A First Principles Approach to Subjective Experience.

Understanding the neural bases of subjective experience remains one of the great challenges of the natural sciences. Higher-order theories of consciousness are typically defended by assessments of neural activity in higher cortical regions during perception, often with disregard to the nature of the neural computations that these regions execute. We have sought to refocus the problem toward identification of those neural computations that are necessary for subjective experience with the goal of defining the sorts of neural architectures that can perform these operations. This approach removes reliance on behaviour and brain homologies for appraising whether non-human animals have the potential to subjectively experience sensory stimuli. Using two basic principles-first, subjective experience is dependent on complex processing executing specific neural functions and second, the structure-determines-function principle-we have reasoned that subjective experience requires a neural architecture consisting of stacked forward models that predict the output of neural processing from inputs. Given that forward models are dependent on appropriately connected processing modules that generate prediction, error detection and feedback control, we define a minimal neural architecture that is necessary (but not sufficient) for subjective experience. We refer to this framework as the hierarchical forward models algorithm. Accordingly, we postulate that any animal lacking this neural architecture will be incapable of subjective experience.

Neural Design Principles for Subjective Experience: Implications for Insects.

How subjective experience is realized in nervous systems remains one of the great challenges in the natural sciences. An answer to this question should resolve debate about which animals are capable of subjective experience. We contend that subjective experience of sensory stimuli is dependent on the brain's awareness of its internal neural processing of these stimuli. This premise is supported by empirical evidence demonstrating that disruption to either processing streams or awareness states perturb subjective experience. Given that the brain must predict the nature of sensory stimuli, we reason that conscious awareness is itself dependent on predictions generated by hierarchically organized forward models of the organism's internal sensory processing. The operation of these forward models requires a specialized neural architecture and hence any nervous system lacking this architecture is unable to subjectively experience sensory stimuli. This approach removes difficulties associated with extrapolations from behavioral and brain homologies typically employed in addressing whether an animal can feel. Using nociception as a model sensation, we show here that the Drosophila brain lacks the required internal neural connectivity to implement the computations required of hierarchical forward models. Consequently, we conclude that Drosophila, and those insects with similar neuroanatomy, do not subjectively experience noxious stimuli and therefore cannot feel pain.

Designing Brains for Pain: Human to Mollusc.

There is compelling evidence that the "what it feels like" subjective experience of sensory stimuli arises in the cerebral cortex in both humans as well as mammalian experimental animal models. Humans are alone in their ability to verbally communicate their experience of the external environment. In other species, sensory awareness is extrapolated on the basis of behavioral indicators. For instance, cephalopods have been claimed to be sentient on the basis of their complex behavior and anecdotal reports of human-like intelligence. We have interrogated the findings of avoidance learning behavioral paradigms and classical brain lesion studies and conclude that there is no evidence for cephalopods feeling pain. This analysis highlighted the questionable nature of anthropometric assumptions about sensory experience with increased phylogenetic distance from humans. We contend that understanding whether invertebrates such as molluscs are sentient should first begin with defining the computational processes and neural circuitries underpinning subjective awareness. Using fundamental design principles, we advance the notion that subjective awareness is dependent on observer neural networks (networks that in some sense introspect the neural processing generating neural representations of sensory stimuli). This introspective process allows the observer network to create an internal model that predicts the neural processing taking place in the network being surveyed. Predictions arising from the internal model form the basis of a rudimentary form of awareness. We develop an algorithm built on parallel observer networks that generates multiple levels of sensory awareness. A network of cortical regions in the human brain has the appropriate functional properties and neural interconnectivity that is consistent with the predicted circuitry of the algorithm generating pain awareness. By contrast, the cephalopod brain lacks the necessary neural circuitry to implement such an algorithm. In conclusion, we find no compelling behavioral, functional, or neuroanatomical evidence to indicate that cephalopods feel pain.

Potential Roles of Dental Pulp Stem Cells in Neural Regeneration and Repair.

