Evolution of scorpion venom

The predominant pharmacologically active components in scorpion venoms are small polypeptide molecules, usually basic in nature. Scorpion venoms and their component elements have been studied for over 35 years; but lately the focus in these studies has shifted from their pharmacological and electrophysiological properties to their molecular structures. This came about due to the realization that many classes of peptides seem to bind to characteristic spots on their targets, which for the most part are ion channels. The peptide toxins also show considerable identity in their arrangement of cysteine residues within the polypeptide chain. These cysteine residues are terribly important because disulphide bonds are one of the major contributors to conformational stability in small peptides.

The first pharmacological studies on scorpion toxins concentrated on their effects on mammalian model systems, so mice were the first ones to draw the short end of the hypodermic syringe, for a while. Peptides were consequently classified into the alpha- and beta-types, where each type bound to its own special site on voltage-gated sodium channels. Eventually, however, it was discovered that some peptides worked against ion channels in insects, and now Sarcophaga argyrostoma blowfly larvae were adopted as the victim model system of choice. All anti-insect toxins induced paralysis, but one class of toxins was found to induce a contraction paralysis, while the other induced a depressant paralysis.

For a while it seemed that all peptides could be neatly docketed in this way, but continuing studies unearthed peptide toxins that were primarily anti-mammal but showed some anti-insect activity too. Subsequently peptides were found that showed comparable anti-insect and anti-mammal activities, one of which – showing anti-insect as well as alpha-type and beta-type anti-mammal activities – was first reported in the paper (Loret et al., 1991) referenced below. This sparked off a line of thought on how the types of peptides found in scorpions in different parts of the world could be used to put forward theories about where scorpions first appeared and how they diverged into the large number of species we see today.

The reason this cross-reactivity of toxins against insect and mammal receptors was such a big deal was this: Before this was discovered, toxins were found to be easily classifiable not only on the basis of their primary peptide sequences and pharmacological activities, but also based on geography. Alpha-type anti-mammal toxins came only from Old World scorpions and beta-type anti-mammal toxins came only from New World scorpions. All anti-insect toxins had been purified from Old World scorpions only. Deviations from this geographical structure brought to light interesting ideas about venom evolution in scorpions.

For example, the toxin mentioned above which had a high effect on mammals but a low effect on insects was a beta-type toxin from Centruroides (a New World species) which was toxic to insects, but 50 times weaker than Old World anti-insect toxins (these studies happened in the 80s). This could indicate that anti-insect activity is just starting to evolve in New World venoms, but has already become established in Old World ones, indicating that the Old World venoms appeared first in their evolutionary history.

The paper referenced below is a report of a toxin from Androctonus australis Hector (pictured below), called AaH IT4. The toxin was purified by successive steps of chromatography, on gel filtration, DEAE-Sephadex and C8 HPLC columns. Toxicities were tested on S. argyrostoma larvae and male C57/BL6 mice, and the ED₅₀ values (for larvae) and LD₅₀ values (for mice) were recorded. Binding assays using ¹²⁵I-iodinated toxins on synaptosomal preparations from cockroaches and rats; radioimmunoassay assays to check for cross-reactions with rabbit antisera against known anti-insect, alpha-type and beta-type toxins; and circular dichroism analyses for structural data were carried out. In addition, sequence analysis and sequence alignment against various other scorpion venoms were carried out.

The experimental data showed that AaH IT4 competed with all three types of toxins for target-binding, and that it cross-reacted with the antibody against a beta-type toxin, indicating some structural similarity with the beta-toxin class. The dendrogram generated from the sequence alignment showed that AaH IT4 is more closely related to beta-type toxins than to either alpha-type or anti-insect toxins (which supports the result of the RIA experiment), although the divergence between AaH IT4 and the beta-type toxin lineage took place a long time ago. This an important point because beta-type toxins come from New World scorpions while AaH IT4 was purified from an Old World toxin, so any sequence similarity suggests that some relationship may exist between the two.

Another point to be made about AaH IT4 is that the sequence analysis shows an absence of the amino acid proline. Proline had previously been found in every purified and studied peptide scorpion toxin, and was suspected to play a role in the stability of their conformations, since proline is more conformationally restricted as compared to the other amino acids. One explanation for the binding of AaH IT4 to three different kinds of binding sites is that the absence of proline allows a certain amount of backbone flexibility which may allow the peptide to switch between conformational states that preferentially bind to each of the target sites.

