Day 70 results from Changing to a youthful phenotype with broccoli sprouts

Here are my Day 70 measurements* to follow up Our model clinical trial for Changing to a youthful phenotype with broccoli sprouts, which had these findings:


Keep in mind that I’m not in the population represented by the clinical trial sample:

  1. My chronological age is above their inclusion range;
  2. My BMI is below their inclusion range; and
  3. I take supplements and meet other exclusion criteria.

I also didn’t take Day 0 measurements.

June 2019 BMI: 24.8

June 2020 BMI: 22.4

2020 IL-6: 1.0 pg / ml. See Part 2 of Rejuvenation therapy and sulforaphane for comparisons.

2020 C-reactive protein: < 1 mg / l.

2019 and 2020 No biological age measurements. Why aren’t epigenetic clocks standard and affordable?


I’ve made four lifestyle “interventions” since last summer:

  1. In July 2019 I started to reduce my consumption of advanced glycation end products after reading Dr. Vlassara’s AGE-Less Diet: How a Chemical in the Foods We Eat Promotes Disease, Obesity, and Aging and the Steps We Can Take to Stop It.
  2. In September I started non-prescription daily treatments of Vitamin D, zinc, and DHEA per clinical trial Reversal of aging and immunosenescent trends.
  3. Also in September, I started non-prescription intermittent quercetin treatments of Preliminary findings from a senolytics clinical trial.
  4. I started eating broccoli sprouts every day eleven weeks ago.

1. Broccoli sprouts oppose effects of advanced glycation end products (AGEs) provided examples of Items 1 and 4 interactions.

2. Two examples of Item 2 treatment interactions with Item 4 are in Reversal of aging and immunosenescent trends with sulforaphane:

  • “The effects of the combined treatment with BSE [broccoli sprout extract] and zinc were always greater than those of single treatments.”
  • “Vitamin D administration decreased tumor incidence and size, and the co-administration with SFN [sulforaphane] magnified the effects. The addition of SFN decreased the activity of histone deacetylase and increased autophagy.”

3. How broccoli sprout compounds may complement three supplements I take was in a 2020 review Central and Peripheral Metabolic Defects Contribute to the Pathogenesis of Alzheimer’s Disease: Targeting Mitochondria for Diagnosis and Prevention:

“The nutrients benefit mitochondria in four ways, by:

  • Ameliorating oxidative stress, for example, lipoic acid;
  • Activating phase II enzymes that improve antioxidant defenses, for example, sulforaphane;
  • Enhancing mitochondrial remodeling, for example, acetyl-l-carnitine; and
  • Protecting mitochondrial enzymes and/or stimulating mitochondrial enzyme activities, for example, enzyme cofactors, such as B vitamins and coenzyme Q10 .

In addition to using mitochondrial nutrients individually, the combined use of mitochondrial nutrients may provide a better strategy for mitochondrial protection.”

The review provided a boatload of mitochondrial multifactorial analyses for Alzheimer’s. But these analyses didn’t include effective mitochondrial treatments of ultimate aging causes. I didn’t see evidence of why, after fifteen years of treating mitochondrial effects with supplements, treating one more effect could account for my Week 9 vastly different experiences.


I nod to An environmental signaling paradigm of aging explanations. Its Section 10 reviewed IL-6, C-reactive protein, senescence, and NF-κB in terms of feedback loops, beginning with:

“It is clear that the increasing number of senescent cells depends on the post-adult developmental stage rather than chronological age. The coincidence that these processes result in particular forms of impairment in old age does not seem to be random as it is present in all mammals, and may be causative of many aspects of aging.”

A derived hypothesis: After sufficient strength and duration, broccoli sprout compounds changed my signaling environment, with appreciable effects beginning in Week 9.

I offered weak supporting evidence in Upgrade your brain’s switchboard with broccoli sprouts where a study’s insufficient one week duration of an insufficient daily 17.3 mg sulforaphane dosage still managed to change a blood antioxidant that may have changed four thalamus-brain-area metabolites. For duration and weight comparisons, I doubled my daily amount of broccoli seeds from one to two tablespoons just before Week 6 (Day 35), and from that point onward consumed a worst-case estimated 30 mg sulforaphane with microwaving 3-day-old broccoli sprouts every day.

