Blackawton bees.

BACKGROUND: Real science has the potential to not only amaze, but also transform the way one thinks of the world and oneself. This is because the process of science is little different from the deeply resonant, natural processes of play. Play enables humans (and other mammals) to discover (and create) relationships and patterns. When one adds rules to play, a game is created. THIS IS SCIENCE: the process of playing with rules that enables one to reveal previously unseen patterns of relationships that extend our collective understanding of nature and human nature. When thought of in this way, science education becomes a more enlightened and intuitive process of asking questions and devising games to address those questions. But, because the outcome of all game-playing is unpredictable, supporting this 'messyness', which is the engine of science, is critical to good science education (and indeed creative education generally). Indeed, we have learned that doing 'real' science in public spaces can stimulate tremendous interest in children and adults in understanding the processes by which we make sense of the world. The present study (on the vision of bumble-bees) goes even further, since it was not only performed outside my laboratory (in a Norman church in the southwest of England), but the 'games' were themselves devised in collaboration with 25 8- to 10-year-old children. They asked the questions, hypothesized the answers, designed the games (in other words, the experiments) to test these hypotheses and analysed the data. They also drew the figures (in coloured pencil) and wrote the paper. Their headteacher (Dave Strudwick) and I devised the educational programme (we call 'i,scientist'), and I trained the bees and transcribed the childrens' words into text (which was done with smaller groups of children at the school's local village pub). So what follows is a novel study (scientifically and conceptually) in 'kids speak' without references to past literature, which is a challenge. Although the historical context of any study is of course important, including references in this instance would be disingenuous for two reasons. First, given the way scientific data are naturally reported, the relevant information is simply inaccessible to the literate ability of 8- to 10-year-old children, and second, the true motivation for any scientific study (at least one of integrity) is one's own curiousity, which for the children was not inspired by the scientific literature, but their own observations of the world. This lack of historical, scientific context does not diminish the resulting data, scientific methodology or merit of the discovery for the scientific and 'non-scientific' audience. On the contrary, it reveals science in its truest (most naive) form, and in this way makes explicit the commonality between science, art and indeed all creative activities. PRINCIPAL FINDING: 'We discovered that bumble-bees can use a combination of colour and spatial relationships in deciding which colour of flower to forage from. We also discovered that science is cool and fun because you get to do stuff that no one has ever done before. (Children from Blackawton)'.

An empirical explanation of the Chubb illusion.

The perceived difference in brightness between elements of a patterned target is diminished when the target is embedded in a similar surround of higher luminance contrast (the Chubb illusion). Here we show that this puzzling effect can be explained by the degree to which imperfect transmittance is likely to have affected the light that reaches the eye. These observations indicate that this 'illusion' is yet another signature of the fundamentally empirical strategy of visual perception, in this case generated by the typical influence of transmittance on inherently ambiguous stimuli.

Target-derived neurotrophic factors regulate the death of developing forebrain neurons after a change in their trophic requirements.

Many neurons die as the normal brain develops. How this is regulated and whether the mechanism involves neurotrophic molecules from target cells are unknown. We found that cultured neurons from a key forebrain structure, the dorsal thalamus, develop a need for survival factors including brain-derived neurotrophic factor (BDNF) from their major target, the cerebral cortex, at the age at which they innervate it. Experiments in vivo have shown that rates of dorsal thalamic cell death are reduced by increasing cortical levels of BDNF and are increased in mutant mice lacking functional BDNF receptors or thalamocortical projections; these experiments have also shown that an increase in the rates of dorsal thalamic cell death can be achieved by blocking BDNF in the cortex. We suggest that the onset of a requirement for cortex-derived neurotrophic factors initiates a competitive mechanism regulating programmed cell death among dorsal thalamic neurons.

Why we see things the way we do: evidence for a wholly empirical strategy of vision.

Many otherwise puzzling aspects of the way we see brightness, colour, orientation and motion can be understood in wholly empirical terms. The evidence reviewed here leads to the conclusion that visual percepts are based on patterns of reflex neural activity shaped entirely by the past success (or failure) of visually guided behaviour in response to the same or a similar retinal stimulus. As a result, the images we see accord with what the sources of the stimuli have typically turned out to be, rather than with the physical properties of the relevant objects. If vision does indeed depend upon this operational strategy to generate optimally useful perceptions of inevitably ambiguous stimuli, then the underlying neurobiological processes will eventually need to be understood within this conceptual framework.