The authors themselves hypothesize that since the evolution of insect-specific toxins is clearly advantageous to scorpions (for whom insects form a major part of the diet), it is possible that AaH IT4 represents the closest approximation available right now to some kind of ancestral toxin sequence/structure, which later diverged into anti-mammal and anti-insect varieties.

I think it’s worth mentioning, in addition, that the isolated AaH IT4 corresponds to 0.06% of the total protein in the venom, so it’s possible that the production of this particular venom component may have undergone some down-regulation over the years, as a result of the development of other, more specialised toxins.

Post-1991, of course, the issue of toxin classification has become even more complicated, and the venoms being studied now include those with anti-arthropod specificity, and the set of recognised targets have grown to include voltage- and ligand-gated potassium- as well as calcium-channels instead of just the initial emphasis on voltage-gated sodium-channels. Peptides with microbial activity have also been reported.

References:

Loret, E., Martin-Eauclaire, M., Mansuelle, P., Sampieri, F., Granier, C., & Rochat, H. (1991). An anti-insect toxin purified from the scorpion Androctonus australis Hector also acts on the .alpha.- and .beta.-sites of the mammalian sodium channel: sequence and circular dichroism study Biochemistry, 30 (3), 633-640 DOI: 10.1021/bi00217a007

Innate Immune Cells: Mediators of the Angiogenic Switch?

The role of Matrix metalloprotease type 9 (MMP-9) in the activation of Vascular Endothelial Growth Factor (VEGF), the induction and maintenance of chronic angiogenesis and early stage tumor growth has been well established. But what is the source of MMP-9 ? In a study published in 2006, Nozawa and co-workers, using the RIP1-Tag2 transgenic mouse as a model, identified two inflammatory cell types, neutrophils and macrophages, as the major sources of MMP-9.

The RIP1-Tag2 transgenic mouse is a well characterized model of multistep carcinogenesis involving the pancreatic islets. Nozawa and co-workers first isolated constituent cell types from a tumor and cell-sorted Gr-1⁺ and Mac-1⁺ cells and then carried out a semi-quantitative RT-PCR (see Fig.1)

Fig. 1: Semi-quantitative RT-PCR. MMP-9⁺ is expressed by Gr-1⁺ and Mac-1⁺cells innate immune cells (Adapted from Nozawa et. al, 2006)

Clearly, MMP-9 was expressed predominantly by innate immune cells positive for the Gr-1 and Mac-1 markers.

In order to determine the localization of these cells , a double label immunohistochemical staining of various leukocyte markers in normal and neoplastic tissue was performed. CD68 and F4/80 are two macrophage markers. Cells positive for both markers were observed inside as well as on the periphery of angiogenic islets and tumors (see Fig. 2). However, the cells inside were MMP-9^- while those along the periphery were MMP-9⁺ 

Fig. 2: MMP-9⁺ macrophages located along the periphery of the angiogenic islet.Macrophages inside the islet were MMP-9^- (Adapted from Nozawa et. al, 2006)

Along similar lines, an antibody against the "7/4" (a neutrophil marker) was used to localize neutrophils. Suprisingly, the situation, this time, was the opposite. 7/4⁺ cells inside the lesions were also MMP-9⁺ while 7/4⁺ cells along the periphery were not (see Fig. 3). All  7/4⁺ cells also displayed polymorphic nuclei, strongly indicating that these cells were neutrophils.

Fig. 3: MMP-9⁺ neutrophils located inside the angiogenic islet. Neutrophils along the periphery of the islet were MMP-9^- (Adapted from Nozawa et. al, 2006)

Neutrophils (7/4⁺ and Gr-1⁺) represented only 0.4% of the total tumor cells and expressed high levels of MMP-9. In contrast, macrophages (CD68⁺) represented 2% of the total tumor cells and expressed low levels of MMP-9.

The researchers then tried to determine the role of these MMP-9⁺ Gr-1⁺ cells that infiltrate angiogenic islets and tumors. An experimental regimen involving daily inoculation of anti-Gr-1 at 7 weeks (when hyperplastic islets start to undergo angiogenesis) was followed.