Maybe a promised “In a submitted study, we will report that peripheral GSH levels may be correlated with cognitive functions” will provide stronger evidence? I’m not holding my breath for relevant studies because:

  • There wouldn’t be potential payoffs for companies to study any broccoli sprout compound connections with research areas such as aging, migraines, etc. Daily clinically-relevant broccoli sprout dosages can be grown for < $500 a year.
  • Sponsors would have to change paradigms, a very-low-probability event. They’d have to explain why enormous resources dedicated to current frameworks haven’t produced effective long-term treatments.

What long-term benefits could be expected if I continue eating broccoli sprouts every day?

The longest relevant clinical trial I’ve seen – referenced in Part 2 of Reversal of aging and immunosenescent trends with sulforaphane – was twelve weeks. Part 2 also provided epigenetic clock examples of changes measured after 9 months, which accelerated from there to the 12-month end-of-trial point.

Reviewing clinical trials of broccoli sprouts and their compounds pointed out:

“Biomarkers of effect need more time than biomarkers of exposure to be influenced by dietary treatment.”


A contrary argument: Perhaps people don’t require long durations to effectively change their signaling environments?

I apparently didn’t start eating an effective-for-me daily broccoli sprouts dosage until Day 35, when I changed from one to two tablespoons of broccoli seeds a day. If so, Weeks 6 through 8 may account for my substantial responses during Week 9.

Could eating broccoli sprouts every day for four weeks dramatically change a person’s signaling environment?

Do you have four weeks and $38 to find out? Two tablespoons of broccoli seeds = 38 g x 30 days = 1.14 kg or 2.5 lbs.

This is what twice-a-day one-tablespoon starting amounts of broccoli seeds look like through three days:


Maintaining the sprouting process hasn’t been a big effort compared with the benefits.

In the absence of determinative evidence, I’ll continue eating broccoli sprouts every day. Several areas of my annual physical have room for improvements. Extending my four lifestyle “interventions” a few more months may also provide hints toward inadequately researched connections.

* Results may not be extrapolatable to other people, to any specific condition, etc.

Too cheap for clinical trials

Let’s compare and contrast a 2019 meta-analysis and a 2017 review of using acetyl-L-carnitine to treat diabetic neuropathy.

A 2019 Brazilian meta-analysis Acetyl‐L‐carnitine for the treatment of diabetic peripheral neuropathy of four previous trials stated:

  • “The risk of bias was high in both trials of different ALC doses and low in the other two trials.
  • No included trial measured the proportion of participants with at least moderate (30%) or substantial (50%) pain relief.
  • At doses greater than 1500 mg/day, ALC reduced pain more than placebo. This subgroup analysis should be viewed with caution as the evidence was even less certain than the overall analysis, which was already of very low certainty.
  • The placebo-controlled studies did not measure functional impairment and disability scores.
  • No study used validated symptom scales.
  • Two studies were funded by the manufacturer of ALC and the other two studies had at least one co-author who was a consultant for an ALC manufacturer.

Authors’ conclusions:

  • We are very uncertain whether ALC causes a reduction in pain after 6 to 12 months treatment in people with DPN, when compared with placebo, as the evidence is sparse and of low certainty.
  • Data on functional and sensory impairment and symptoms are lacking, or of very low certainty.
  • The evidence on adverse events is too uncertain to make any judgements on safety.”

A 2017 Italian review Effects of acetyl-L-carnitine in diabetic neuropathy and other geriatric disorders stated:

“A long history of diabetes mellitus and increasing age are associated with the onset of diabetic neuropathy, a painful and highly disabling complication with a prevalence peaking at 50% among elderly diabetic patients. The management of diabetic neuropathy is extremely difficult: in addition to the standard analgesics used for pain control, common treatments include opioids, anticonvulsants, antidepressants, and local anesthetics, alone or in combination. Such therapies still show a variable, often limited efficacy, however.

Many patients do not spontaneously report their symptoms to physicians, but, if asked, they often describe having experienced a persistent and non-abating pain for many years. The prevalence of painful symptoms is just as high in patients with mild neuropathy as in those with more advanced DPN.