This review summarizes current advances in dental pulp stem cells (DPSCs) and their potential applications in the nervous diseases. Injured adult mammalian nervous system has a limited regenerative capacity due to an insufficient pool of precursor cells in both central and peripheral nervous systems. Nerve growth is also constrained by inhibitory factors (associated with central myelin) and barrier tissues (glial scarring). Stem cells, possessing the capacity of self-renewal and multicellular differentiation, promise new therapeutic strategies for overcoming these impediments to neural regeneration. Dental pulp stem cells (DPSCs) derive from a cranial neural crest lineage, retain a remarkable potential for neuronal differentiation, and additionally express multiple factors that are suitable for neuronal and axonal regeneration. DPSCs can also express immunomodulatory factors that stimulate formation of blood vessels and enhance regeneration and repair of injured nerve. These unique properties together with their ready accessibility make DPSCs an attractive cell source for tissue engineering in injured and diseased nervous systems. In this review, we interrogate the neuronal differentiation potential as well as the neuroprotective, neurotrophic, angiogenic, and immunomodulatory properties of DPSCs and its application in the injured nervous system. Taken together, DPSCs are an ideal stem cell resource for therapeutic approaches to neural repair and regeneration in nerve diseases.

Epidermal YAP2-5SA-ΔC Drives ß-Catenin Activation to Promote Keratinocyte Proliferation in Mouse Skin In Vivo.

The epidermis is a highly regenerative tissue. YAP is a pivotal regulator of stem/progenitor cells in tissue regeneration, including in the epidermis. The molecular mechanisms downstream of YAP that activate epidermal cell proliferation remain largely unknown. We found that YAP and ß-catenin co-localize in the nuclei of keratinocytes in the regenerating epidermis in vivo and in proliferating HaCaT keratinocytes in vitro. Inactivation of YAP in HaCaT keratinocytes resulted in reduced activated ß-catenin and reduced keratinocyte numbers in vitro. In addition, we found that in the hyperplastic epidermis of YAP2-5SA-ΔC mice, the mutant YAP2-5SA-ΔC protein was predominantly localized in the keratinocyte nuclei and caused increased expression of activated nuclear ß-catenin. Accordingly, ß-catenin transcriptional activity was elevated in the skin of live YAP2-5SA-ΔC/TOPFLASH mice. Lastly, loss of ß-catenin in basal keratinocytes of YAP2-5SA-ΔC/K14-creERT/CtnnB1-/- mice resulted in reduced proliferation of basal keratinocytes and a striking rescue of the hyperplastic abnormalities. Taken together, our work shows that YAP2-5SA-ΔC drives ß-catenin activity to promote basal keratinocyte proliferation in the mouse skin in vivo. Our data shine new light on the etiology of regenerative dermatological disorders and other human diseases that display increased YAP and ß-catenin activity.

Fish do not feel pain and its implications for understanding phenomenal consciousness.

Phenomenal consciousness or the subjective experience of feeling sensory stimuli is fundamental to human existence. Because of the ubiquity of their subjective experiences, humans seem to readily accept the anthropomorphic extension of these mental states to other animals. Humans will typically extrapolate feelings of pain to animals if they respond physiologically and behaviourally to noxious stimuli. The alternative view that fish instead respond to noxious stimuli reflexly and with a limited behavioural repertoire is defended within the context of our current understanding of the neuroanatomy and neurophysiology of mental states. Consequently, a set of fundamental properties of neural tissue necessary for feeling pain or experiencing affective states in vertebrates is proposed. While mammals and birds possess the prerequisite neural architecture for phenomenal consciousness, it is concluded that fish lack these essential characteristics and hence do not feel pain.

Activity-dependent expression of neuronal PAS domain-containing protein 4 (npas4a) in the developing zebrafish brain.

In rodents, the Npas4 gene has recently been identified as being an important regulator of synaptic plasticity and memory. Homologs of Npas4 have been found in invertebrate species though their functions appear to be too divergent for them to be studied as a proxy for the mammalian proteins. The aim of this study, therefore, was to ascertain the suitability of the zebrafish as a model organism for investigating the function of Npas4 genes. We show here that the expression and regulation of the zebrafish Npas4 homolog, npas4a, is remarkably similar to that of the rodent Npas4 genes. As in mammals, expression of the zebrafish npas4a gene is restricted to the brain where it is up-regulated in response to neuronal activity. Furthermore, we also show that knockdown of npas4a during embryonic development results in a number of forebrain-specific defects including increased apoptosis and misexpression of the forebrain marker genes dlx1a and shha. Our work demonstrates that the zebrafish is a suitable model organism for investigating the role of the npas4a gene and one that is likely to provide valuable insights into the function of the mammalian homologs. Furthermore, our findings highlight a potential role for npas4a in forebrain development.

