Phenotyping clonal populations of glioma stem cell reveals a high degree of plasticity in response to changes of microenvironment.

The phenotype of glioma-initiating cells (GIC) is modulated by cell-intrinsic and cell-extrinsic factors. Phenotypic heterogeneity and plasticity of GIC is an important limitation to therapeutic approaches targeting cancer stem cells. Plasticity also presents a challenge to the identification, isolation, and propagation of purified cancer stem cells. Here we use a barcode labelling approach of GIC to generate clonal populations over a number of passages, in combination with phenotyping using the established stem cell markers CD133, CD15, CD44, and A2B5. Using two cell lines derived from isocitrate dehydrogenase (IDH)-wildtype glioblastoma, we identify a remarkable heterogeneity of the phenotypes between the cell lines. During passaging, clonal expansion manifests as the emergence of a limited number of barcoded clones and a decrease in the overall number of clones. Dual-labelled GIC are capable of forming traceable clonal populations which emerge after as few as two passages from mixed cultures and through analyses of similarity of relative proportions of 16 surface markers we were able to pinpoint the fate of such populations. By generating tumour organoids we observed a remarkable persistence of dominant clones but also a significant plasticity of stemness marker expression. Our study presents an experimental approach to simultaneously barcode and phenotype glioma-initiating cells to assess their functional properties, for example to screen newly established GIC for tumour-specific therapeutic vulnerabilities.

The assembly of developing motor neurons depends on an interplay between spontaneous activity, type II cadherins and gap junctions.

A core structural and functional motif of the vertebrate central nervous system is discrete clusters of neurons or 'nuclei'. Yet the developmental mechanisms underlying this fundamental mode of organisation are largely unknown. We have previously shown that the assembly of motor neurons into nuclei depends on cadherin-mediated adhesion. Here, we demonstrate that the emergence of mature topography among motor nuclei involves a novel interplay between spontaneous activity, cadherin expression and gap junction communication. We report that nuclei display spontaneous calcium transients, and that changes in the activity patterns coincide with the course of nucleogenesis. We also find that these activity patterns are disrupted by manipulating cadherin or gap junction expression. Furthermore, inhibition of activity disrupts nucleogenesis, suggesting that activity feeds back to maintain integrity among motor neurons within a nucleus. Our study suggests that a network of interactions between cadherins, gap junctions and spontaneous activity governs neuron assembly, presaging circuit formation.

Distance-dependent gradient in NMDAR-driven spine calcium signals along tapering dendrites.

Neurons receive a multitude of synaptic inputs along their dendritic arbor, but how this highly heterogeneous population of synaptic compartments is spatially organized remains unclear. By measuring N-methyl-d-aspartic acid receptor (NMDAR)-driven calcium responses in single spines, we provide a spatial map of synaptic calcium signals along dendritic arbors of hippocampal neurons and relate this to measures of synapse structure. We find that quantal NMDAR calcium signals increase in amplitude as they approach a thinning dendritic tip end. Based on a compartmental model of spine calcium dynamics, we propose that this biased distribution in calcium signals is governed by a gradual, distance-dependent decline in spine size, which we visualized using serial block-face scanning electron microscopy. Our data describe a cell-autonomous feature of principal neurons, where tapering dendrites show an inverse distribution of spine size and NMDAR-driven calcium signals along dendritic trees, with important implications for synaptic plasticity rules and spine function.

Rapid Bidirectional Reorganization of Cortical Microcircuits.

Mature neocortex adapts to altered sensory input by changing neural activity in cortical circuits. The underlying cellular mechanisms remain unclear. We used blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI) to show reorganization in somatosensory cortex elicited by altered whisker sensory input. We found that there was rapid expansion followed by retraction of whisker cortical maps. The cellular basis for the reorganization in primary somatosensory cortex was investigated with paired electrophysiological recordings in the periphery of the expanded whisker representation. During map expansion, the chance of finding a monosynaptic connection between pairs of pyramidal neurons increased 3-fold. Despite the rapid increase in local excitatory connectivity, the average strength and synaptic dynamics did not change, which suggests that new excitatory connections rapidly acquire the properties of established excitatory connections. During map retraction, entire excitatory connections between pyramidal neurons were lost. In contrast, connectivity between pyramidal neurons and fast spiking interneurons was unchanged. Hence, the changes in local excitatory connectivity did not occur in all circuits involving pyramidal neurons. Our data show that pyramidal neurons are recruited to and eliminated from local excitatory networks over days. These findings suggest that the local excitatory connectome is dynamic in mature neocortex.