Through the donation of acetyl groups, ALC exerts a positive action on mitochondrial energy metabolism. ALC has cytoprotective, antioxidant, and antiapoptotic effects in the nervous system.

ALC has also been proposed for the treatment of other neurological and psychiatric diseases, such as mood disorders and depression, dementia, Alzheimer’s disease, and Parkinson’s disease, given that synaptic energy states and mitochondrial dysfunctions are core factors in their pathogenesis. Compared to other treatments, ALC is safe and extremely well tolerated.”

“In nerve injury, the mGlu2 receptor overexpressed by ALC binds the glutamate, reducing its concentration in the synapses with an analgesic effect. ALC may improve nerve regeneration and damage repair after primary nerve trauma.”


Where will the money come from to realize what the 2017 review promised, as well as provide what the 2019 meta-analysis required?

Do we prefer the current “limited efficacy” treatments of “opioids, anticonvulsants, antidepressants, and local anesthetics?”

Who will initiate clinical trials of a multiple of the normal dietary supplement dose (500 mg at $.25 a day, retail)? How profitable is a product whose hypothetical effective dosage for diabetic neuropathy (3000 mg) sells for only $1.50 a day?

A drug that countered effects of a traumatizing mother

This 2019 US rodent study concerned transmitting poor maternal care to the next generation:

“The quality of parental care received during development profoundly influences an individual’s phenotype, including that of maternal behavior. Infant experiences with a caregiver have lifelong behavioral consequences.

Maternal behavior is a complex behavior requiring the recruitment of multiple brain regions including the nucleus accumbens, bed nucleus of the stria terminalis, ventral tegmental area, prefrontal cortex, amygdala, and medial preoptic area. Dysregulation within this circuitry can lead to altered or impaired maternal responsiveness.

We administered zebularine, a drug known to alter DNA methylation, to dams exposed during infancy to the scarcity-adversity model of low nesting resources, and then characterized the quality of their care towards their offspring.

  1. We replicate that dams with a history of maltreatment mistreat their own offspring.
  2. We show that maltreated-dams treated with zebularine exhibit lower levels of adverse care toward their offspring.
  3. We show that administration of zebularine in control dams (history of nurturing care) enhances levels of adverse care.
  4. We show altered methylation and gene expression in maltreated dams normalized by zebularine.

These findings lend support to the hypothesis that epigenetic alterations resulting from maltreatment causally relate to behavioral outcomes.”

“Maternal behavior is an intergenerational behavior. It is important to establish the neurobiological underpinnings of aberrant maternal behavior and explore treatments that can improve maternal behavior to prevent the perpetuation of poor maternal care across generations.”


The study authors demonstrated intergenerational epigenetic effects, and missed an opportunity to also investigate transgenerational epigenetically inherited effects. They cited reference 60 for the first part of the above quotation, but the cited reviewer misused the transgenerational term by applying it to grand-offspring instead of the great-grand-offspring.

There were resources available to replicate the study authors’ previous findings, which didn’t show anything new. Why not use such resources to uncover evidence even more applicable to humans by extending experiments to great-grand-offspring that would have no potential germline exposure to the initial damaging cause?

Could a study design similar to A limited study of parental transmission of anxiety/stress-reactive traits have been integrated? That study’s thorough removal of parental behavior would be an outstanding methodology to confirm by falsifiability whether parental behavior is both an intergenerational and a transgenerational epigenetic inheritance mechanism.

Rodent great-grand-offspring can be studied in < 9 months. It takes > 50 years for human studies to reach the great-grand-offspring transgenerational generation.

  • Why not attempt to “prevent the perpetuation of poor maternal care across generations?”
  • Isn’t it a plausible hypothesis that humans “with a history of maltreatment mistreat their own offspring?”
  • Isn’t it worth the extra effort to extend animal research to investigate this unfortunate chain?

https://www.nature.com/articles/s41598-019-46539-4 “Pharmacological manipulation of DNA methylation normalizes maternal behavior, DNA methylation, and gene expression in dams with a history of maltreatment”

Going off the rails with the biomarker paradigm

This 2018 US government rodent study used extreme dosages to achieve its directed goals of demonizing nicotine and extolling the biomarker paradigm:

“This study examined whether adolescent nicotine exposure alters adult hippocampus-dependent learning, involving persistent changes in hippocampal DNA methylation and if choline, a dietary methyl donor, would reverse and mitigate these alterations.

