellular differentiation because the methyl mark on cytosine residues conferred by DNMT is known to be the most stable epigenetic modification due to its covalent nature. During the past decade, accumulating reports have presented observations of active demethylation [22-28]. Although there are several conceivable mechanisms for demethylation and evidence supporting them [21], these remain inconclusive. Nucleotide excision repair-based mechanism involving growth arrest and DNA damage-inducible 45 (Gadd45α or Gadd45β) proteins is considered a candidate [22,29-31]. Gadd45 proteins are known as stress-inducible non-enzymatic proteins that regulate cell cycle arrest and promote the DNA repair reaction, coupling the deamination and glycosylation, and then filling in the bare cytosine. Another candidate is the ten-eleven translocation 1 (TET1) protein, that has been shown to convert 5-methylcytosine of DNA to 5-hydroxymethylcytosine, raising the possibility that DNA demethylation may be a TET1-mediated process [32]. Further, all three TET proteins (Tet1, Tet2, and Tet3) have been reported to catalyze a similar reaction [33].

Approximately 60%-70% of CpGs in the mammalian genome are highly methylated, the exception being the CpG-dense area in the vicinity of a promoter, which is called the CpG island and displays generally low, but tissue-specific methylation levels [4]. Given that cytosine methylation in the promoter represses the transcription of the gene, there are two modes of repression: (1) methyl cytosine can repel transcriptional activators, or (2) attract transcriptional repressors that have methyl cytosine-binding domains and recruit proteins such as histone deacetylase, which facilitate the formation of the silent chromatin state [34,35]. These methyl cytosine-binding proteins include methyl CpG-binding domains (MBDs) and methyl CpG-binding protein 2(MeCP2). In humans, the MeCP2 mutation is the well-known cause of autism spectrum disorder, Rett Syndrome [36], indicating the importance of proper interpretation of methyl cytosine marks by MeCP2.

Cytosine methylation patterns undergo dynamic change in the central nervous system

It was a long-held belief that cytosine methylation patterns would be cell-type specific and stable. The only opportunity to change the methylation pattern was thought to be during the cell division period when DNA is newly synthesized with bare cytosine residues. Unexpectedly, even though postmitotic neurons are terminally differentiated and no longer proliferate, DNA methyltransferases were found to be abundantly expressed in neurons [7], and their enzymatic activity was also significant in the brain [8]. These findings raised the possibility that DNA methylation patterns might be dynamically changing in the brain for some unknown roles. Accumulating evidence has shown that cytosine methylation could be altered in postmitotic neurons by neural activity [25,26,37] or during behavioral change in response to external signals [23,24,38].

A well-known example of this is cytosine methylation pattern formation by early life experience. Maternal care is known to affect the stress response in adulthood in rats. Weaver et al. showed that this enduring effect is mediated by a cytosine methylation change on specific gene loci [23,39]. High levels of maternal licking and grooming and arched-back nursing (High-LG) demethylate the promoter of the glucocorticoid receptor (GR) gene exon 1₇ in the hippocampus, and this change persists into adulthood [23]. This change can be reversed by treating the brain with L-methionine, so that the GR promoter is highly methylated, causing the low GR expression levels and high stress response as seen in the offspring of low-LG mothers [40]. This demonstrates that methylation change plays a key role in behavioral control through regulating GR expression. In addition, methylation of the glutamic acid decarboxylase 1 (GAD1) promoter, which is negatively correlated with mRNA levels, is decreased in the offspring of high-LG mothers [41]. Early life stress such as maltreatment or repeated maternal separation, which causes defective behavior in adulthood, also changes the methylation level of brain-derived neurotrophic factor (BDNF) or arginine vasopressin (AVP) [42,43]. In humans, higher methylation levels at the glucocorticoid receptor gene (Nr3c1) exon 1₇ promoter was found in the brains of suicide victims having a history of childhood abuse compared to those without such a history [44]. These findings show that DNA methylation patterns are shaped by experiences during the critical period of postnatal brain development, which persists throughout the lifespan.