The expression pattern of EVA1C, a novel Slit receptor, is consistent with an axon guidance role in the mouse nervous system.

The Slit/Robo axon guidance families play a vital role in the formation of neural circuitry within select regions of the developing mouse nervous system. Typically Slits signal through the Robo receptors, however they also have Robo-independent functions. The novel Slit receptor Eva-1, recently discovered in C. elegans, and the human orthologue of which is located in the Down syndrome critical region on chromosome 21, could account for some of these Robo independent functions as well as provide selectivity to Robo-mediated axon responses to Slit. Here we investigate the expression of the mammalian orthologue EVA1C in regions of the developing mouse nervous system which have been shown to exhibit Robo-dependent and -independent responses to Slit. We report that EVA1C is expressed by axons contributing to commissures, tracts and nerve pathways of the developing spinal cord and forebrain. Furthermore it is expressed by axons that display both Robo-dependent and -independent functions of Slit, supporting a role for EVA1C in Slit/Robo mediated neural circuit formation in the developing nervous system.

Growth cone dynamics in the zebrafish embryonic forebrain are regulated by Brother of Cdo.

During development of the embryonic zebrafish brain, the differential expression of axon guidance molecules directs the growth of axons along defined neuronal tracts. Neurons within the dorsorostral cluster of the presumptive telencephalon project axons ventrally along the supraoptic tract. Brother of Cdo (Boc) is a known axon guidance molecule that is expressed in a broad band lying ventral to the dorsorostral cluster of neurons. Loss of Boc function has previously been shown to perturb the development of the supraoptic tract. We have used live cell imaging of individual growth cones within the living zebrafish embryo to determine how Boc regulates the growth cone dynamics and axon guidance within the supraoptic tract. A plasmid construct encoding elavl3-eGFP was injected into early embryos to selectively label a small number of neurons while the expression of Boc was knocked down by injection of antisense morpholino oligonucleotides. Time-lapse imaging of growth cones within the living embryos revealed that loss of Boc significantly affected the morphology of growth cones in comparison to axons within control embryos. Growth cones navigating along the supraoptic tract in the absence of Boc extended significantly longer filopodia in the rostrocaudal direction. These results indicate that Boc acts to restrict axons and their filopodia within the narrow pathway of the supraoptic tract. The highly selective nature of these pathfinding defects reveal that Boc is likely to be one of many molecules that coordinate the trajectory of axons within the supraoptic tract.

Distinct expression patterns of syndecans in the embryonic zebrafish brain.

Axon pathfinding in the neuroepithelium of embryonic brain is dependent on a variety of short and long range guidance cues. Heparan sulfate proteoglycans such as syndecans act as modulators of these cues and their importance in neural development is highlighted by their phylogenetic conservation. In Drosophilia, a single syndecan is present on the surface of axon growth cones and is required for chemorepulsive signalling during midline crossing. Understanding the role of syndecans in the vertebrate nervous system is challenging given that there are four homologous genes, syndecans 1-4. We show here that syndecans 2-4 are expressed in the zebrafish embryonic brain during the major period of axon growth. These genes show differing expression patterns in the brain which provides putative insights into their functional specificity.

Frizzled-3a and Wnt-8b genetically interact during forebrain commissural formation in embryonic zebrafish.

The commissural plate forms the rostral surface of the embryonic vertebrate forebrain and provides a cellular substrate for forebrain commissural axons. We have previously reported that the Wnt receptor frizzled-3a (fzd3a) restricts the expression of the chemorepulsive guidance ligand slit2 to a discrete domain of neuroepithelial cells in the commissural plate of embryonic zebrafish. Loss of Fzd3a function perturbed slit2 expression and disrupted the formation of glial bridges which guide the formation of forebrain commissures. We now show that Wnt8b is also necessary for anterior commissural formation as well as for patterning of slit2 expression at the midline. Knock down of Wnt8b produced the same phenotype as loss of Fzd3a which suggested that these genes were acting together to regulate axon guidance. Simultaneous sub-threshold knock down of both Fzd3a and Wnt8b led to a greater than additive increase in the penetrance of the mutant phenotype which indicated that these two genes were indeed interacting. We have shown here that Fzd3a/Wnt8b signaling is essential for normal patterning of the commissural plate and that loss-of-function in either receptor or ligand causes Slit2-dependent defects in glial bridge morphology which indirectly attenuated axon midline crossing in the embryonic vertebrate forebrain.