Mice were chronically treated with nicotine (12.6mg/kg/day) starting at post-natal day 23 (pre-adolescent), p38 (late adolescent), or p54 (adult) for 12 days followed by a 30-day period during which they consumed either standard chow or chow supplemented with choline (9g/kg).

Our gene expression analyses support this model and point to two particular genes involved in chromatin remodeling, Smarca2 and Bahcc1. Both Smarca2 and Bahcc1 showed a similar inverse correlation pattern between promoter methylation and gene expression.

Our findings support a role for epigenetic modification of hippocampal chromatin remodeling genes in long-term learning deficits induced by adolescent nicotine and their amelioration by dietary choline supplementation.”


Let’s use the average weight of a US adult male – published by the US Centers for Disease Control as 88.8 kg – to compare the study’s dosages with human equivalents:

  1. Nicotine at “12.6mg/kg/day” x 88.8 kg = 1119 mg. The estimated lower limit of a lethal dose of nicotine has been reported as between 500 and 1000 mg!
  2. Choline at “9g/kg” x 88.8 kg = 799 g. The US National Institutes of Health published the Tolerable Upper Intake Levels for Choline as 3.5 g!!

Neither of these dosages are even remotely connected to human realities:

  1. The human-equivalent dosage of nicotine used in this study would probably kill an adult human before the end of 12 days.
  2. What effects would an adult human suffer from exceeding the choline “Tolerable Upper Intake Level” BY 228 TIMES for 30 days?

Isn’t the main purpose of animal studies to help humans? What’s the justification for performing animal studies simply to promote an agenda?


A funding source of this study was National Institute on Drug Abuse (NIDA) Identification of Biomarkers for Nicotine Addiction award (T-DA-1002 MG). Has the biomarker paradigm been institutionalized to the point where research proposals that don’t have biomarkers as goals aren’t funded?

https://www.sciencedirect.com/science/article/pii/S107474271830193X “Choline ameliorates adult learning deficits and reverses epigenetic modification of chromatin remodeling factors related to adolescent nicotine exposure” (not freely available)

How do memories transfer?

This 2018 Chinese study electronically modeled the brain’s circuits to evaluate memory transfer mechanisms:

“During non-rapid-eye-movement (NREM) sleep, thalamo-cortical spindles and hippocampal sharp wave-ripples have been implicated in declarative memory consolidation. Evidence suggests that long-term memory consolidation is coordinated by the generation of:

  • Hierarchically nested hippocampal ripples (100-250 Hz),
  • Thalamo-cortical spindles (7-15 Hz), and
  • Cortical slow oscillations (<1 Hz)

enabling memory transfer from the hippocampus to the cortex.

Consolidation has also been demonstrated in other brain tasks, such as:

  • In the acquisition of motor skills, where there is a shift from activity in prefrontal cortex to premotor, posterior parietal, and cerebellar structures; and
  • In the transfer of conscious to unconscious tasks, where activity in initial unskilled tasks and activity in skilled performance are located in different regions, the so-called ‘scaffolding-storage’ framework.

By separating a neural circuit into a feedforward chain of gating populations and a second chain coupled to the gating chain (graded chain), graded information (i.e. information encoded in firing rate amplitudes) may be faithfully propagated and processed as it flows through the circuit. The neural populations in the gating chain generate pulses, which push populations in the graded chain above threshold, thus allowing information to flow in the graded chain.

In this paper, we will describe how a set of previously learned synapses may in turn be copied to another module with a pulse-gated transmission paradigm that operates internally to the circuit and is independent of the learning process.”