After 1 week of injection, most neutrophils disappeared from the pancreatic islets of the transgenic mice (see Fig.4)

Fig. 4: Left: Control; Right: Following 1 week of treatment with anti-Gr-1, MMP-9⁺ neutrophils located inside the angiogenic islet disappeared (Adapted from Nozawa et. al, 2006)

After two weeks of injection, the neutrophil population rebounded in neoplastic islets. Incredibly, none of the rebound neutrophils were MMP-9⁺ (see Fig. 5)!

Fig. 5: Left: Control; Right: Following 2 weeks of treatment with anti-Gr-1, neutrophils rebounded back into the islets. However, none of the rebound neutrophils were MMP-9⁺  (Adapted from Nozawa et. al, 2006)

In a short 2 week prevention trial (week7-week9), carried out to assess angiogenic switching frequency, the anti-Gr-1 antibody regimen, reduced the number of angiogenic islets by 57%!

Furthermore, immunostaining for VEGF:VEGF-R2 complex with GVM39 (red) and for endothelial cells with Meca-32 (green) showed a decrease in bioactive VEGF:VEGF-R2 interaction, consequent to the depletion of infiltrating MMP-9⁺ neutrophils (see Fig.6)

Fig. 6: Left: Control; Right: Short intervention trials lead to reduced VEGF:VEGF-R2 interactions. Endothelial cells are stained with an antibody directed against Meca-32 (a pan-endothelial cell marker)  (Adapted from Nozawa et. al, 2006)

Taken together, the results from this paper indicate that subtle infiltration by innate immune cells (such as neutrophils) may play a role in the progression of neoplasias towards angiogenic tumors.

Reference:

Nozawa H, Chiu C, & Hanahan D (2006). Infiltrating neutrophils mediate the initial angiogenic switch in a mouse model of multistage carcinogenesis. Proceedings of the National Academy of Sciences of the United States of America, 103 (33), 12493-8 PMID: 16891410

Alan Turing-May He Finally Rest in Peace

Gordon Brown's official statement on Alan Turing

2009 has been a year of deep reflection - a chance for Britain, as a nation, to commemorate the profound debts we owe to those who came before. A unique combination of anniversaries and events have stirred in us that sense of pride and gratitude which characterise the British experience. Earlier this year I stood with Presidents Sarkozy and Obama to honour the service and the sacrifice of the heroes who stormed the beaches of Normandy 65 years ago. And just last week, we marked the 70 years which have passed since the British government declared its willingness to take up arms against Fascism and declared the outbreak of World War Two. So I am both pleased and proud that, thanks to a coalition of computer scientists, historians and LGBT activists, we have this year a chance to mark and celebrate another contribution to Britain’s fight against the darkness of dictatorship; that of code-breaker Alan Turing.

Turing was a quite brilliant mathematician, most famous for his work on breaking the German Enigma codes. It is no exaggeration to say that, without his outstanding contribution, the history of World War Two could well have been very different. He truly was one of those individuals we can point to whose unique contribution helped to turn the tide of war. The debt of gratitude he is owed makes it all the more horrifying, therefore, that he was treated so inhumanely. In 1952, he was convicted of ‘gross indecency’ - in effect, tried for being gay. His sentence - and he was faced with the miserable choice of this or prison - was chemical castration by a series of injections of female hormones. He took his own life just two years later.

Thousands of people have come together to demand justice for Alan Turing and recognition of the appalling way he was treated. While Turing was dealt with under the law of the time and we can’t put the clock back, his treatment was of course utterly unfair and I am pleased to have the chance to say how deeply sorry I and we all are for what happened to him. Alan and the many thousands of other gay men who were convicted as he was convicted under homophobic laws were treated terribly. Over the years millions more lived in fear of conviction.

I am proud that those days are gone and that in the last 12 years this government has done so much to make life fairer and more equal for our LGBT community. This recognition of Alan’s status as one of Britain’s most famous victims of homophobia is another step towards equality and long overdue.

But even more than that, Alan deserves recognition for his contribution to humankind. For those of us born after 1945, into a Europe which is united, democratic and at peace, it is hard to imagine that our continent was once the theatre of mankind’s darkest hour. It is difficult to believe that in living memory, people could become so consumed by hate - by anti-Semitism, by homophobia, by xenophobia and other murderous prejudices - that the gas chambers and crematoria became a piece of the European landscape as surely as the galleries and universities and concert halls which had marked out the European civilisation for hundreds of years. It is thanks to men and women who were totally committed to fighting fascism, people like Alan Turing, that the horrors of the Holocaust and of total war are part of Europe’s history and not Europe’s present.