An increasing number of reports have shown the dynamic nature of cytosine methylation in the brain. Social avoidance behavior that was induced by chronic social defeat stress given in adulthood coincided with demethylation of the stress hormone corticotrophin-releasing factor (Crf) gene and increase of its mRNA in the mouse hypothalamus [45]. There are studies showing that DNA methylation levels change according to age [46-48]. Electroconvulsive treatment into the hippocampal dentate gyrus area, which induces neuronal activity by direct electrical current injection in vivo, decreases the methylation level of specific regulatory regions of BDNF and fibroblast growth factor-1 (FGF) genes, correlatively increasing their mRNA and protein expression levels [22]. In addition, remarkable works done by JD Sweatt and his colleagues showed that contextual fear conditioning also induces methylation change in neural plasticity-related genes such as BDNF, reelin, PP1, and calcineurin [24,38,49].

In a rodent primary neuron culture, high potassium-induced neuronal depolarization demethylates the BDNF exon IV promoter (according to the new nomenclature) [50], correlating with a corresponding increase in mRNA after neural activity [25,26]. MeCP2 basally represses BDNF expression, and is phosphorylated and dissociated from the BDNF promoter in response to neural activity, as the promoter is demethylated. Other repressing chromatin complexes are also detached, whereas phospho-CREB, the transcriptional activator, binds to the promoter. Picrotoxin-induced chronic network activity also causes methylation changes in specific loci of the BDNF and reelin promoter, which in turn alters the miniature excitatory postsynaptic current (mEPSC) frequency [37].

Methylation levels in the BDNF gene region are dynamically changed in postmitotic neurons both in vitro and in vivo as has been reported in several studies from different laboratories. The rodent BDNF gene has nine exons, the individual mRNAs of which are differentially regulated according to the tissue and brain region [50]. Epigenetic mechanisms have been reported to control the transcription of the BDNF gene, and DNA methylation seems to participate in the regulation. However, according to different reports, the specific promoters undergoing changes in methylation are quite diverse. Chronic network activity caused by picrotoxin treatment of cultured neurons [37] or contextual exposure to the living animals demethylates the promoter of exon I [38]. The methylation level of this exon I promoter was also shown to be correlated with object recognition memory task performance [51]. The exon IV promoter is demethylated in the hippocampus by high potassium treatment that induces membrane depolarization [25,26], as well by contextual fear-conditioning given to animals [38]. The promoter is methylated in the prefrontal cortex by maltreatment in the early postnatal period [42], but is not affected by electroconvulsive treatment (ECT) to the dentate gyrus of the hippocampus [22]. ECT demethylates the promoter of the coding exon IX [22], which is methylated by early life maltreatment [42]. Collectively, it seems that the regulatory elements of the BDNF gene are differentially methylated or demethylated according to age or brain region, and the types of upstream signals.

As stated above, many studies have shown that external stimuli can alter the DNA methylation levels of behaviorally important genes in the brain. Mostly, the DNA methylation level is negatively correlated with mRNA or protein level. DNMT inhibitors can reverse the increased methylation and decreased transcription, which also blocks behavioral plasticity, suggesting the biological importance of these changes. Therefore, we would like to describe the contribution of methyl change to learning and memory in the next part.

DNA methylation contributes to synaptic or behavioral long-term plasticity

Alteration of neuronal gene expression pattern is required for long-term memory formation or for synaptic plasticity which is thought to underlie learning. Since DNA methylation is involved in neural activity-induced transcriptional changes, methylation might be important in the process of in vivo long-term memory formation. This key hypothesis was first examined by JD Sweatt and his colleagues. They showed by a series of elegant experiments that DNMT activity is required for associative memory formation and induction of long-term potentiation (LTP) [24,52]. The DNMT inhibitor 5-azadeoxycytidine (5-AZA), or zebularin infusion into the hippocampus immediately after contextual fear conditioning reverses the methylation and downregulation of PP1, a memory suppressor gene, impairing the formation of a fear memory [24]. Interestingly, methylation levels in the hippocampus return to baseline in 24 hrs, although the fear memory is maintained and is still dependent on the hippocampus at that time point [24]. This indicates that in the hippocampus, DNA methylation might not be a mechanism of contextual fear memory maintenance, but is a regulatory mechanism of transient gene expression. However, we cannot exclude the possibility that the methylation state of other genes might be persistently changed in hippocampal neurons during the time the memory is dependent on the hippocampus. Not only fear conditioning but also other types of learning, such as cocaine-induced conditioned place preference memory, were recently shown to require DNA methylation in the hippocampus [53].