An empirical explanation of color contrast.

For reasons not well understood, the color of a surface can appear quite different when placed in different chromatic surrounds. Here we explore the possibility that these color contrast effects are generated according to what the same or similar stimuli have turned out to signify in the past about the physical relationships between reflectance, illumination, and the spectral returns they produce. This hypothesis was evaluated by (i) comparing the physical relationships of reflectances, illuminants, and spectral returns with the perceptual phenomenology of color contrast and (ii) testing whether perceptions of color contrast are predictably changed by altering the probabilities of the possible sources of the stimulus. The results we describe are consistent with a wholly empirical explanation of color contrast effects.

The relevance of visual perception to cortical evolution and development.

The quality of brightness--perhaps the simplest visual attribute we perceive--appears to be determined probabilistically. In this empirical conception of the perception of light, the stimulus-induced activity of visual cortical neurons does not encode the retinal image or the properties of the stimulus per se, but associations (percepts) determined by the relative probabilities of the possible sources of the stimulus. If this theory is correct, the rationale for the prolonged postnatal construction of visual circuitry--and the evolution of this visual scheme--is to strengthen and/or create by activity-dependent feedback the empirically determined association on which vision depends.

The effects of color on brightness.

Observation of human subjects shows that the spectral returns of equiluminant colored surrounds govern the apparent brightness of achromatic test targets. The influence of color on brightness provides further evidence that perceptions of luminance are generated according to the empirical frequency of the possible sources of visual stimuli, and suggests a novel way of understanding color contrast and constancy.

Basic fibroblast growth factor promotes subplate cell survival in explant cultures of embryonic mouse cortex.

Subplate neurons form a transient layer immediately below the embryonic cortex and die early in postnatal life. It has been suggested that trophic factors, perhaps coming from cortical afferents, maintain the initial survival of these cells. Later withdrawal of these factors may cause subplate cell death. We tested whether basic fibroblast growth factor (bFGF) has survival-promoting effects on subplate cells in organotypic cultures from the late embryonic mouse cortex. We found that the survival of subplate cells was promoted by adding bFGF to the cultures. By contrast, there was no effect of bFGF on the survival of overlying cortical neurons. These results indicate that bFGF may have a role in the regulation of subplate cell survival and death in vivo.

An empirical explanation of the cornsweet effect.

A long-standing puzzle in vision is the assignment of illusory brightness values to visual territories based on the characteristics of their edges (the Craik-O'Brien-Cornsweet effect). Here we show that the perception of the equiluminant territories flanking the Cornsweet edge varies according to whether these regions are more likely to be similarly illuminated surfaces having the same material properties or unequally illuminated surfaces with different properties. Thus, if the likelihood is increased that these territories are surfaces with similar reflectance properties under the same illuminant, the Craik-O'Brien-Cornsweet effect is diminished; conversely, if the likelihood is increased that the adjoining territories are differently reflective surfaces receiving different amounts of illumination, the effect is enhanced. These findings indicate that the Craik-O'Brien-Cornsweet effect is determined by the relative probabilities of the possible sources of the luminance profiles in the stimulus.

Effects of the thalamus on the development of cerebral cortical efferents in vitro.