So on behalf of the British government, and all those who live freely thanks to Alan’s work I am very proud to say: we’re sorry, you deserved so much better.

Gordon Brown

Aging by Epigenetics

We know that organismal longevity and aging is caused by a lot of interacting factors such as nuclear and mitochondrial genome mutations, shortened telomeres, oxidative damage to DNA and other macromolecules, senescence, apoptosis and many more. This review discusses another possible determinant of aging that is 'epigenetics'. We have seen in the earlier post that epigenetics refers to changes in the DNA and histones which are heritable through cell divisions, but do not involve any change in the sequence of the DNA. 

Chromatin is broadly divided into two types, euchromatin and heterochromatin. Euchromatin is decondensed during interphase and is relatively transcriptionally active. Heterochromatin on the other hand remains compact and condensed and is transcriptionally inactive. It is further divided into constitutive and facultative. Constitutive heterochromatin is present in the telomer and centromere and appears to be fixed or irreversible throughout the life time of an organism. Facultative heterochromatin on the other hand, is made as a part of regulated cell differentiation process or other changes in cell phenotype. For example, a single X chromosome is silenced in female mammalian cells for dosage compensation.

However, despite this apparent clear distinction between euchromatin and heterochromatin, it is now being understood that chromatin structure is highly dynamic and stochastic. Essential processes, such as DNA replication, transcription and repair all involve disruption of the very compact structure of DNA. The proteins bound to the chromatin are also not static but exhibit relatively high 'on' and 'off' binding states even in the 'fixed' heterochromatin. It has also been showed that, formation of heterochromatin depends on a degree of transcription, which contributes to heterochromatinization through the RNAi pathway. Thus the telomeric heterochromatin is also shown to be transcribed. Thus heterochromatin is not a static entity.

Is aging associated with epigenetic changes?

The enzymes Histone acetyl teransferases (HATs) and Histone deacetylases (HDACs) respectively determine the steady-state level of histone acetylation. In S.cerevisiae, inactivation of a HDAC, Sir2, decreases replicative lifespan while activation extends it. The anti aging effects of Sir2 in yeast are due to translocation of a Sir2 containing protein complex from telomeres to ribosomal DNA (rDNA) repeats. These repeats are prone to recombination to form extrachromosomal rDNa circles (ERCs), which curtail yeast lifespan. However, Sir2 mediated histone deactylation and heterochromatization, prevents formation of ERCs and thus extends lifespan of yeast. Thus, this epigenetic redistribution counteracts organismal aging. Orthologs of Sir2 have been found in many species like nematodes, flies and even mice. Thus its anti aging role seems to have been conserved throughout evolution.

In mammals, it has been seen that there is a reduction in genomic DNA methylation, with age. It occurs mostly at repetitive DNA sequences, predominantly in regions of constitutive heterochromatin. Since methylation induces silencing of genes, this change will promote deheterochromatinization of these regions. However DNA methylation increases at specific sites called the CpG islands. These are CG rich sequences, some of them present in the promoter regions, which can get methylated. Methylation also increases in the histone 4 on the lysine 20 residue (H4K20) in rat liver and kidney, with age. Like DNA methylation H4K20 methylation is also linked to gene repression, supporting the notion that heterochromatin accumulates in some sites atleast with aging.

One of the histone chaperones, HIRA, shows increased levels of expression in aging baboon skin. This is shown to have a evolutionary conserved role in formation of heterochromatin.

These observations suggest that mammalian aging is associated with chromatin remodelling. In particular, there is a global decrease in DNA methylation, but an increase at specific sites on the genome.

Cellular senescence, is characterized by irreversible proliferation arrest. This may arise due to excessive cell divisions and shortening of the telomere length. Due to this, most human cells have a finite proliferative lifespan. Senescent cells or molecular markers of senescent phenotype increase during aging. Cellular senescence is also well established tumor suppression process, because it can prevent proliferation and neoplastic progression of cells harboring neoplastic lesions. Senescent cells also show chromatin remodeling. Many senescent cells form domains of facultative heterochromatin, called Senescence Associated Heterochromatin Foci (SAHF), which are visibly more condensed that interphase chromatin. These foci have been proposed to silence proliferation promoting genes. Accumulation of SAHF has been associated with aging. Formation of SAHF requires presence of HIRA, which has been shown to be upregulated in aging baboon skin. Also it seems that in cellular senescence too, the constitutive heterochromatin regions are deheterochromatinized. Thus like tissue aging, cellular senescence also is accompanied by redistribution of heterochromatin, from constitutive heterochromatin to other normally euchromatic sites.