Since DNA methyltransferase inhibitor drugs have a non-specificity problem [54], genetic manipulation of DNMT provides another valuable strategy for understanding the causal relationship. Genetic approaches targeting DNMTs have been performed to assess their importance in synaptic function or learning and memory.

Conventional DNMT1, or DNMT3a and DNMT3b deletion causes genomic hypomethylation and embryonic lethality [6,55], indicating that proper DNA methylation is required for normal development, but make it impossible to study the function of this modification in adulthood using these mice. DNMT1 ablation in neuronal precursor cells eventually causes global DNA hypomethylation, cell death during early postnatal development, and neonatal lethality [56]. Conditional DNMT3a knockout in neuronal precursor cells leads to a specific methylation pattern change, neuromuscular function abnormalities, and premature death [57]. These findings suggest that an appropriate DNA methylation pattern is required for postnatal neuronal survival or function, but cannot be used to evaluate the contribution of DNA methylation to adult brain function.

To assess the role of DNA methylation in the mature brain, DNMT1 has been deleted specifically in the precursors of postnatal excitatory neurons in the dorsal forebrain in Emx1, a promoter-driven conditional mutant mouse line, which survived into adulthood but showed abnormal development of somatosensory barrel cortex and impaired thalamocortical long-term potentiation [58]. When DNMT1 is deleted under the control of the calmodulin-kinase IIα (CaMKIIα) promoter [56], neither the DNA methylation level of endogenous retroviral repeats nor neuronal survival is affected. Interestingly, double knockout of DNMT1 and DNMT3a, mediated by the CaMKIIα-cre system results in smaller cell sizes, impaired hippocampal LTP, enhanced LTD, and deficits in spatial and contextual fear memory formation, whereas the single knockout lines of each gene display no abnormalities [59]. Immune function-related genes are upregulated and the global or specific DNA methylation levels are decreased in the double knockout mouse forebrain. In particular, Stat1, which is involved in neural plasticity and the interferon pathway, is upregulated at the mRNA level and decreases at the methylation level in NeuN-positive neurons. These results suggest that DNA methylation is important for synaptic plasticity and learning, probably through affecting the expression of plasticity-related genes. Another implication is that DNMT1 and DNMT3a play complementary roles in postmitotic excitatory neurons, although they have distinct enzymatic properties. Starting from this interesting finding, numerous questions arise. The detailed molecular mechanisms of how their roles are redundant and how their deficiency causes demethylation of specific sequences are elusive. Since the absence of DNMT was prolonged in the abovementioned study, the behavioral effect and the gene expression pattern appearing in the microarray might reflect only chronic influence and not the inducible role of DNMTs during learning.

Although some genes, including BDNF, are demethylated after learning, with a corresponding increase in mRNA, no study has shown if DNA demethylation is necessary for memory consolidation. This is because the demethylation mechanism remains unclear. A recent interesting report described how activity-induced gene Gadd45b is required for activity-dependent upregulation of BDNF [22]. The role of Gadd45b for DNA demethylation might be challenged in the learning and memory paradigm in the future.