BDNF promotes axon branching of retinal ganglion cells via miRNA-132 and p250GAP.

A crucial step in the development of the vertebrate visual system is the branching of retinal ganglion cell (RGC) axons within their target, the superior colliculus/tectum. A major player in this process is the neurotrophin brain-derived neurotrophic factor (BDNF). However, the molecular basis for the signaling pathways mediating BDNF action is less well understood. As BDNF exerts some of its functions by controlling the expression of microRNAs (miRNAs), we investigated whether miRNAs are also involved in BDNF-mediated retinal axon branching. Here, we demonstrate that the expression pattern of miRNA-132 in the retina is consistent with its involvement in this process, and that BDNF induces the upregulation of miRNA-132 in retinal cultures. Furthermore, in vitro gain-of-function and loss-of-function approaches in retinal cultures reveal that miRNA-132 mediates axon branching downstream of BDNF. A known target of miRNA-132 is the Rho family GTPase-activating protein, p250GAP. We find that p250GAP is expressed in RGC axons and mediates the effects of miRNA-132 in BDNF-induced branching. BDNF treatment or overexpression of miRNA-132 leads to a reduction in p250GAP protein levels in retinal cultures, whereas the overexpression of p250GAP abolishes BDNF-induced branching. Finally, we used a loss-of-function approach to show that miRNA-132 affects the maturation of RGC termination zones in the mouse superior colliculus in vivo, while their topographic targeting remains intact. Together, our data indicate that BDNF promotes RGC axon branching during retinocollicular/tectal map formation via upregulation of miRNA-132, which in turn downregulates p250GAP.

Emergent properties of the optic tectum revealed by population analysis of direction and orientation selectivity.

How local circuits within the brain process visual information has classically been addressed at the single neuron level. Such reductionist approaches, however, struggle to capture the full scope of functional properties associated with even "simple" brain nuclei. Using population functional calcium imaging, we aim to describe how local circuits within the zebrafish optic tectum process visual information. Specifically, how are previously identified direction-selective (DS) and orientation-selective (OS) retinal ganglion cell (RGC) inputs (Nikolaou et al., 2012) represented in tectal cells? First, we identify an emergent population of DS tectal cell with a direction preference not explicitly present in any one of the RGC inputs. Second, this is associated with a striking shift from a tiled and triangular representation of directional space (RGC inputs) into an overlapping cardinal representation by tectal cell populations. Third, and in contrast, we find that orientation space is represented similarly in both the RGC input and tectal cell populations illustrating feature-dependent differences in how tectal circuits process their inputs. Finally, we identify OS and two populations of DS cells at the superficial border of the tectal neuropil, one of which is an emergent population. This study, together with our previous one (Nikolaou et al., 2012), demonstrate that direction-selectivity is established in both the retina and tectum.

A systems-based dissection of retinal inputs to the zebrafish tectum reveals different rules for different functional classes during development.

We have examined the form, diversity, and organization of three functional classes of retinal inputs to the zebrafish optic tectum during development. Our systems-based approach was to analyze data from populations of retinal ganglion cells labeled with a presynaptic targeted calcium indicator, synaptophysin GCaMP3 (SyGCaMP3). Collectively, our findings provide an insight as to the degree of visual encoding during retino-tectal development and how it dynamically evolves from a nascent and noisy presynaptic neural-scape to an increasingly complex and refined representation. We report five key features: (1) direction-selective inputs are developmentally invariant; (2) orientation-selective inputs exhibit highly dynamic properties over the same period, with changes in their functional characteristics and spatial organization; (3) inputs defined as anisotropic are an early dominant functional class, with heterogeneous response profiles, which progressively diminish in incidence and spatial extent; (4) dark rearing selectively affects the orientation-selective responses: both functional characteristics and relative spatial distributions; and (5) orientation-selective inputs exhibit four subtypes, two more than previously identified in any species. Our approach was to label RGC axon terminals with an indicator of activity and quantitatively characterize coherent response properties to different visual stimuli. Its application in the zebrafish, given its small size and the accessibility of the tectum, has enabled a quick yet robust assessment of multiple functional populations of responses.