Netrin-1 is required for efficient neural tube closure.

During neural tube formation, neural plate cells migrate from the lateral aspects of the dorsal surface towards the midline. Elevation of the lateral regions of the neural plate produces the neural folds which then migrate to the midline where they fuse at their dorsal tips, generating a closed neural tube comprising an apicobasally polarized neuroepithelium. Our previous study identified a novel role for the axon guidance receptor neogenin in Xenopus neural tube formation. We demonstrated that loss of neogenin impeded neural fold apposition and neural tube closure. This study also revealed that neogenin, via its interaction with its ligand, RGMa, promoted cell-cell adhesion between neural plate cells as the neural folds elevated and between neuroepithelial cells within the neural tube. The second neogenin ligand, netrin-1, has been implicated in cell migration and epithelial morphogenesis. Therefore, we hypothesized that netrin-1 may also act as a ligand for neogenin during neurulation. Here we demonstrate that morpholino knockdown of Xenopus netrin-1 results in delayed neural fold apposition and neural tube closure. We further show that netrin-1 functions in the same pathway as neogenin and RGMa during neurulation. However, contrary to the role of neogenin-RGMa interactions, neogenin-netrin-1 interactions are not required for neural fold elevation or adhesion between neuroepithelial cells. Instead, our data suggest that netrin-1 contributes to the migration of the neural folds towards the midline. We conclude that both neogenin ligands work synergistically to ensure neural tube closure.

Yap controls stem/progenitor cell proliferation in the mouse postnatal epidermis.

Tissue renewal is an ongoing process in the epithelium of the skin. We have begun to examine the genetic mechanisms that control stem/progenitor cell activation in the postnatal epidermis. The conserved Hippo pathway regulates stem cell turnover in arthropods through to vertebrates. Here we show that its downstream effector, yes-associated protein (YAP), is active in the stem/progenitor cells of the postnatal epidermis. Overexpression of a C-terminally truncated YAP mutant in the basal epidermis of transgenic mice caused marked expansion of epidermal stem/progenitor cell populations. Our data suggest that the C-terminus of YAP controls the balance between stem/progenitor cell proliferation and differentiation in the postnatal interfollicular epidermis. We conclude that YAP functions as a molecular switch of stem/progenitor cell activation in the epidermis. Moreover, our results highlight YAP as a possible therapeutic target for diseases such as skin cancer, psoriasis, and epidermolysis bullosa.

HuC-eGFP mosaic labelling of neurons in zebrafish enables in vivo live cell imaging of growth cones.

The field of axon guidance is taking advantage of the powerful genetic and imaging tools that are now available to visualise growth behaviour in living cells, both in vivo and in real time. We have developed a method to visualise individual neurons within the living zebrafish embryo which provides exceptional cellular resolution of growth cones and their filopodia. We generated a DNA construct in which the HuC promoter drives expression of eGFP. Injection of the plasmid into single cell fertilised zebrafish egg resulted in mosaic expression of eGFP in neurons throughout the developing embryo. By manipulating the concentration of injected plasmid, it was possible to optimise the numbers of neurons that expressed the construct so that individual growth cones could be easily visualised. We then used time-lapse high magnification widefield epifluorescence microscopy to visualise the growth cones as they were exploring their environment. Growth cones both near the surface of the embryo as well as deep within the developing brain of embryos at 20 h post fertilisation were clearly imaged. With time-lapse sequence imaging with intervals between frames as frequent as 1 s there was minimal loss of fluorescence intensity and the dynamic nature of the growth cones became evident. This method therefore provides high magnification, high resolution time-lapse imaging of living neurons in vivo and by use of widefield epifluorescence rather than confocal it is a relatively inexpensive microscopy method.

Chemoattractant axon guidance cues regulate de novo axon trajectories in the embryonic forebrain of zebrafish.