Consequences of age-associated epigenetic changes

Studies going back to 1960s indicate that aging is associated with cell aneuploidy. Proper chromosome segregation is dependent on pericentromeric heterochromatin sturcture. Hence cecreased methylation and deheterochromatinization may lead to faulty chromosome segregation and associated aneuploidy. Aneuploidy confers various altered cell phenotypes including a proliferative impairment, which might contribute to decreased tissue renewal capacity with age. It may also give rise to cancer, for which age is the biggest risk factor. Some of the age associated changes in gene expression may also occur due to epigenetics. Methylation of CpG islands is a well described mode of silencing some tumor suppressor genes. A tissue wide age associated methylation of CpG may be an early causative event in the development of neoplasms.

From the discussion it is apparent that chromatin does undergo change with aging in organisms as diverse as yeast and mammals. However with the exception of Sir2 in yeast, the extent to which this impacts the aging process is not yet defined. Some age associated epigenetic alterations in mammals like formation of SAHF, might extend life span by suppressing age associated diseases like cancer. In sum the effects of chromatin on aging are likely to be complex. A pre requisite to properly understanding the contribution of epigenetics to aging is to better understand the specific cell, tissue and system wide malfunctions that are responsible for aging phenotypes like osteoporosis, sarcopenia, cancer and many others. Then it will be feasible to dissect out the contribution to each phenotype of each candidate epigenetic determinant, like global methylation, CpG island methylation and SAHF.

Reference

Sedivy JM, Banumathy G, & Adams PD (2008). Aging by epigenetics--a consequence of chromatin damage? Experimental cell research, 314 (9), 1909-17 PMID: 18423606

Fake Hands and Tables-The Malleability of the Body Image

In " The Classical Rubber Hand Illusion", we discussed the original experiments of Botvinick and Cohen (1998). Their hypothesis for the rubber hand illusion was that vision has higher reliability and spatial acquity than proprioception, so the brain gives more weight to visual information. People would thus localize a body part to it's apparent visual location, particularly when the visible location falls within the possible range dictated by proprioception. Some support for this theory lies in the fact that placing the fake hand perpendicular to the real occluded hand destroys the illusion that the fake hand is one's own.

Armel and Ramachandran  (2003) reported a closely related but bizarre illusion. The subject is made to place his or her real hand on a table and the hand is hidden from view. However, instead of stroking a fake rubber hand, the researchers simply stroked and tapped the table in precise synchrony for a minute. Astonishingly, subjects reported sensations arising from the table surface, despite the fact that it bears no physical resemblance to a hand. Whereas Botvinick and Cohen interpret their result in terms of resolving incongruities between visual versus proprioceptive location of the hand, Armel and Ramachandran's experiment would lead one to argue that the illusion arises mainly  from Bayesian logic of all perception; the brain's remarkable ability to detect statistical correlations in sensory inputs in constructing useful perceptual representations of the world, including one's own body. It is especially intriguing that the bizarre perceptual representation (assimilating the table into the body image) is resistant to the "top-down" knowledge of the absurdity of the situation!

To measure the extent to which subjects incorporated the external objects into their body image, they were asked to rate the vividness of the illusion. The experimenters also recorded the skin conductance response (SCR), to provide an objective test of whether the table had indeed become informationally coupled with the subject's body image. If the external objects became integrated into the body's image, would they be aroused when the table (or a fake hand for that matter) was 'injured'?