In vivo magnetic resonance imaging of acute brain inflammation using microparticles of iron oxide.

Multiple sclerosis is a disease of the central nervous system that is associated with leukocyte recruitment and subsequent inflammation, demyelination and axonal loss. Endothelial vascular cell adhesion molecule-1 (VCAM-1) and its ligand, alpha4beta1 integrin, are key mediators of leukocyte recruitment, and selective inhibitors that bind to the alpha4 subunit of alpha4beta1 substantially reduce clinical relapse in multiple sclerosis. Urgently needed is a molecular imaging technique to accelerate diagnosis, to quantify disease activity and to guide specific therapy. Here we report in vivo detection of VCAM-1 in acute brain inflammation, by magnetic resonance imaging in a mouse model, at a time when pathology is otherwise undetectable. Antibody-conjugated microparticles carrying a large amount of iron oxide provide potent, quantifiable contrast effects that delineate the architecture of activated cerebral blood vessels. Their rapid clearance from blood results in minimal background contrast. This technology is adaptable to monitor the expression of endovascular molecules in vivo in various pathologies.

Real-time electrochemical monitoring of brain tissue oxygen: a surrogate for functional magnetic resonance imaging in rodents.

Long-term in-vivo electrochemistry (LIVE) enables real-time monitoring and measurement of brain metabolites. In this study we have simultaneously obtained blood oxygenation level dependent (BOLD) fMRI and amperometric tissue O(2) data from rat cerebral cortex, during both increases and decreases in inspired O(2) content. BOLD and tissue O(2) measurements demonstrated close correlation (r=0.7898) during complete (0%) O(2) removal, with marked negative responses occurring ca. 30s after the onset of O(2) removal. Conversely, when the inspired O(2) was increased (50, 70 and 100% O(2) for 1min) similar positive rapid changes (ca. 15s) in both the BOLD and tissue O(2) signals were observed. These findings demonstrate, for the first time, the practical feasibility of obtaining real-time metabolite information during fMRI acquisition, and that tissue O(2) concentration monitored using an O(2) sensor can serve as an index of changes in the magnitude of the BOLD response. As LIVE O(2) sensors can be used in awake animals performing specific behavioural tasks the technique provides a viable animal surrogate of human fMRI experimentation.

Neither in vivo MRI nor behavioural assessment indicate therapeutic efficacy for a novel 5HT(1A) agonist in rat models of ischaemic stroke.

BACKGROUND: 5HT1A agonists have previously been shown to promote recovery in animal models of stroke using ex vivo outcome measures which have raised the hopes for a potential clinical implementation. The purpose of this study was to evaluate the potential neuroprotective properties of a novel 5HT1A agonist DU123015 in 2 different models of transient focal ischaemic stroke of varying severities using both in vivo neuroimaging and behavioural techniques as primary outcome measures. For these studies, the NMDA receptor antagonist MK-801 was also utilized as a positive control to further assess the effectiveness of the stroke models and techniques used. RESULTS: In contrast to MK-801, no significant therapeutic effect of DU123015 on lesion volume in either the distal MCAo or intraluminal thread model of stroke was found. MK-801 significantly reduced lesion volume in both models; the mild distal MCAo condition (60 min ischaemia) and the intraluminal thread model, although it had no significant impact upon the lesion size in the severe distal MCAo condition (120 min ischaemia). These therapeutic effects on lesion size were mirrored on a behavioural test for sensory neglect and neurological deficit score in the intraluminal thread model. CONCLUSION: This study highlights the need for a thorough experimental design to test novel neuroprotective compounds in experimental stroke investigations incorporating: a positive reference compound, different models of focal ischaemia, varying the duration of ischaemia, and objective in vivo assessments within a single study. This procedure will help us to minimise the translation of less efficacious compounds.

