Multicellular "hotspots" harbor high-grade potential in lower-grade gliomas.

BACKGROUND: Lower-grade gliomas may be indolent for many years before developing malignant behavior. The mechanisms underlying malignant progression remain unclear. METHODS: We collected blocks of live human brain tissue donated by people undergoing glioma resection. The tissue blocks extended through the peritumoral cortex and into the glioma. The living human brain tissue was cut into ex vivo brain slices and bathed in 5-aminolevulinic acid (5-ALA). High-grade glioma cells avidly take up 5-ALA and accumulate high levels of the fluorescent metabolite, Protoporphyrin IX (PpIX). We exploited the PpIX fluorescence emitted by higher-grade glioma cells to investigate the earliest stages of malignant progression in lower-grade gliomas. RESULTS: We found sparsely distributed "hot-spots" of PpIX-positive cells in living lower-grade glioma tissue. Glioma cells and endothelial cells formed part of the PpIX hotspots. Glioma cells in PpIX hotspots were IDH1 mutant and expressed nestin suggesting they had acquired stem-like properties. Spatial analysis with 5-ALA-conjugated quantum dots indicated that these glioma cells replicated adjacent to blood vessels. PpIX hotspots were formed in the absence of angiogenesis. CONCLUSION: Our data show that PpIX hotspots represent microdomains of cells with high-grade potential within lower-grade gliomas and identify locations where malignant progression could start.

New strategies for managing adult gliomas.

Gliomas are hard to treat. Their prognosis has improved little over the past few decades. Fundamental therapeutic challenges such as treatment resistance, malignant progression, and tumour recurrence persist. New strategies are needed to advance the management and treatment of gliomas. Here, we focus on where those new strategies could emerge. We consider how recent advances in our understanding of the biology of adult gliomas are informing new approaches to their treatment.

Ex vivo ultrasonic samples of human brain tumors in the molecular era.

BACKGROUND: Gliomas are composed of multiple clones of tumor cells. This intratumor heterogeneity contributes to the ability of gliomas to resist treatment. It is vital that gliomas are fully characterized at a molecular level when a diagnosis is made to maximize treatment effectiveness. METHODS: We collected ultrasonic tissue fragments during glioma surgery. Large tissue fragments were separated in the operating theater and bathed continuously in oxygenated artificial cerebrospinal fluid to keep them alive. The ex vivo tissue fragments were transferred to a laboratory and incubated in 5-aminolevulinic acid (5-ALA). 5-ALA is metabolized to Protoporphyrin IX (PpIX), which accumulates in glioma cells and makes them fluorescent. The molecular and neuropathological features of the PpIX fluorescent ultrasonic tissue fragments were studied. RESULTS: We show that PpIX fluorescence can rapidly identify tissue fragments infiltrated by glioma in the laboratory. Ultrasonic tissue fragments from the tumor core provided molecular and neuropathological information about the glioma that was comparable to the surgical biopsy. We characterized the heterogeneity within individual gliomas by studying ultrasonic tissue fragments from different parts of the tumor. We found that gliomas exhibit a power relationship between cellular proliferation and tumor infiltration. Tissue fragments that deviate from this relationship may contain foci of more malignant glioma. The methylation status of the O 6-methylguanine DNA methyltransferase gene promoter varied within each glioma. CONCLUSIONS: Ex vivo ultrasonic tissue fragments can be rapidly screened for glioma infiltration. They offer a viable platform to characterize heterogeneity within individual gliomas, thereby enhancing their diagnosis and treatment.

Rewiring the connectome: Evidence and effects.

Neuronal connections form the physical basis for communication in the brain. Recently, there has been much interest in mapping the "connectome" to understand how brain structure gives rise to brain function, and ultimately, to behaviour. These attempts to map the connectome have largely assumed that connections are stable once formed. Recent studies, however, indicate that connections in mammalian brains may undergo rewiring during learning and experience-dependent plasticity. This suggests that the connectome is more dynamic than previously thought. To what extent can neural circuitry be rewired in the healthy adult brain? The connectome has been subdivided into multiple levels of scale, from synapses and microcircuits through to long-range tracts. Here, we examine the evidence for rewiring at each level. We then consider the role played by rewiring during learning. We conclude that harnessing rewiring offers new avenues to treat brain diseases.