The cerebral cortex is a multilayered tissue, with each layer differing in its cellular composition and connections. Axons from deep layer neurons project subcortically, many to the thalamus, whereas superficial layer neurons target other cortical areas. The mechanisms that regulate the development of this pattern of connections are not fully understood. Our experiments examined the potential of the thalamus to attract and/or select neurites from appropriate cortical layers. First, we cocultured murine cortical slices in close proximity to thalamic explants in collagen gels. The amount of neurite outgrowth from deep layer cells was enhanced by, but not attracted to, the thalamic explants. Second, we cocultured cortical slices in contact with thalamic or cortical explants to test for laminar specificity of connections. Specificity was apparent after culture for about a week, in that deep cortical layers contained the highest proportions of corticothalamic cells and superficial cortical layers contained the highest proportions of corticocortical cells. After shorter culture of only a few days, however, specificity was not apparent and there were larger numbers of corticothalamic projections from the superficial layers than after a week. To study how the early nonspecific pattern of corticothalamic connections was transformed into the more specific pattern, we labeled corticothalamic cells early, after 2 days, but let the cultures survive for 8 days. On day 8, the nonspecific pattern of early-labeled cells was still seen. We conclude that although the thalamus does not block the initial entry of inappropriate axons from the superficial layers, many of these axons are subsequently lost. This suggests that contact-mediated interactions between cortical axons and the thalamus allow cortical efferents from appropriate layers to be distinguished from those arising in inappropriate layers. This may contribute to the development of layer-specific cortical connections in vivo.

An empirical basis for Mach bands.

Mach bands, the illusory brightness maxima and minima perceived at the initiation and termination of luminance gradients, respectively, are generally considered a direct perceptual manifestation of lateral inhibitory interactions among retinal or other lower order visual neurons. Here we examine an alternative explanation, namely that Mach bands arise as a consequence of real-world luminance gradients. In this first of two companion papers, we analyze the natural sources of luminance gradients, demonstrating that real-world gradients arising from curved surfaces are ordinarily adorned by photometric highlights and lowlights in the position of the illusory bands. The prevalence of such gradients provides an empirical basis for the generation of this perceptual phenomenon.

Mach bands as empirically derived associations.

If Mach bands arise as an empirical consequence of real-world luminance profiles, several predictions follow. First, the appearance of Mach bands should accord with the appearance of naturally occurring highlights and lowlights. Second, altering the slope of an ambiguous luminance gradient so that it corresponds more closely to gradients that are typically adorned with luminance maxima and minima in the position of Mach bands should enhance the illusion. Third, altering a luminance gradient so that it corresponds more closely to gradients that normally lack luminance maxima and minima in the position of Mach bands should diminish the salience of the illusion. Fourth, the perception of Mach bands elicited by the same luminance gradient should be changed by contextual cues that indicate whether the gradient is more or less likely to signify a curved or a flat surface. Because each of these predictions is met, we conclude that Mach bands arise because the association elicited by the stimulus (the percept) incorporates these features as a result of past experience.

A role for neurotrophins in the survival of murine embryonic thalamic neurons.

The mechanisms that determine whether developing CNS neurons live or die are poorly understood. We studied the role of the neurotrophins and fibroblast growth factors in the survival of embryonic thalamic neurons in culture. Dissociated embryonic dorsal thalamic neurons cultured at high density in defined serum-free medium survived and grew neurites. As in vivo, they expressed all the neurotrophins, fibroblast growth factor-1 and their high-affinity tyrosine kinase receptors. The survival of these cells was reduced by the addition of the protein kinase inhibitor K252a at concentrations that block neurotrophin receptor activity but not the activity of other tyrosine kinase receptors. In low-density cultures, most dorsal thalamic neurons died, but their survival was increased by co-culture with thalamic explants or with most of the neurotrophins and fibroblast growth factor-1 added singly. These results indicate that thalamic neurons have remarkably promiscuous trophic responses to a battery of neurotrophins and fibroblast growth factors. They suggest that neurotrophins endogenous to the early embryonic thalamus may be required to promote the survival of its neurons.

Influences of the thalamus on the survival of subplate and cortical plate cells in cultured embryonic mouse brain.

The afferent and efferent connections of the cerebral neocortex develop simultaneously toward the end of embryogenesis. At this stage, the neocortex comprises two main cell-dense layers: the thicker and more superficial cortical plate (future layers 2-6) and the thinner underlying subplate. Many early thalamocortical projections temporarily innervate the subplate before leaving it to locate their ultimate targets in the overlying cortical plate. The subplate then disappears. In this study, we performed in vitro experiments on late embryonic murine brain to test whether the thalamus can influence the survival of cortical plate and subplate cells at this stage. In isolated organotypic cortical explants from embryonic day 19 mice, most of the cells that had formed the subplate died. Coculture with a thalamic explant prevented this loss; coculture with additional cortical or cerebellar explants did not. By contrast, many cells in or destined for the cortical plate survived even in isolated cortical explants; coculture with a thalamic explant did not alter the numbers of these cells that survived. Our results suggest that the thalamus provides trophic support for subplate cells but not for late embryonic cortical plate cells. In vivo, a loss of thalamic-derived trophic support for the subplate late in embryogenesis, consequent on the movement of thalamocortical axons into the cortical plate, may contribute to subplate death.