Sensory inputs from whisking movements modify cortical whisker maps visualized with functional magnetic resonance imaging.

Rodents vary the frequency of whisking movements during exploratory and discriminatory behaviors. The effect of whisking frequency on whisker cortical maps was investigated by simulating whisking at physiological frequencies and imaging the whisker representations with blood oxygen level-dependent (BOLD) functional magnetic resonance imaging. Repetitive deflection of many right-sided whiskers at 10 Hz evoked a positive BOLD response that extended across contralateral primary somatosensory cortex (SI) and secondary somatosensory cortex (SII). In contrast, synchronous deflection of 2 adjacent whiskers (right C1 and C2) at 10 Hz evoked separate positive BOLD responses in contralateral SI and SII that were predominantly located in upper cortical layers. The positive BOLD responses were separated and partially surrounded by a negative BOLD response that was mainly in lower cortical layers. Two-whisker representations varied with the frequency of simulated whisking. Positive BOLD responses were largest with 7-Hz deflection. Negative BOLD responses were robust at 10 Hz but were weaker or absent with 7-Hz or 3-Hz deflection. Our findings suggest that sensory inputs attributable to the frequency of whisking movements modify whisker cortical representations.

Multi-modal characterisation of the neocortical clip model of focal cerebral ischaemia by MRI, behaviour and immunohistochemistry.

The neocortical clip model of focal cerebral ischaemia has previously been used with success in neuroprotection studies. To further improve its translational qualities, we have characterised this model using a combination of serial Magnetic Resonance Imaging (MRI), neurological assessment, the bilateral asymmetry test (BAT) and immunohistochemistry. The right MCA was occluded in spontaneously hypertensive rats for 0, 60 and 120 min. MRI was performed pre-surgery, 1, 3 and 7 days post-surgery. Behavioural assessment was performed 2 days before and 3 and 7 days post-surgery whilst neurological deficits were monitored daily. Neuroimaging results showed that 0 min of MCA occlusion did not produce a lesion, whereas occlusion for 60 min produced a lesion that remained stable over time. Occlusion for 120 min caused a more severe lesion 1 day post-surgery, but decreased by 7 days. Behaviour, neurological scores and histological lesion volumes correlated strongly with MRI lesion volume. Immunohistochemistry revealed neuronal loss, astrogliosis and macrophage infiltration in lesioned cortices. The neocortical clip model produced ischaemic lesions that are restricted to cortical territories of the MCA. The duration of occlusion dictates lesion severity which may prove useful for probing therapeutic interventions at different stages of stroke progression. The correlation of MRI with two different behavioural measures and post-mortem histology strengthens the basis for MRI providing an in vivo surrogate marker for structural and behavioural deficits caused by a cortical stroke.

Geniculo-Cortical Projection Diversity Revealed within the Mouse Visual Thalamus.

The mouse dorsal lateral geniculate nucleus (dLGN) is an intermediary between retina and primary visual cortex (V1). Recent investigations are beginning to reveal regional complexity in mouse dLGN. Using local injections of retrograde tracers into V1 of adult and neonatal mice, we examined the developing organisation of geniculate projection columns: the population of dLGN-V1 projection neurons that converge in cortex. Serial sectioning of the dLGN enabled the distribution of labelled projection neurons to be reconstructed and collated within a common standardised space. This enabled us to determine: the organisation of cells within the dLGN-V1 projection columns; their internal organisation (topology); and their order relative to V1 (topography). Here, we report parameters of projection columns that are highly variable in young animals and refined in the adult, exhibiting profiles consistent with shell and core zones of the dLGN. Additionally, such profiles are disrupted in adult animals with reduced correlated spontaneous activity during development. Assessing the variability between groups with partial least squares regression suggests that 4-6 cryptic lamina may exist along the length of the projection column. Our findings further spotlight the diversity of the mouse dLGN--an increasingly important model system for understanding the pre-cortical organisation and processing of visual information. Furthermore, our approach of using standardised spaces and pooling information across many animals will enhance future functional studies of the dLGN.

Noninvasive competition for endoscopic ultrasound.