Time Cognition: Entangled Neuronal Firing.

The neuroscience of time frequently focuses on either measuring short time intervals (sensory timing) or reproducing them (motor timing); during cognition, the two are integrated. New experiments using a combined sensory and motor timing task suggest that neuronal firing during the sensory and motor phases are linked.

Time in Cortical Circuits.

Time is central to cognition. However, the neural basis for time-dependent cognition remains poorly understood. We explore how the temporal features of neural activity in cortical circuits and their capacity for plasticity can contribute to time-dependent cognition over short time scales. This neural activity is linked to cognition that operates in the present or anticipates events or stimuli in the near future. We focus on deliberation and planning in the context of decision making as a cognitive process that integrates information across time. We progress to consider how temporal expectations of the future modulate perception. We propose that understanding the neural basis for how the brain tells time and operates in time will be necessary to develop general models of cognition. SIGNIFICANCE STATEMENT: Time is central to cognition. However, the neural basis for time-dependent cognition remains poorly understood. We explore how the temporal features of neural activity in cortical circuits and their capacity for plasticity can contribute to time-dependent cognition over short time scales. We propose that understanding the neural basis for how the brain tells time and operates in time will be necessary to develop general models of cognition.

Delayed and Temporally Imprecise Neurotransmission in Reorganizing Cortical Microcircuits.

Synaptic neurotransmission is modified at cortical connections throughout life. Varying the amplitude of the postsynaptic response is one mechanism that generates flexible signaling in neural circuits. The timing of the synaptic response may also play a role. Here, we investigated whether weakening and loss of an entire connection between excitatory cortical neurons was foreshadowed in the timing of the postsynaptic response. We made electrophysiological recordings in rat primary somatosensory cortex that was undergoing experience-dependent loss of complete local excitatory connections. The synaptic latency of pyramid-pyramid connections, which typically comprise multiple synapses, was longer and more variable. Connection strength and latency were not correlated. Instead, prolonged latency was more closely related to progression of connection loss. The action potential waveform and axonal conduction velocity were unaffected, suggesting that the altered timing of neurotransmission was attributable to a synaptic mechanism. Modeling studies indicated that increasing the latency and jitter at a subset of synapses reduced the number of action potentials fired by a postsynaptic neuron. We propose that prolonged synaptic latency and diminished temporal precision of neurotransmission are hallmarks of impending loss of a cortical connection.

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.

Pansynaptic enlargement at adult cortical connections strengthened by experience.

Behavioral experience alters the strength of neuronal connections in adult neocortex. These changes in synaptic strength are thought to be central to experience-dependent plasticity, learning, and memory. However, it is not known how changes in synaptic transmission between neurons become persistent, thereby enabling the storage of previous experience. A long-standing hypothesis is that altered synaptic strength is maintained by structural modifications to synapses. However, the extent of synaptic modifications and the changes in neurotransmission that the modifications support remain unclear. To address these questions, we recorded from pairs of synaptically connected layer 2/3 pyramidal neurons in the barrel cortex and imaged their contacts with high-resolution confocal microscopy after altering sensory experience by whisker trimming. Excitatory connections strengthened by experience exhibited larger axonal varicosities, dendritic spines, and interposed contact zones. Electron microscopy showed that contact zone size was strongly correlated with postsynaptic density area. Therefore, our findings indicate that whole synapses are larger at strengthened connections. Synaptic transmission was both stronger and more reliable following experience-dependent synapse enlargement. Hence, sensory experience modified both presynaptic and postsynaptic function. Our findings suggest that the enlargement of synaptic contacts is an integral part of long-lasting strengthening of cortical connections and, hence, of information storage in the neocortex.

Epileptic high-frequency network activity in a model of non-lesional temporal lobe epilepsy.

