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

Polycomb group proteins in Drosophila

In the earlier post titled ' Role of polycomb and trithorax in developmental regulation' we have seen that the main function of the polycomb group proteins (PcGs) is to repress target gene expression, their major targets being the homeobox (Hox) genes. These proteins bind to Polycomb Response Elements (PREs), their target sequences on the DNA. The recruitment of the PcGs to these sites occurs due to covalent histone modifications in the DNA. Here we look into this aspect in some detail.

Till date three distinct PcG complexes have been discovered in Drosophila Polycomb repressor complex 1 and 2 (PRC1 and 2) and Pho repressive complex (Pho RC). All three contain multiple subunits encoded by PcG genes that are crucial for Hox gene silencing. PRC2 is known to have histone methyl transferase activity. It has H3K27 methylase activity. The PRC1 subunit inhibits nucleosome remodelling and transcription and brings about chromatin compaction. The PRC1 and 2 have various catalytic and non catalytic subunits which play a role in silencing target sites. The Pho repressive complex contains Pho and dSfmbt. Pho subunit has a distinct sequence specific DNA binding activity which is essential for targeting. dSfmbt binds to H3 or H4 tail peptides which are mono or di methylated at H3K9 or H4K20 which is crucial for repression.

The above figure depicts a speculative model for long range interactions between the PRE tethered PcG complexes and the methylated nucleosomes in the flanking chromatin. PRC1, PRC2 and PhoRC are locally bound to the PREs whereas trimethylation at H3K27, H3K9 and H4K20 is observed over an extended domain emcompassing the promoter and the coding regions.

Left : In case of PRC1 the Pc chromadomain (red triangle) would dock onto the nucleosomes that are tri methylated at H3K27 and through such bridging interactions will bring them in proximity to the other PRE bound PcG proteins. This may permit the other PRC1 subunits to block remodelling of the target nucleosomes or facilitate methylation of the neighbouring hypomethylated (pink) nucleosomes by PRC2.

Right : In case of PhoRC, the MBT (Malignant Brain Repeats) of dSfmbt (pink triangle) would interact with mono or dimethylated nucleosomes at H3K9 or H4K20.This bridging interaction would permit PRC2 to efficiently tri methylate H3K27 in hypomethylated nucleosomes.

Its is also possible that as yet unindentified HMTases which tri methylate these histones are also localized at the PREs.Similarly proteins with specificity for binding to other mrthyl-lysine modifications in histones might also be localized to the PREs.Various proteins have been purified which are associated to PcG complexes that are strictly essential for silencing. Pcl (Polycomb like) has been shown to be associated with PRC2 and mutations in this protein affect PRC2 functioning. Another protein zeste is a component of PRC1 and it has DNA binding activity.

As described above biochemical purification of PRC1, PRC2 and PhoRC suggests that these three complexes are separate entities. However since they co localize at the PREs various studies are aimed at finding out the physical interactions between subunits from the different complexes which may explain how PRC1 and PRC2 are localized to the PREs. Different models have been put forward based on various studies. In particular Wang et al (2004), reported that E(z) and Esc (subunits of PRC2) directly interact with Pho, leading to the proposal that Pho directly tethers PRC2 to the PREs. the PRE tethered PRC2 then locally trimethylates H3K27 thereby creating binding sites for the chromodomain of the PRC1 subinut Pc. More recent studies however challenge this model. First quantitative ChIp studies by two different labs suggest that PREs at the Ubx gene are infact devoid of nucleosomes.(V Pirrotta). Moreover PREs constitue of hypersensitive sites providing additional evidence that PRE DNA is not packaged into nucleosomes. Second studies by Mohd Sarip et al (2002), suggested that Pho can directly interact with PRC1 subunits, and that in in vitro studies Pho and PRC1 can co assemble on a naked PRE DNA template in absence of nucleosomes. This assembly assumes a conformation that is difficult to reconcile with the conformation of a nucleosome core particle. Thus available evidence suggests that not only PhoRC, but also PRC1 and PRC2 localize onto the PRE through interactions with Pho and other DNA binding proteins and not through covalent histone modifications or interactions with nucleosomes.