Effects of subcortical structures on the growth of cortical neurites in vitro.

Cortical efferents grow from deep cortical layers to innervate numerous subcortical structures late in embryogenesis. The mechanisms that control their development are poorly understood. We co-cultured organotypic embryonic cortical explants with other tissues, maintaining a distance between them to avoid contact-mediated interactions. At embryonic day 15, when the cortical plate comprises only cells of the deep cortical layers, outgrowth from cultured cortex was stimulated by co-cultured subcortical structures, but not by additional cortex or liver. These data support the hypothesis that diffusible factors from subcortical structures, and not from the cortex itself, enhance cortical efferent growth.

The stimulation of thalamic neurite outgrowth by cortex-derived growth factors in vitro: the influence of cortical age and activity.

Recent in vitro experiments have provided useful insights into the development of connections between the thalamus and the cortex. While most of these previous studies focused on neurite guidance and target recognition, our experiments used a serum-free culture system to examine the possible roles of unidentified diffusible cortex-derived growth factors. We demonstrated that occipital cortical explants release diffusible growth factors that enhance neurite outgrowth from explants of the posterior thalamus (the region around the developing lateral geniculate nucleus). The amount of thalamic outgrowth was dependent on the age of the cocultured cortical slices. Our results suggest that there is an overall increase in the release of cortex-derived growth factors during the first three postnatal weeks in mice; this parallels known postnatal increases in the production of several identified growth factors. We found evidence for two peaks in the release of cortex-derived growth factors during the general upward trend, the first at around postnatal day 6 (shortly after thalamocortical innervation of layer 4) and a second between postnatal days 14 and 18 (just after eye-opening). The increased release of cortex-derived growth factors was not found when cortical slices were from mice that had been dark-reared from birth, suggesting that neural activity may be important for enhancing release. Other regions of the central nervous system, including the cerebellum and medulla, were also capable of stimulating some thalamic outgrowth; neither additional explants of the thalamus nor hepatic explants enhanced outgrowth. Fibroblast growth factor is one substance that is distributed preferentially among those tissues that were stimulatory in our experiments. Its level of transcription is known to increase in the brain during the first three postnatal weeks and to be influenced by neural activity. At low doses, fibroblast growth factor greatly increased outgrowth from isolated posterior thalamic explants. Nerve growth factor, another candidate molecule, was less effective. Overall, our results complement the in vivo observations of others on the synthesis of identified growth factors in the cortex and the factors that influence their production. They suggest that growth factors may influence thalamic neurons, and indicate that fibroblast growth factor, and possibly nerve growth factor, are two candidates for molecules mediating the in vitro effects.

The roles of growth factors and neural activity in the development of the neocortex.

Previous research on primarily the peripheral nervous system has shown that soluble growth factors help control key developmental events by contributing to dynamic autocrine and paracrine signalling systems. Much less is known about the roles of these substances in neocortical development. Using cell and tissue culture paradigms, we have demonstrated that soluble growth factors are produced by the neocortex and its subcortical targets, and that these tissues can respond to them. There are several possible functions for these factors in neocortical development in vivo: they may initiate axonal growth from neocortical neurons and/or their afferents; accelerate or guide that growth; and/or play a role in the later refinement of connections. Although none of these possibilities can be excluded, the existing evidence strengthens the hypothesis that soluble growth factors are important for the early postnatal growth and refinement of neocortical connections, when their levels of release may be regulated by neocortical activity. At present we do not know which growth factors are involved in these processes, but the results of preliminary experiments indicate that neurotrophins and fibroblast growth factor are prime candidates.

Post-natal development of tonic activity and membrane excitability in mouse medial vestibular nucleus neurones.