If a finger of the fake hand is bent backwards to seem painful, does the subject register an SCR? To what extent is the fake hand assimilated into the subject's body image? To address this, after ca. 2.5 minutes of the touching procedure, both the real and the fake fingers were lifted, but only the fake finger was bent backwards into a 'painful position'. SCR was recorded at this point and a free response description and intensity rating of the illusion were obtained. The control for this experiment was a 'delayed synchrony' condition wherein touch to the real and fake hands were identical, the only difference being, that the touch to the real hand was delivered 1 sec after the touch on the fake hand. Mean intensity and mean SCRs showed that subjects identified with the fake hand more in the condition where the touch was synchronized rather than the one where the touch was synchronized but delayed (see Fig. 1)

Fig. 1: Mean Intensity ratings (gray bars) and SCR (black circles) in the first experiment where synchronous touch to the real hand was delayed by a second in the control condition. The error bars indicate one SEM (Adapted from Armel and Ramachandran, 2003)

Would subjects still experience the illusion if the form of the external object was manipulated? To explore this, a barren table was stroked  and tapped in the same manner and in the same relative location (see Fig.2 ). 

Fig. 2: Form manipulation where subjects received the table condition (Adapted from Armel and Ramachandran, 2003)

Band-aids were placed on both the real hand and the table and subjects were told that the band-aid would be pulled off the table but not off their real hands. At the end of the 2.5 min touching period, in lieu of pulling back a fake finger, the band-aid on the table was partially pulled off. In the control condition for this experiment, the real hand was made visible by removing the occluder. Subjects were instructed to look back and forth between their real hands and the table. Furthermore, the real hand and the location where the table was touched were close together so that they could be seen simultaneously even while looking at one or the other. The band-aid was pulled off the table while the subject viewed it. To ensure that the subject was only looking at the table, the experimenter occluded the subject's real finger at this time. In a comparison of the conditions in which the table and real hand were touched with the partition in place or removed, intensity ratings and SCR were significantly different (see Fig. 3). However, the same experiment (using a band-aid) carried out with a fake rubber hand in lieu of the table is more effective at inducing the illusion in terms of intensity ratings but only marginally so for SCR (see Fig. 3)

Fig. 3: Mean Intensity ratings (gray bars) and SCR (black circles) in the second experiment where form of the external object was manipulated. The error bars indicate one SEM (Adapted from Armel and Ramachandran, 2003)

Would subjects still experience the illusion if the form of the external object was manipulated? Each subject viewed touch to a fake hand in a 'realistic' location in one condition and then to a distant fake hand in another (see Fig. 4).

Fig. 4: Location manipulation where subjects received the distant-hand condition (Adapted from Armel and Ramachandran, 2003)

The fake arm was extended so that it lay 3 feet beyond the real hand. In the distant fake hand manipulation, a fake finger was bent back for a painful stimulus. In the control condition, the touch applied to the fake and real hands was asynchronous i.e.  touch was random and there was no correlation (see Table 1 at the end for summary of conditions for all experiments). Mean intensity and mean SCR showed that subjects identified with the fake hand and the distant fake hand, more in the conditions where touch was synchronized than when not synchronized. However, the 'anatomically correct' fake hand condition was more effective than the distant fake hand condition (both with synchronous touch, see Fig. 5)  

Fig. 5: Mean Intensity ratings (gray bars) and SCR (black circles) in the second experiment where the location of the fake hand was manipulated. The error bars indicate one SEM (Adapted from Armel and Ramachandran, 2003)

The so-called body image appears to be highly malleable. Despite  it's appearance of durability, it can be profoundly altered by stimulus contingencies and correlations that one encounters. Taken together, these experiments illustrate an important principle underlying perception: that the mechanisms of perception may be involved in extracting statistical correlations. Further investigations will further our understanding of phenomena such as body-dysmorphic disorder and anorexia nervosa

Table 1: Experimental design for all experiments. For a given experiment, each subject received all conditions in one of different possible orders (Adapted from Armel and Ramachandran, 2003)

Reference:

Armel, K., & Ramachandran, V. (2003). Projecting sensations to external objects: evidence from skin conductance response Proceedings of the Royal Society B: Biological Sciences, 270 (1523), 1499-1506 DOI: 10.1098/rspb.2003.2364

Epigenetics - Implications for behavioral neuroendocrinology

Individuals vary in their sociosexual behavior and reactivity. How an organism interacts with the environment to produce these variations has been a focus in psychology since its inception as a scientific discipline.There is now no question that cumulative experiences throughout life history interact with genetic predispositions to shape the individual's behavior. Recent evidence suggests that events in the past generations may also influence how an individual responds to events in their own life history. Epigenetics is the study of how the environment can affect the genome of an individual and its descendants all without changing the sequence of the DNA.