The development of axon tracts in the early vertebrate brain is controlled by combinations of soluble, membrane-bound and extracellular matrix molecules. How these multiple and sometimes conflicting guidance cues are integrated in order to establish stereotypical pathways remains to be determined. We show here that when interactions between the chemoattractive signal Netrin1a and its receptor Dcc are suppressed using a loss-of-function approach, a novel axon trajectory emerges in the dorsal diencephalon. Axons arising from a subpopulation of telencephalic neurons failed to project rostrally into the anterior commissure in the absence of either Netrin1a or Dcc. Instead these axons inappropriately exited the telencephalon and ectopically coursed caudally into virgin neuroepithelium. This response was highly specific since loss-of-function of Netrin1b, a paralogue of Netrin1a, generated a distinct phenotype in the rostral brain. These results show that a subpopulation of telencephalic neurons, when freed from long-range chemoattraction mediated by Netrin1a-Dcc interactions, follow alternative instructive cues that lead to creation of an ectopic axon bundle in the diencephalon. This work provides insight into how integration of multiple guidance signals defines the initial scaffold of axon tracts in the embryonic vertebrate forebrain.

Repulsive guidance molecule A (RGMa): a molecule for all seasons.

RGMa (repulsive guidance molecule a) was the first identified molecule that possessed the necessary functional activity to repulse and steer growth cones to their target in the brain. By binding to its neogenin receptor, RGMa caused the collapse of growth cones and encouraged axons to grow along specific trajectories in vitro. Although originally characterized in 1990, RGMa was not conclusively shown to mediate axon guidance in vivo for another 12 years. Loss-of-function analysis in mice revealed that RGMa may play a more important role in neural tube morphogenesis. RGMa has now emerged as a molecule with pleiotropic roles involving cell adhesion, cell migration, cell polarity and cell differentiation which together strongly influence early morphogenetic events as well as immune responses. RGMa can be regarded as a molecule for all seasons.

Frizzled-3a and slit2 genetically interact to modulate midline axon crossing in the telencephalon.

The anterior commissure forms the first axon connections between the two sides of the embryonic telencephalon. We investigated the role of the transmembrane receptor Frizzled-3a in the development of this commissure using zebrafish as an experimental model. Knock down of Frizzled-3a resulted in complete loss of the anterior commissure. This defect was accompanied by a loss of the glial bridge, expansion of the slit2 expression domain and perturbation of the midline telencephalic-diencephalic boundary. Blocking Slit2 activity following knock down of Frizzled-3a effectively rescued the anterior commissure defect which suggested that Frizzled-3a was indirectly controlling the growth of axons across the rostral midline. We have shown here that Frizzled-3a is essential for normal development of the commissural plate and that loss-of-function causes Slit2-dependent defects in axon midline crossing in the embryonic vertebrate forebrain. These data supports a model whereby Wnt signaling through Frizzled-3a attenuates expression of Slit2 in the rostral midline of the forebrain. The absence of Slit2 facilitates the formation of a midline bridge of glial cells which is used as a substrate for commissural axons. In the absence of this platform of glia, commissural axons fail to cross the rostral midline of the forebrain.

Novel roles of the chemorepellent axon guidance molecule RGMa in cell migration and adhesion.

The repulsive guidance molecule A (RGMa) is a contact-mediated axon guidance molecule that has significant roles in central nervous system (CNS) development. Here we have examined whether RGMa has novel roles in cell migration and cell adhesion outside the nervous system. RGMa was found to stimulate cell migration from Xenopus animal cap explants in a neogenin-dependent and BMP-independent manner. RGMa also stimulated the adhesion of Xenopus animal cap cells, and this adhesion was dependent on neogenin and independent of calcium. To begin to functionally characterize the role of specific domains in RGMa, we assessed the migratory and adhesive activities of deletion mutants. RGMa lacking the partial von Willebrand factor type D (vWF) domain preferentially perturbed cell adhesion, while mutants lacking the RGD motif affected cell migration. We also revealed that manipulating the levels of RGMa in vivo caused major migration defects during Xenopus gastrulation. We have revealed here novel roles of RGMa in cell migration and adhesion and demonstrated that perturbations to the homeostasis of RGMa expression can severely disrupt major morphogenetic events. These results have implications for understanding the role of RGMa in both health and disease.