Role of CpG methylation for maintaining long-lasting memory

Persistence is one of the most enigmatic features of memory. The prevailing hypothesis is that memory is encoded by the altered synaptic strength in the complex neuronal circuits; however, the detailed mechanism remains elusive. The DNA modification hypothesis of memory storage was first proposed by J.S. Griffith and H.R. Mahler in 1969 [60]. Since molecular turnover is a naturally continual process, DNA might be the one storage molecule that could maintain the learned information for the lifetime. In 1984, F. Crick [61] postulated that the maintenance molecule to overcome the dissipation of acquired changes would form multimers or at least dimers with each monomer having modified (+) or unmodified (-) modes. Even if one (+) component is exchanged by a newly synthesized (-) molecule by natural turnover, the hypothetic maintenance enzyme will quickly convert it to the modified (+) mode. In this way, once the (+)(+) conformation is acquired, the (+)(+) conformation would be maintained, which matches the feature of DNMT1. In 1999, R. Holliday indicated the cytosine methylation state of specific gene loci as a candidate crucial mechanism of memory storage [62].

It was only recently that a first report by J.D. Sweatt and his colleagues appeared demonstrating that DNA methylation is required for the maintenance of memory [49]. Contextual fear memory formation and its initial maintenance depend on the hippocampus, but it is generally believed that memory undergoes systems consolidation over approximately 3 weeks, so that the remote memory becomes dependent on the prefrontal cortex, including the anterior cingulate cortex (ACC), and independent of the hippocampus [63,64]. To test whether memory maintenance requires DNA methylation, Miller et al. [49] looked at the ACC region rather than the hippocampus. After contextual fear conditioning, hypermethylation of the calcineurin (CaN) gene was maintained for at least 30 days. A correlative decrease of calcineurin mRNA and protein also persisted for at least a month. When a DNMT inhibitor was injected into the ACC 29 days after training, DNA methylation on CaN decreased and 30-day memory was impaired. These findings suggest that the DNA methylation and demethylation processes are ongoing in the ACC region and that this dynamic balance is required for memory maintenance. It would be worth testing whether the same mechanism is applicable to other types of long-lasting memories such as conditioned taste aversion. Similarly, retrieval of conditioned place preference memory was recently reported to depend on DNA methylation in the prelimbic cortex [53].

One might ask how the modification of DNA in the nucleus that has a cell-wide effect could be involved in maintaining a specific memory [65]. A neuron has thousands of synapses connecting to a number of other neurons, and there is a high possibility that it participates in multiple memories through different synapses. It seems quite certain that modification of DNA in the nucleus itself cannot differentially affect each synapse without synapse-specific changes. Therefore, we believe that DNA methylation in itself is insufficient to store the memory. However, as recent evidence suggests, it is likely that maintaining the DNA methylation pattern by balanced methylation-demethylation activity is required for memory maintenance [49]. After a learning experience, the profile of synaptic strength or property in a participating neuron would be changed (Figure 2). To maintain this altered pattern of connections, neurons would need to contain certain amounts of their gene products, which could be stably controlled at the transcriptional level by CpG methylation at the regulatory element. If an imbalance is induced by DNMT inhibitor, the neuron would lose its capacity to maintain the strength of connections, impairing the memory storage [49]. If we are correct in our hypothesis that a neuron participates in multiple memories and that DNA methylation is required to maintain the entire synaptic properties of a neuron, DNMT inhibitor injection into a specific brain region would affect the different types of memory stored in that region. In addition, this persistent change should be subtle, such that the element could still be responsive to the upcoming signals to encode another memory. Conversely, there might be a limit to the number of memories a single neuron can participate in.

Figure 2. DNA methylation in learning and memory. Upper: One neuron (central green circle) has a number of connections with other neurons (peripheral circles). Lower: Each small circle represents a CpG site in a regulatory element of the gene. Filled circles indicate methylated CpGs and the white circles unmethylated CpGs. When the animal has a new experience ("Memory formation" state), some connections are activated (red dashed lines). Transient waves of gene upregulation or downregulation are required for memory formation and could be mediated by temporal modifications of DNA methylation. Memory suppressor genes (Gene B), such as PP1, are transcriptionally downregulated through DNA methylation, and plasticity-inducing genes, such as BDNF or reelin (Gene A), are upregulated with DNA demethylation. The methylation states of these genes are restored to the baseline level after memory consolidation. When the memory has been stabilized ("Memory maintenance" state), the neurons exhibit an altered profile of connection strength (compared to "Before learning" state in upper panel). We expect that the gene expression patterns in neurons need to be different from those before learning in order to maintain this modified combination of connection strengths. Maintaining this altered gene expression pattern might involve a stable change in DNA methylation. Calcineurin is a known example for Gene D that is increased in cytosine methylation and decreased in mRNA level. However, Gene C with decreased methylation and increased mRNA level that is associated with learning and memory has not been discovered in adult animals.