The early stages of life, beginning before birth and up to weaning in mammals, are the time of maximum neuronal plasticity.It is during this early period that hormones and genotype predispose an individual's responses to future experiences throughout the life cycle. Suites of genes underlie the fundamental plasticity of an organism, particularly during development and life history transitions. How do these gene networks interact with the experiences that cumulate during an individual's life history?

An important interface between the environment (both internal and external) is that of epigenetic modifications. Exactly how these modifications occur is still relatively unknown, but recent studies indicate that origin of such effects may occur at in the previous generations. That is, experience of previous generations may modify regulatory factors affecting gene expression without changing the sequence of the DNA, but the physiology and behavior of the organism may be substantially influenced. Thus understanding how such events really occur will enhance our understanding of how the environment influences the relationship between genotype and behavior during sensitive periods of development.

Molecular vs Molar epigenetics

There have been several reviews recently as to the origin of the field of epigenetics, all of which recognize the multiple roots of the current tree of research. The debates in the 16th-17th century pitted preformationism against epigenesis, with a central question of how a multicellular organism develops from a single cell, the zygote. The former camp believed that adult characters were present fully formed in the egg and simply unfolded during growth, while the latter held that traits arose due to interactions between the multiple constituent parts of the zygote. Spawned after the resolution of this conflict were two different groups, one rooted in anatomy and geology, which later became the broad science of biology, and the other focused on sensation, perception and mind, which ultimately became the study of psychology. Thus though both have a common origin, they evolved very differently both in perspective and substance. These the author labels respectively as, 'Molecular epigenetics' and 'Molar epigenetics'.

Molar epigenetics arises from historical literature in psychilogy, particularly functionalism. Rather than measuring sensory processes, the functionalists focused on the organism itself and development of behavior in relation to its natural environment. Molecular epigenetics arose from molecular biology and modern genetics and its emphasis was on gene regulation and its developmental significance.Thus the object of study in Molecular epigenetics is gene expression during embryogenesis, while in Molar epigenetics, it is on the individual's interactions with the biotic and physical environment usually after birth.

Molecular epigenetics

Conrad H. Waddington proposed the term 'epigenetics' from classical embryology, ascribing it to the study of processes by which genotype gives rise to a phenotype. His concept was central to what became the modern era of epigenetics when Robin Holliday proposed a molecular model of heritable gene activation and inactivation during development by DNA methylation and demethylation respectively. Since then the term epigenetics is used to connote the study of change in gene expression without changing the DNA sequence. There are several mechanisms that can achieve this end, such as DNA methylation and modification of histones by processes of methylation, deacetylation and phosphorylation. While DNA methylation is clearly involved in genomic imprinting, the signal for the imprint is still not known.

Molar epigenetics

Early comparitive psychologists were also interested in epigenetics, but from the perspective of interaction of an organism's heredity and the nature of species typical behaviors or 'instincts'. Work principally by Konrad Lorenz and Niko Tinbergen emphasized that such behaviors were a product of natural selection, the result of genes acting in the brain to generate behaviors that were unlearned and innate. Thus, these studies laid the foundation of psychobiology, a field that focuses on how experiences cumulate throughout life to shape the way in which an individual interacts with its environment.

It is necessary to emphasize that, an approach that integrates both Molecular and Molar epigenetics, will be required to reveal the mechanisms that underlie behavioral evolution.

Context dependent vs Germline dependent epigenetic modification 

In context dependent epigenetic modification, the change is transmitted within a generation, within an individual's own lifetime, including the interaction of parent and young. An example of this is exposure to endocrine disrupting chemical in utero during childhood, in which case the disease manifests itself later. The extent to which the modification is perpetuated is by the simple presence of the environmental factor, that brings about the epigenetic modification. The modification is present until the effect of the factor is present. Later it declines and to reestablish the effect the individual has to be exposed to the same agent again. This effect is called context dependent epigenetic modification. In germline dependent modification, the genetic imprint is independent of the original causative agent. Here the epigenetic modification is transferred to the subsequent generations because, the change in the epigenome has been incorporated in the germline. Thus the effect is manifest in each generation without the need for re-exposure. The DNA methylation of heritable epialleles are passed through to subsequent generations without being erased as occurs normally during gametogenesis or shortly after fertilization.