The study had neither been peer-reviewed, nor were the mechanisms tested in living beings.

https://www.biorxiv.org/content/early/2018/07/27/351114 “A Mechanism for Synaptic Copy between Neural Circuits”

A mid-year selection of epigenetic topics

Here are the most popular of the 65 posts I’ve made so far in 2018, starting from the earliest:

The pain societies instill into children

DNA methylation and childhood adversity

Epigenetic mechanisms of muscle memory

Sex-specific impacts of childhood trauma

Sleep and adult brain neurogenesis

This dietary supplement is better for depression symptoms than placebo

The epigenetic clock theory of aging

A flying human tethered to a monkey

Immune memory in the brain

The lack of oxygen’s epigenetic effects on a fetus

The lack of oxygen’s epigenetic effects on a fetus

This 2018 Loma Linda review subject was gestational hypoxia:

“Of all the stresses to which the fetus and newborn infant are subjected, perhaps the most important and clinically relevant is that of hypoxia. This review explores the impact of gestational hypoxia on maternal health and fetal development, and epigenetic mechanisms of developmental plasticity with emphasis on the uteroplacental circulation, heart development, cerebral circulation, pulmonary development, and the hypothalamic-pituitary-adrenal axis and adipose tissue.

An understanding of the specific hypoxia-induced environmental and epigenetic adaptations linked to specific organ systems will enhance the development of target-specific inhibition of DNA methylation, histone modifications, and noncoding RNAs that underlie hypoxia-induced phenotypic programming of disease vulnerability later in life.

A potential stumbling block to these efforts, however, relates to timing of the intervention. The greatest potential effect would be accomplished at the critical period in development for which the genomic plasticity is at its peak, thus ameliorating the influence of hypoxia or other stressors.

With future developments, it may even become possible to intervene before conception, before the genetic determinants of the risk of developing programmed disease are established.”

Table 3 “Antenatal hypoxia and developmental plasticity” column titles were Species | Offspring Phenotypes of Disorders and Diseases | Reference Nos.

Hypoxia phenotypes


This review was really an ebook, with 94 pages and 1,172 citations in the pdf file. As I did with Faith-tainted epigenetics, I read it with caution toward recognizing 1) the influence of the sponsor’s biases, 2) any directed narrative that ignored evidence contradicting the narrative, and 3) any storytelling.

Can you match the meaning of the review’s last sentence (“intervene before conception” quoted above) with the meaning of any sentence in its cited reference Developmental origins of noncommunicable disease: population and public health implications? I can’t.

One review topic that was misconstrued was transgenerational epigenetic inheritance of hypoxic effects. The “transgenerational” term was used inappropriately by several of the citations, and no cited study provided evidence for gestational hypoxic effects through the F3 great-grandchild generation.

One omitted topic was gestational hypoxic effects of caffeine. The first paper that came up for my PubMed search of “caffeine pregnancy hypoxia” was an outstanding 2017 Florida rodent review Long-term consequences of disrupting adenosine signaling during embryonic development that had this paragraph and figure:

“One substance that fetuses are frequently exposed to is caffeine, which is a non-selective adenosine receptor antagonist. We discovered that in utero alteration in adenosine action leads to adverse effects on embryonic and adult murine hearts. We find that cardiac A1ARs [a type of adenosine receptor] protect the embryo from in utero hypoxic stress, a condition that causes an increase in adenosine levels. 

After birth in mice, we observed that in utero caffeine exposure leads to abnormal cardiac function and morphology in adults, including an impaired response to β-adrenergic stimulation. Recently, we observed that in utero caffeine exposure induces transgenerational effects on cardiac morphology, function, and gene expression.”

The timing of in utero caffeine treatment leads to differences in adult cardiac function, gene expression, and phenotype. Exposure to caffeine from E6.5–9.5 leads the F1 generation to develop dilated cardiomyopathy with decrease % FS and increased Myh7 expression. In utero caffeine exposure from E10.5–13.5 leads to a hypertrophic cardiomyopathy in the F2 generation along with increased % FS and decreased Myh7 expression

Why was this review and its studies omitted? It was on target for both gestational hypoxia and transgenerational epigenetic inheritance of hypoxic effects!

It was alright to review smoking, cocaine, methamphetamine, etc., but the most prevalent drug addiction – caffeine – couldn’t be a review topic?


The Loma Linda review covered a lot, but I had a quick trigger due to the sponsor’s bias. I started to lose “faith” in the reviewers after reading the citation for the review’s last sentence that didn’t support the statement.

My “faith” disappeared after not understanding why a few topics were misconstrued and omitted. Why do researchers and sponsors ignore, misrepresent, and not continue experiments through the F3 generation to produce evidence for and against transgenerational epigenetic inheritance? Where was the will to follow evidence trails regardless of socially acceptable beverage norms?