This conclusion then raises a major question with regards to the role of histone modification (trimethylation) in PcG silencing. Quantitative ChIp analysis that compared the Ubx gene in its off and on states in Drosophila larvae suggest that trimethylation in the promoter and coding region is essential for PcG silencing. In the 'off' state extensive trimethylation is present throughout the upstream control, promoter and coding regions but in the 'on' state this methylation is restricted only to the upstream control region and not seen in the promoter and coding regions.This led to the proposal that trimethylation at H3K9, H3K27 and H4K20 in the promoter and coding regions is required to demarcate the chromatin interval that is targeted for repression by the PRE-tethered PcG protein complexes. In this context, an important aspect about the relationship between the PREs and histone modifications of the target genes comes from studies with PRE reporter genes in Drososphila, in which the PRE DNA was flanked with FRT ( Flip Recombinase Target) sites and could thus be deleted from the reporter gene. This study indicated that excision of the PRE DNA from the silenced reporter gene, caused loss of PcG silencing, even when the excision was done late in development. Thus although the PcG proteins appear to repress transcription by methylation in promoter and coding regions, silencing depends on the continuous presence of the PREs and the PcGs tethered to them.

Although much progress has been made towards understanding the mechanism of PcG silencing, much remains to be learned. It is still not understood how the PcG proteins are targeted to the PREs beacuse Pho binding sites alone do not make a PRE. ChIp studies may also help elucidate additional DNA binding PcG proteins.Also there may be many additional PcG complexes and PRC1, PRC2 and PhoRC may represent only the most stable or most abundant PcG complexes. Also it seems likely that these findings of PcG silencing in Drosophila will prove important in inderstanding the mechanism of PcG silencing in mammals, given the recent discovery that mammalian PcG proteins seem to be acting as global repressors of developmental control genes in embryonic stem cells.

Reference:

MULLER, J., & KASSIS, J. (2006). Polycomb response elements and targeting of Polycomb group proteins in Drosophila Current Opinion in Genetics & Development, 16 (5), 476-484 DOI: 10.1016/j.gde.2006.08.005

Evidence for Systemic Spread prior to the Establishment of Primary Tumors

It is widely believed that metastasis is a late event in cancer progression. This view is based on several clinical and experimental observations. First, most cancer patients die from metastasis and not the primary disease. Second, early surgery is often the only cure. Third, somatic genetic changes accumulate during local progression (Fearon and Vogelstein, 1990) which was extrapolated to systemic progression. Fourth, repeated rounds of in vivo selection led to cell lines with increased metastasis formation (Kang et. al 2003; Minn et. al 2005). However, in a recent study titled Systemic Spread is an Early Step in Breast Cancer, published in Cell in 2008, Hüsemann, Geigl, Schubert et. al report that tumor cells can disseminate systemically from the earliest epithelial alterations in HER-2 and PyMT transgenic mice and from ductal carcinoma in situ in women. 

The mouse model they work with is the BALB/c mice transgenic for the activated rat HER-2/neu gene (BALB-Neu T mice). This model mimics progression and gene expression profiles of human breast cancer. Females hemizygous for the rat HER-2 gene under control of the MMTV (mouse mammary tumor virus) promoter develop  invasive mammary cancer while their HER-2 negative siblings (wild-type BALB/c mice) remain tumor free. Typically, in the BALB-Neu T model, the mammary epithelia starts to express the oncogene at about weeks 3 to 4 (which coincides with the onset of puberty). Hyperplasia can be detected microscopically at weeks 7-9. Progress to in situ  carcinomas occurs between weeks 14-18 and at weeks 23-30, invasive cancers become apparent (see Fig. 1a)

Fig. 1a; Left Panel: Whole mount of the mammary gland at week 9 showing absence of tumor in branching ductal tree; Middle Panel: Histological Section at week 9 showing side buds displaying the morphology of atypical ductal hyperplasia; Right Panel: Histology of invasive cancer at week 30 (Adapted from Hüsemann, Geigl, Schubert et. al (2008))

The principle goal was to determine when cells expressing the HER-2 transgene disseminate. The authors chose the lung and bone marrow as the sites to look out for metastasis since the HER-2 receptor is not expressed in either of these two organs. In addition to HER, they also used anti-cytokeratin (CK) antibodies to check whether disseminated cells were epithelial in origin. Surprisingly in BALB-Neu T mice, CK⁺ and HER-2⁺ cells became detectable 4-9 weeks when meticulous analysis could only detect atypical ductal hyperplasia (ADH) (see Fig. 1b) 

Fig. 1b; Left Panel: Increase in tumor area at primary site. Triangles indicate mean value, whiskers indicate 95% confidence interval and solid line indicates best fitted curve; Right Panel: Number of CK⁺ cells (red dots) and HER⁺ cells (blue dots) per 5000 bone marrow cells. Triangles, whiskers and solid line indicate same. Note the presence of disseminated cells in the bone marrow even when the primary tumor area is zero.(Adapted from Hüsemann, Geigl, Schubert et. al (2008)) 