Endoscopic ultrasonography (EUS) is one of the most significant developments in gastrointestinal (GI) imaging in recent years. EUS now plays a key role in the pretreatment staging of GI tract tumors and in the investigation of benign pancreaticobiliary pathology. It has not replaced conventional cross-sectional imaging (eg, ultrasound, CT, and MRI), but it has distinct properties and capabilities. EUS is most beneficial when used in a complementary fashion with cross-sectional and radionuclide imaging in the management of patients with GI tract disease. This article reviews the role of noninvasive imaging modalities in several clinical situations where EUS plays a prominent role.

Zebrafish brain mapping--standardized spaces, length scales, and the power of N and n.

Mapping anatomical and functional parameters of the zebrafish brain is moving apace. Research communities undertaking such studies are becoming ever larger and more diverse. The unique features, tools, and technologies associated with zebrafish are propelling them as the 21st century model organism for brain mapping. Uniquely positioned as a vertebrate model system, the zebrafish enables imaging of anatomy and function at different length scales from intraneuronal compartments to sparsely distributed whole brain patterns. With a variety of diverse and established statistical modeling and analytic methods available from the wider brain mapping communities, the richness of zebrafish neuroimaging data is being realized. The statistical power of population observations (N) within and across many samples (n) projected onto a standardized space will provide vast databases for data-driven biological approaches. This article reviews key brain mapping initiatives at different levels of scale that highlight the potential of zebrafish brain mapping. By way of introduction to the next wave of brain mappers, an accessible introduction to the key concepts and caveats associated with neuroimaging are outlined and discussed.

Parametric functional maps of visual inputs to the tectum.

How features of the visual scene are encoded in the population activity of retinal ganglion cells (RGCs) targeting specific regions of the brain is not well understood. To address this, we have used a genetically encoded reporter of presynaptic function (SyGCaMP3) to record visually evoked activity in the population of RGC axons innervating the zebrafish tectum. Using unbiased voxel-wise analysis of SyGCaMP3 signals, we identify three subtypes of direction-selective and two subtypes of orientation-selective retinal input. Composite parametric functional maps generated across many larvae show laminar segregation of direction- and orientation-selective responses and unexpected retinotopic biases in the distribution of functional subtypes. These findings provide a systematic description of the form, organization, and dimensionality of visual inputs to the brain and will serve as a platform for understanding emergent properties in tectal circuits associated with visually driven behavior.

Lipid peptide nanocomplexes for gene delivery and magnetic resonance imaging in the brain.

Gadolinium-labelled nanocomplexes offer prospects for the development of real-time, non-invasive imaging strategies to visualise the location of gene delivery by MRI. In this study, targeted nanoparticle formulations were prepared comprising a cationic liposome (L) containing a Gd-chelated lipid at 10, 15 and 20% by weight of total lipid, a receptor-targeted, DNA-binding peptide (P) and plasmid DNA (D), which electrostatically self-assembled into LPD nanocomplexes. The LPD formulation containing the liposome with 15% Gd-chelated lipid displayed optimal peptide-targeted, transfection efficiency. MRI conspicuity peaked at 4h after incubation of the nanocomplexes with cells, suggesting enhancement by cellular uptake and trafficking. This was supported by time course confocal microscopy analysis of transfections with fluorescently-labelled LPD nanocomplexes. Gd-LPD nanocomplexes delivered to rat brains by convection-enhanced delivery were visible by MRI at 6 h, 24 h and 48 h after administration. Histological brain sections analysed by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) confirmed that the MRI signal was associated with the distribution of Gd(3+) moieties and differentiated MRI signals due to haemorrhage. The transfected brain cells near the injection site appeared to be mostly microglial. This study shows the potential of Gd-LPD nanocomplexes for simultaneous delivery of contrast agents and genes for real-time monitoring of gene therapy in the brain.

Neuroadaptive responses to citalopram in rats using pharmacological magnetic resonance imaging.