If the context in which an individual is nurtured affects its behavior as an adult, it is likely that the activities of the neural circuitry underlying this behavior is also affected. This should apply to both context dependent and germ line dependent epigenetic modifications.

Transgenerational epigenetic imprint on the nuclear genome and its effect on behavior and brain

Two critical elements of demonstrating a germline dependent epigenetic modification are that, first a single exposure of the environmental factor that is never again repeated, and second the number of generations since that exposure. A new model system was used in which an endocrine disrupting chemical (EDC) reprograms methylation patterns that are then incorporated into the germline and hence transmitted to future generations. In this model system the exposure of gestating female rats to pesticide methoxychlor or fungicide vinclozolin during the period of embryonic sex determination, induces an epigenetic transgenerational phenotype through reprogramming the germline in a sex specific manner. Specifically, in each generation males whose ancestor have been treated underwent progressive spermatogonial apoptosis, decreased sperm count and motility and as the animals aged, adult onset disease is accelerated, including cancer and immune cell defects.

Studies were carried out to determine if this altered epigenome also influences male/partner preference behavior. For these studies F3 descendants of females were used which were treated with dimethylsulphoxide buffer alone (control) or with vinclozolin (EDC treated). Partner preference was carried out by placing an individual (male or female) in a testing arena. At either ends was a small cage containing the stimulus rats separated by a wire mesh barrier to allow exchange of visual, olfactory and tactile cues. All males were tested with both types of females as stimulus and vice versa. Behaviors directed to the stimulus animals included time spent in contact with the wire mesh during which the animals often touched noses through the mesh, grooming, aimless walking and sniffing, standing on hind paws and sniffing with nose pointed upwards, contacting the walls of the test cage and time spent in the center of the test arena.

The results were clear cut and sex specific. The females discriminate and prefer males who do not have a history of exposure, while males do not exhibit such a preference. In social engagements in rodents time is spent in mutual facial investigation. Males investigate females equally, while females spend more time investigating males from the control lineage. It is known that pheromones from the vomeronasal organ and urine are involved in mate recognition in rodents. Methylation analysis revealed that Major Urinary Protein 4 (MUP4) is one of the candidate imprinted like genes in the vinclozolin treated lineage. This MUP group of gene products binds to and releases male specific pheromones in rodents. Also males and females both explored odors of the opposite sex than familiar odors. These behaviors may reflect differences in pattern of gene expression in different brain areas.Using gene micro arrays specific ares of the brain like the hippocampus , amygdala and the whole brain have been studied in both control and test males. Of the altered genes only a limited number shows similar changes in all the three regions an some of these have been implicated in schizophrenia, autism and depression.

Cytoplasmic genes like the mitochondrial genes also have CpG islands and polymorphisms in these genes plays a significant role in adaptive evolution. These genes are important for determining sperm quality and motility and mutations result in decrease in human sperm motility. In this regard males of all five generations stemming from females treated during pregnancy with either methoxychlor or vinclozolin have decreased sperm motilty and numbers. The mitochondrial genome is also vitally involved in aging and mutations are involved in onset of age related phenotypes, including reduced fertility. Genes encoding for cytochrome oxidase were tested in transgenerationally imprinted rats. (CO is used as a measure of brain activity). None of the constituent genes of this mito-nuclear gene product, seem to be altered in the three regions of the brain. However, nuclear respiratory factor 2 which modulates CO activity, shows an increased expression in the whole brain, perhaps accounting for the behavioral differences observed between the mice of the two lineages. Thus the relative fitness of the specific mito-nuclear genotype combinations is dependent on the modified DNA environment in which they persist. Thus EDCs could act via epigenetically modifying mitochondrial DNA as well as nuclear DNA, and influence epistatic interactions between cytoplasmic and nuclear genes.

The linking of Molar with Molecular epigenetics, extensive knowledge of hormones which play a role in organizing and activating brain-behavior mechanisms and the predisposition in neuroscience research to use molecular methods to understand cellular function and development should facilitate the incorporation of epigenetics in neuroendocrinological research.

Reference:

CREWS, D. (2008). Epigenetics and its implications for behavioral neuroendocrinology Frontiers in Neuroendocrinology, 29 (3), 344-357 DOI: 10.1016/j.yfrne.2008.01.003