The review acquired the taint of storytelling with the reviewers’ assertion:

“..timing of the intervention. The greatest potential effect would be accomplished at the critical period in development for which the genomic plasticity is at its peak, thus ameliorating the influence of hypoxia or other stressors.”

Contradictory evidence was in the omitted caffeine study’s graphic above which described two gestational critical periods where an “intervention” had opposite effects, all of which were harmful to the current fetus’ development and/or to following generations. Widening the PubMed link’s search parameters to “caffeine hypoxia” and “caffeine pregnancy” returned links to human early life studies that used caffeine in interventions, ignoring possible adverse effects on future generations.

This is my final curation of any paper sponsored by this institution.

https://www.physiology.org/doi/abs/10.1152/physrev.00043.2017 “Gestational Hypoxia and Developmental Plasticity” (not freely available) Thanks to coauthor Dr. Xiang-Qun Hu for providing a copy.

“Transgenerationally” inherited epigenetic effects of fetal alcohol exposure

The fourth paper of Transgenerational epigenetic inheritance week was a 2016 German rodent study of of improperly-termed “transgenerational” epigenetic effects of alcohol:

“We investigated 2 generations of offspring born to alcohol-treated mothers. Here, we show that memory impairment and reduced synthesis of acetylcholine occurs in both F1 (exposed to ethanol in utero) and F2 generation (never been exposed to ethanol). Effects in the F2 generation are most likely consequences of transgenerationally transmitted epigenetic modifications in stem cells induced by alcohol.

The results further suggest an epigenetic trait for an anticholinergic endophenotype associated with cognitive dysfunction which might be relevant to our understanding of mental impairment in neurodegenerative disorders such as Alzheimer’s disease and related disorders.”

F0 generation mothers modeled human fetal alcohol syndrome. They were exposed to ethanol gradually up to 20%, then mated. The 20% ethanol intake level was maintained until the F1 generation pups were born, then gradually diminished to 0%. After a ten-day wait, an eight-week handling and shaping period started, followed by five weeks of behavioral testing.

The F1 children and F2 grandchildren started an eight-week handling and shaping period after young adulthood, followed by five weeks of behavioral testing. The F1 children were mated after behavioral testing.

The F0 parents showed no significant differences in working memory and reference memory compared with controls. Both the F1 children and F2 grandchildren were significantly impaired in the same tests compared with controls, with the F1 children performing worse than the F2 grandchildren. No sex-dependent differences were noted.

After behavioral impairments due to intergenerational epigenetic modifications were established, the F2 grandchildren received treatments to ascertain the contribution of cholinergic dysfunction in their behavioral impairments. It was confirmed, as an acetylcholine esterase inhibitor that crosses the blood-brain barrier almost completely erased working-memory and reference-memory performance deficits.

Items in the Discussion section included:

  • A dozen studies from 2014-2016 were cited for epigenetic mechanisms of inheritance stemming from parental alcohol consumption; and
  • Transgenerational inheritance of alcohol-induced neurodevelopmental deficits may involve epigenetic mechanisms that are resistant to developmental clearance.

As argued in Transgenerational effects of early environmental insults on aging and disease and A review of epigenetic transgenerational inheritance of reproductive disease, testing of F3 great-grandchildren born of F2 grandchild females was needed in order to establish transgenerational vs. intergenerational results. A F3 generation was necessary to control for the variable of F2 direct germline exposure.

http://www.neurobiologyofaging.org/article/S0197-4580(16)30303-7/pdf “Transgenerational transmission of an anticholinergic endophenotype with memory dysfunction” (not freely available)

Epigenetic effects of diet, and reversing DNA methylation

This 2015 French review focused on:

“The role of maternal health and nutrition in the initiation and progression of metabolic and other disorders.

The effects of various in utero exposures and maternal nutritional status may have different effects on the epigenome. However, critical windows of exposure that seem to exist during development need to be better defined.

The epigenome can be considered as an interface between the genome and the environment that is central to the generation of phenotypes and their stability throughout the life course.”