RATIONALE: The majority of psychoactive compounds, including antidepressants in clinical practice, were discovered largely by serendipity. The underlying neuropharmacological mechanisms of action of these compounds leading to resolution of depressive symptomatology are targets of the current research. Pharmacological magnetic resonance imaging (phMRI), a rapidly developing advancement of blood oxygenation level dependent (BOLD) contrast offers the potential to localize the regional sites of action in the CNS. OBJECTIVE: Acute and chronic effects of the clinically effective selective serotonin reuptake inhibitor (SSRI) citalopram were examined for changes in BOLD contrast using phMRI in rats. To pharmacologically characterize the specific involvement of the 5-HT(1A) receptors, citalopram was co-administered with a highly selective 5-HT(1A) receptor antagonist WAY100635. RESULTS: Acute citalopram treatment (10 and 20 mg/kg i.p.) produced a widespread and dose-dependent activation throughout the whole brain. Following 14 days of chronic daily administration of citalopram (20 mg/kg i.p.), localized effects were observed; regions integral in the therapeutic antidepressant effects included the hypothalamus, hippocampus, and cortical regions, suggesting desensitization of serotonergic receptors in the midbrain contributing to elevated levels of 5-HT. Co-administration with WAY100635 (0.3 mg/kg s.c.) increased BOLD activation in the frontal cortex and decreased BOLD contrast in the hypothalamus, hippocampus, and hindbrain structures. CONCLUSION: The present findings highlight the adaptive nature of responses to citalopram which exhibits regional and pharmacological specificity. These findings translate well to the clinical findings and suggest that this approach may offer the opportunity to develop more efficacious antidepressants with a faster clinical response.

Quantitative manganese tract tracing: dose-dependent and activity-independent terminal labelling in the mouse visual system.

At concentrations sufficient for visualisation using MRI, manganese (Mn) is believed to behave as a calcium analogue. This study examines different concentrations of Mn for enhanced MR tract tracing. The premise of activity-dependent axonal transport was also examined by partial or complete blockade of retinal ganglion cell activity. Quantitative T(1) maps and semi-quantitative normalised signal intensities in the superior colliculi facilitated assessment of applied intraocular concentrations and activity dependence, respectively. Varying the concentration of applied Mn revealed a non-monotonic profile, with optimal, unfavourable and undesirable effects noted: 25 mM proved optimal, showing a maximal decrease in T(1), whereas 400 mM was associated with no terminal-field enhancement. The estimated vitreal concentration for optimal transport of Mn (2 mM) is substantially lower than that used in previous studies of the mouse. Both the partial blockade of inputs to 50% of retinal ganglion cells by a mGluR6 glutamate agonist and the complete blockade of all retinal ganglion cell activity with tetrodotoxin failed to decrease the relative enhancement in the superior colliculus. The failure to prevent axonal transport of Mn by blocking activity (and therefore theoretically the intracellular influx) appeared to be paradoxical. The optimal vitreal concentration of Mn has previously been shown to facilitate massive intracellular uptake of Mn, competitively blocking calcium, and 1 mM Mn blocks neurotransmission pre-synaptically. These results suggest that, at concentrations required for optimal Mn-enhanced MRI tract tracing in the visual system of the mouse, the uptake and transport of Mn may be dominated by passive mechanisms, which may also block neurotransmission.

A method for removing global effects in small-animal functional MRI.

Global effects in functional MRI are temporal modulations in signal intensity resulting from various scanner and subject phenomena. These effects contribute to the overall variance, reducing the effect size associated with an experimental paradigm. Statistical estimations that include an approximation for concurrent global effects will reduce the residual error within the model and so improve statistical power of the study. Conventionally, estimates of global effects are derived from mean intracerebral signal intensities, but these may be prone to contributions from localised experimentally evoked signal changes. In such cases, inaccurate estimates of global effects may result in erroneous inferences of neural modulations based on statistical artefact. A novel, computationally simple, method of estimating global effects is proposed using muscle tissue acquired within the same acquisition volume. Quantitative improvements in sensitivity are reported for a somatosensory stimulation paradigm using global muscle signal intensities as a covariate of no-interest. The method is independent of local neurogenic signal changes and, under particular experimental conditions, may be more representative of true global effects. The utility of this strategy to applications in small-animal functional MRI that evoke systemic physiological changes as a result of the experimental manipulation is critically discussed.