The development of tonic activity and membrane excitability of MVN neurones was examined using extracellular and intracellular recordings in slices prepared from mice at various stages in the first post-natal month. The tonic spontaneous discharge rates of MVN cells as post-natal day 5 (P5) were typically below 5 imp/s, and gradually increased to reach adult values of 11-20 imp/s by P30. While most MVN cells at P5 were electrophysiologically immature, by P10-P15 they had developed overshooting sodium spikes and pacemaker conductances which generated a steady discharge of spontaneous action potentials. From the earliest stages when tonic activity was observed, immature forms of the adult Type A and Type B action potential shapes could be recognized in tonically active cells. There was a marked rostro-caudal gradient in the time course of the maturation of MVN neurones, with cells in the rostral part of the MVN firing at higher rates and having more mature action potential shapes than caudally located cells.

Evidence that molecules influencing axonal growth and termination in the developing geniculocortical pathway are conserved between divergent mammalian species.

The general architecture of the visual system is similar for all species of mammal. To determine if the development of connections in the visual system might be under the influence of conserved molecules, we co-cultured explants of the murine lateral geniculate nucleus with slices from either murine or feline occipital cortex. Neurite outgrowth from embryonic murine geniculate explants was significantly enhanced by slices of newborn mouse occipital cortex or kitten visual cortex or by medium previously conditioned by these slices. Slices of similar volume but from sites other than occipital cortex had less or no effect on the murine geniculate explants. Fibers from murine geniculate explants grew freely on cortical slices from the kitten. They terminated mainly in layer 4 and also in layer 6, in both murine and feline visual and frontal cortical slices, irrespective of whether they entered through the white matter or pial side. Only the deep layers of the kitten's cortex sent projections to co-cultured murine geniculate explants. We suggest that the diffusible factors released by the cortex that stimulate the growth of axons from the lateral geniculate nucleus and the molecules that mark specific cortical laminae as targets for ingrowing afferents, are conserved in divergent species. We also found that murine geniculate axons grew freely on feline cerebellar slices. It is known from previous co-culture experiments that rodent geniculate axons are inhibited on rodent cerebellum and we suggest that the inhibitory factors involved are not conserved.

Growth-promoting interactions between the murine neocortex and thalamus in organotypic co-cultures.

The aim of this study was to assess whether developing cerebral cortex produces diffusible factors that can affect the growth of thalamic cells and, if so, what the role of these factors might be during the formation of thalamocortical connections. We studied interactions between cultured organotypic explants from mice maintained in defined serum-free medium. First, we cultured explants of embryonic dorsolateral thalamus in isolation from any other tissue; after culture, these explants were viewed intact and then sectioned. We estimated the numbers of healthy and pyknotic cells before and after culture, and the rates of mitosis in the explants during culture (using bromodeoxyuridine). Based on these data, we concluded that the majority of cells in the thalamic explants survived, although significant numbers of pyknotic cells did accumulate. Thalamic explants extended either very few or no neurites when cultured alone. We then cultured explants of embryonic thalamus near to explants from other tissues. A gap was always maintained between the explants, and we measured the length and density of neurite outgrowth from each thalamic explant. Slices of embryonic cortex promoted a small but significant increase in the amount of growth from thalamic explants. Postnatal cortex stimulated much more profuse neurite outgrowth; postnatal cerebellum had less of an effect, and postnatal medulla or liver had none. We showed that there was significantly more outgrowth from thalamic explants cultured in medium that had been preconditioned with cortical slices than from thalamic explants cultured in control medium, confirming that diffusible factors were produced by the cortex. The survival and mitotic rates of thalamic cells were unaffected by co-culture with the cortex. We conclude that the developing cortex releases diffusible factors that stimulate the growth of thalamic neurites and that other regions of the brain may also release the same substance(s). The lack of a specific source of thalamic growth promoting factor(s) argues against a role for these factors in guiding thalamic axons to specific targets; indeed, we were unable to demonstrate any chemotropic guidance of thalamic axons towards cortical explants in collagen gels. Since postnatal cortex has a more potent stimulatory effect than prenatal cortex, it seems possible that, in vivo, the cortical-derived factors act mainly on thalamocortical axons that have located their targets and are in the process of arborizing and refining their connections.