Further, lung micrometastasis from mammary tissue was confirmed by demonstration of mammary-specific alpha casein and lactalbumin transcripts (see Fig. 1c)

Fig. 1c; Left Panel: Lung micrometastasis at week 27, detected using anti-HER-2 antibody;Right Panel: Eight HER-2 ⁺ and two samples of normal lung tissues, analyzed for mammary gland specific transcripts. "+" indicates normal mammary gland and "-" indicates mock control (Adapted from Hüsemann, Geigl, Schubert et. al (2008)) 

Despite exponential growth at the primary tumor site, the number of CK⁺ cells and HER-2⁺cells in the bone marrow rose marginally over the course of time. Cells singly positive for HER-2 and CK were not congruent. Not all tumor cells expressed both markers (see Fig. 1d)

Fig. 1d; Left Panel: CK⁺, HER-2^- cell; Middle Panel: CK^-, HER-2⁺ cell; Right Panel: CK⁺, HER-2⁺ cell (Adapted from Hüsemann, Geigl, Schubert et. al (2008))

This suggests, either the existence of heterogeneous tumor cell populations that disseminate to distant sites or different cellular states of the disseminated tumor cells (DTCs)

More Evidence for Early Dissemination

Could the HER-2⁺, CK⁺ cells have disseminated from extra-mammary tissues expressing the transgene? The authors took mammary gland fragments from 3-12 week old transgenic mice (displaying only atypical ductal hyperplasia) and transplanted the them into 3 week old wild-type siblings.  Wild type bone marrow was screened at different time points (see Fig. 2)

Fig. 2; Left Panel: Week 14 post-transplantation, CK⁺ cells in bone marrow of wild-type siblings; Right Panel: HER-2⁺ cells in the bone marrow (Adapted from Hüsemann, Geigl, Schubert et. al (2008))

Although CK⁺ and HER-2⁺ cells were observed in the bone marrow, the levels were lower than transgenic BALB-Neu T mice, but were definitely above rare false positives. Additionally, just as in the transgenic mice, there was no significant increase in disseminated cells from ADH stages to invasive cancer, in the wild-type siblings. 

The malignant nature of these DTCs was established by Comparative Genomics Hybridization (see Fig. 3)

Fig. 3; Top Panel: Karyogram (left) and CGH profile (right) of sorted mouse metaphase of a single HER-2⁺ cell disseminated from a transplanted mammary gland and subsequently isolated from bone marrow of a recipient wild-type mouse; Bottom Panel: Sorting of mouse metaphase of a single leukocyte in a balanced CGH profile; Green and red bars indicate genomic gains and losses respectively. (Adapted from Hüsemann, Geigl, Schubert et. al (2008))

Evidence that early disseminated cells can grow into metastases

The onset of metastasis relative to primary tumor growth was assessed . Histological sections of the lungs were analyzed and micrometastases could be detected from weeks 20 to 21 onward, a time point at which mostly in-situ carcinomas are present at the primary sites (see Fig. 4)

Fig. 4; Progression of lung metastasis in BALB-Neu T mice, with and without the removal of the primary tumor. Left Panel: Increase in tumor area over time in BALB-Neu T mice (same as Fig. 1b); Right Panel: Size of the largest lung metastasis detected in individual mice. Blue squares indicate average size of metastases from non-operated mice at various time points and red triangles indicate average size from operated animals at 10-15 weeks after surgery. Whiskers indicate 95% confidence intervals (Adapted from Hüsemann, Geigl, Schubert et. al (2008))

Since metastases need time to grow, their increase in size paralleling that of the primary lesion supports the conclusion that, at least in some cases, founder cells of metastasis had disseminated earlier and had started to proliferate.

Some other experiments conducted by the group, demonstrate that bone marrow disseminated cells that do not grow into metastases can be released from growth arrest. Also, there does not appear to be an association between the number of disseminated cells and the stage of the tumor. Large tumors do not necessarily seed more.

Overall the paper presents some compelling evidence for the occurrence of dissemination, even before the primary tumor is established. This would indeed force the field to rethink it's current strategies in combating cancer.