Conclusions and perspectives

Contrary to the traditional view, an ever-growing number of reports indicate that cytosine methylation in neurons seems to change quite dynamically after birth. Recent studies have shown that cytosine methylation is important for both memory formation and memory maintenance (Figure 2). In addition to its critical involvement in the transcriptional regulation of genes controlling memory consolidation processes, cytosine methylation has also been shown to be required for retaining the memory. However, the detailed mechanism is hardly known and many questions remain. What might be the upstream pathway of temporal regulation of DNMTs or the demethylation machinery? What other genes and how many genes in the genome will be affected during a behavioral learning process? Which mechanism would confer target specificity? There is skepticism that experience-mediated DNA methylation changes in neurons are too small to be of biological importance, although it is statistically significant. Clear answer to the questions about mechanism will help to evaluate whether the observed methylation changes in the brain are indeed biologically significant.

Moreover, most studies on the role of DNA methylation in the brain have analyzed the DNA methylation state using whole tissue lysates by methods such as bisulfite sequencing, methylation-specific PCR (MSP), or methyl CpG-specific immunoprecipitation. Due to the high heterogeneity of cell types in the brain, it is difficult to determine the type of cells in which the methylation level is changed and the number of cells that undergo the changes. In addition, the extent of methylation change could be blurred by the mixed population of cells. This problem might be solved by utilizing a recently reported nucleus analysis protocol [45,66]. Cell type-specific nuclei are labeled by transgenic expression of a fluorescence protein or by immunostaining. Using flow cytometry, only the target nuclei are separated from the whole tissue-purified nuclei, and these can then be analyzed in terms of DNA methylation. This method might be utilized to determine whether the detected changes after learning occur in neurons or astrocytes, or in other specific subpopulation of neurons. Interesting results are expected from labeling the activated neurons using reporter proteins driven by an immediate early gene promoter [67,68]. Analysis of the methylation pattern in this specific set of neurons would provide valuable information regarding the activity-induced regulation of cytosine methylation.

Another potentially useful means of detecting DNA methylation state at the cell level is by using the MSP-ISH technique [69]. Although to date there have been an insufficient number of studies using this method, possibly due to the methodological difficulties, the concept of observing the methylation pattern at the cell level could be used in the future to examine how many cells have the changes, and to analyze other factors such as neuronal structure concomitantly with methylation. It might be also combined with the various tracing methods used to examine neuronal connections.

Furthermore, it seems that cytosine methylation in neurons would be modulated delicately and dynamically for behavioral plasticity, which is distinct from the conspicuous cytosine methylation change in developmental or disease states. Conceivably, extensive change in DNA methylation patterns could cause abnormal or pathological states for the neuron; therefore, there might be a mechanism of neuron-specific tight regulation of DNA methylation.

Since DNA methylation is related to long-term behavioral alteration, it might be a good therapeutic target for treating long-term behavioral disorders. L-Methionine treatment in adulthoods has been shown to reverse the behavioral effect of lack of maternal care [40]. The content of diet, such as folate or vitamin C, is closely related to cytosine methylation levels [70,71]. DNMT inhibitors, such as 5-AZA, which are currently used for cancer treatment, might be utilized as treatment for behavioral disorders.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

NKY, SHB and BKK conceived of the review and drafted the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This work was supported by the National Creative Research Initiative Program of the Korean Ministry of Science and Technology. NKY is supported by BK21 fellowship.

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