The reviewer used the term “transgenerational” to refer to effects that were more appropriately termed parental or intergenerational. Per the definition in A review of epigenetic transgenerational inheritance of reproductive disease, for the term to apply there needed to be evidence in at least the next 2 male and/or 3 female generations of:

“Altered epigenetic information between generations in the absence of continued environmental exposure.”

The review had separate sections for animal and human studies.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4663595/ “Impact of Maternal Diet on the Epigenome during In Utero Life and the Developmental Programming of Diseases in Childhood and Adulthood”


I arrived at the above review as a result of it citing the 2014 Harvard Reversing DNA Methylation: Mechanisms, Genomics, and Biological Functions. I’ll quote a few items from that review’s informative “Role of DNA demethylation in neural development” section:

“Distinct parts of mammalian brains, including frontal cortex, hippocampus, and cerebellum, all exhibit age-dependent acquisition of 5hmC [an oxidized derivative of 5mC [methylation of the fifth position of cytosine]].

In fact, the genome of mature neurons in adult central nervous system contains the highest level of 5hmC of any mammalian cell-type (~40% as abundant as 5mC in Purkinje neurons in cerebellum). These observations indicate that 5mC oxidation and potentially DNA demethylation may be functionally important for neuronal differentiation and maturation processes.

A comprehensive base-resolution analyses of 5mC and 5hmC in mammalian frontal cortex in both fetal and adult stages indicate that non-CpG methylation (mCH) and CpG hydroxymethylation (hCG) drastically build up in cortical neurons after birth, coinciding with the peak of synaptogenesis and synaptic pruning in the cortex. This study demonstrated that mCH could become a dominant form of cytosine modifications in adult brains, accounting for 53% in adult human cortical neuronal genome.

In mature neurons, intragenic mCH is preferentially enriched at inactive non-neuronal lineage-specific genes, indicating a role in negative regulation of the associated transcripts. By contrast, genic hCG is positively correlated with gene expression levels.”

Brain-region-specific energy metabolism affected the social competitiveness of highly-anxious rats

This 2015 Swiss rodent study found:

Mitochondrial function in the nucleus accumbens, a brain region relevant for motivation and depression, is a critical mediating factor in the subordinate status displayed by high-anxious rats.

Treatment with nicotinamide, an amide form of vitamin B3 that boosts mitochondrial respiration, into the NAc [nucleus accumbens] of high-anxious rats at a time point before the social encounter and at a dose that increased accumbal mitochondrial respiration, abolished the disadvantage of high-anxious animals to become dominant against low-anxious animals.

Our findings highlight a key role for brain energy metabolism in social behavior and point to mitochondrial function in the nucleus accumbens as a potential marker and avenue of treatment for anxiety-related social disorders.”

The researchers handled individual differences of the outbred subjects by separating them into high-, intermediate-, and low-anxiety categories according to their responses on two tests. The high- and low-anxiety subjects were matched by weight, age, and social experience.

Here are a few examples of the researchers thoroughly ruling out confounding factors:

“Differences in social competitiveness are not related to overall differences in social motivation or sociability.


Although social competition did significantly increase corticosterone compared with baseline levels, there were no significant differences between anxiety groups at either time point.


Microinfusion of either ROT, MA, or 3NP [mitochondrial respiration inhibitors] reduced the success of treated animals to win the social contest.

Importantly, these treatments did not induce side effects on social investigation or auto-grooming during social competition, or alter locomotor activity, anxiety, or sociability in additional experiments.

Furthermore, these inhibitor treatments did not produce neurotoxic effects, as the drugs were infused at low doses and we confirmed the absence of lesion and neuronal death.

The effects of complex I or complex II inhibition on social competition were specific for the NAc, as infusions of the same inhibitors into the BLA [basolateral amygdala] had no effect on social dominance and did not affect general locomotor activity.

We further showed that, unlike infusion of muscimol [a GABA receptor agonist] in the BLA that interferes with BLA-dependent auditory fear conditioning, 3NP did not affect conditioning in this task, discarding that neuronal inactivation could be a general mechanism whereby impairing mitochondrial function would affect putative functions from the affected brain region.


The impact of mitochondrial function in social competition described here is not mediated by oxidative stress.”

http://www.pnas.org/content/112/50/15486.full “Mitochondrial function in the brain links anxiety with social subordination”