Reference:

Hüsemann, Y., Geigl, J., Schubert, F., Musiani, P., Meyer, M., Burghart, E., Forni, G., Eils, R., Fehm, T., & Riethmüller, G. (2008). Systemic Spread Is an Early Step in Breast Cancer Cancer Cell, 13 (1), 58-68 DOI: 10.1016/j.ccr.2007.12.003

 

Designed and Designoid Objects

Part 2 of the 5 part Royal Institution Christmas Lectures for Children, 1991. With sheer clarity and brilliance, Richard Dawkins puts forward the concept of objects that are designed and "look designed"

Look Mom, Three Hands -The Classical Rubber Hand Illusion

Most illusions are not only fun to experience but are also interesting to study in depth for what they can reveal about perceptual purposes. One of the most interesting illusions discovered in recent times in the rubber hand illusion. It was first reported in a paper in Nature in 1998, titled "Rubber hands 'feel' touch that eyes see" by Matthew Botvinick and Jonathan Cohen.  So how does it work? A subject is seated with the left harm resting on a table while a standing screen is positioned besides the arm to hide it from the subject's view. A life-sized rubber model of a left hand and arm is placed on the table, directly in front of the patient. The experimenter uses two paintbrushes to stroke the rubber hand and the real hidden hand, synchronizing the timing of strokes as closely as possible.

In the original study by Botvinick and Cohen, subjects were asked to complete a two part questionnaire that asked for an open description of their experience and also to affirm or deny the occurrence of nine specific perceptual effects (see Fig. 1).

Fig. 1: Questionnaire includes nine statements presented in random order. Subjects indicated their response on a seven-step visual analogue scale ranging from 'agree strongly (+++)' to 'disagree strongly (---)'. Points indicate mean response and bars indicate response range. Underlined questions show a significant tendency to evoke an affirmative response (Adapted from Botvinick and Cohen (1998))

Most subjects indicated that they seemed to feel the touch not of the hidden brush, but that of the viewed brush, as though the rubber hand had sensed the touch. It was hypothesized that the illusion may arise due to a spurious reconciliation of visual and tactile inputs while distorting position sense (proprioception). In a second experiment, subjects were exposed to the illusion for a prolonged period and were then probed for distortion in proprioceptive information. Before and after the viewing period, subjects completed a series of three intermanual reaches. With eyes closed, the right index finger was drawn along a straight edge, until it was judged to be aligned with the index finger of the hidden left hand. The authors found that the subjects' reaches after experiencing the illusion were displaced towards the rubber hand, the magnitude of displacement varying in proportion to the reported duration of the illusion (see Fig. 2)

Fig. 2: Results of reaching experiment. x-axis indicates the percentage of 30-min viewing period during which the illusion was experienced. The y-axis indicates displacement of the three reaches made after the viewing period from the three made before. Data is fitted with a least-squares regression line (adapted from Botvinick and Cohen (1998)) 

In more systematic explorations by Tsakiris and Haggard (2005), it was shown that the drift, while indicating the position of the real left hand, in the direction of the fake rubber hand not only depends upon synchrony of strokes, but also on the position of the rubber hand (spatial congruency) as well as visual characteristics of the hand (body top-down effects) (see Fig. 3 for experimental setup)

Fig. 3: Participants saw in different conditions (a) a rubber hand in a congruent position, (b) a rubber hand in  incongruent position, or (c) a wooden stick. The participant's left hand was out of view for the whole duration of the experiment (Adapted from Tsakiris and Haggard (2005))

The congruent posture elicits the maximum proprioceptive drift while having the rubber hand in an incongruent position or replacing the rubber hand by a wooden stick does not (see Fig. 4).

Fig. 4: Mean proprioceptive drift towards the rubber hand. Error bars indicate standard error. Asterix indicates significant difference between synchronous and asynchronous stimulation (Adapted from Tsakiris and Haggard (2005))

The body is distinguished from other objects as belonging to the self by participating in inter-modal perceptual correlations. In the experiments of Botvinick and Cohen, subjects who referred the tactile sensation to the rubber hand also reported experiencing the rubber hand as belonging to themselves. Indeed eight out of ten subjects employed terms of ownership in the free descriptions (Botvinick and Cohen (1998))

The rubber hand illusion presents a very intriguing case. It shows that certain forms of inter-modal correlations may be sufficient for self attribution even in the face of contradicting signals from other sensory modalities.

References

Botvinick M, & Cohen J (1998). Rubber hands 'feel' touch that eyes see. Nature, 391 (6669) PMID: 9486643

Tsakiris M, & Haggard P (2005). The rubber hand illusion revisited: visuotactile integration and self-attribution. Journal of experimental psychology. Human perception and performance, 31 (1), 80-91 PMID: 15709864