High-frequency cortical activity, particularly in the 250-600 Hz (fast ripple) band, has been implicated in playing a crucial role in epileptogenesis and seizure generation. Fast ripples are highly specific for the seizure initiation zone. However, evidence for the association of fast ripples with epileptic foci depends on animal models and human cases with substantial lesions in the form of hippocampal sclerosis, which suggests that neuronal loss may be required for fast ripples. In the present work, we tested whether cell loss is a necessary prerequisite for the generation of fast ripples, using a non-lesional model of temporal lobe epilepsy that lacks hippocampal sclerosis. The model is induced by unilateral intrahippocampal injection of tetanus toxin. Recordings from the hippocampi of freely-moving epileptic rats revealed high-frequency activity (>100 Hz), including fast ripples. High-frequency activity was present both during interictal discharges and seizure onset. Interictal fast ripples proved a significantly more reliable marker of the primary epileptogenic zone than the presence of either interictal discharges or ripples (100-250 Hz). These results suggest that fast ripple activity should be considered for its potential value in the pre-surgical workup of non-lesional temporal lobe epilepsy.

Sensory experience and cortical rewiring.

Adult primary sensory cortex is not hard wired, but adapts to sensory experience. The cellular basis for cortical plasticity involves a combination of functional and structural changes in cortical neurons and the connections between them. Functional changes such as synaptic strengthening have been the focus of many investigations. However, structural modifications to the connections between neurons play an important role in cortical plasticity. In this review, the authors focus on structural remodeling that leads to rewiring of cortical circuits. Recent work has identified axonal remodeling, growth of new dendritic spines, and synapse turnover as important structural mechanisms for experience-dependent plasticity in mature cortex. These findings have begun to unravel how rewiring occurs in adult neocortex and offer new insights into the cellular mechanisms for learning and memory.

Altered sensory experience induces targeted rewiring of local excitatory connections in mature neocortex.

Experience-dependent plasticity in adulthood is slower than during development. Previous experience can accelerate adult cortical plasticity. However, the contributions of functional synaptic changes and modifications in neuronal structure to the acceleration of adult cortical plasticity remain unclear. If structural remodeling was important then it should be exhibited by neuronal connections that have altered during plasticity. We trimmed rodents' whiskers to induce experience-dependent plasticity and reconstructed pairs of layer 2/3 (L2/3) pyramidal neurons after electrophysiological recording. We reported recently that local excitatory connections strengthen without a change in synapse number in cortex with retained sensory input (spared) (Cheetham et al., 2007). Here, we show that strengthened connections are rewired. The rewiring involves remodeling of the axonal arbor of excitatory connections with only minor changes in postsynaptic dendritic trees. The axonal remodeling resulted in a greater length of presynaptic axon close to postsynaptic dendrites at existing local excitatory connections in spared cortex. In control cortex, the length of axon close to dendrite in unconnected pairs of L2/3 pyramidal neurons was similar to that in synaptically connected pairs of L2/3 pyramidal neurons. This finding suggests that the probability of forming a synapse and, therefore, establishing a connection, is not driven solely by the length of axon close to dendrite. The axonal remodeling that we describe is not associated with altered synapse number, but instead increases the number of sites where synapses could be formed between synaptically connected neurons with minimal structural changes. This enables rapid and cost-efficient rewiring of local excitatory connections when re-exposed to similarly altered sensory experience in adulthood.

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.

Sensory experience alters cortical connectivity and synaptic function site specifically.

Neocortical circuitry can alter throughout life with experience. However, the contributions of changes in synaptic strength and modifications in neuronal wiring to experience-dependent plasticity in mature animals remain unclear. We trimmed whiskers of rats and made electrophysiological recordings after whisker cortical maps have developed. Measurements of miniature EPSPs suggested that synaptic inputs to layer 2/3 pyramidal neurons were altered at the junction of deprived and spared cortex in primary somatosensory cortex. Whole-cell recordings were made from pairs of synaptically connected pyramidal neurons to investigate possible changes in local excitatory connections between layer 2/3 pyramidal neurons. The neurons were filled with fluorescent dyes during recording and reconstructed in three dimensions using confocal microscopy and image deconvolution to identify putative synapses. We show that sensory deprivation induces a striking reduction in connectivity between layer 2/3 pyramidal neurons in deprived cortex without large-scale, compensatory increases in the strength of remaining local excitatory connections. A markedly different situation occurs in spared cortex. Connection strength is potentiated, but local excitatory connectivity and synapse number per connection are unchanged. Our data suggest that alterations in local excitatory circuitry enhance the expansion of spared representations into deprived cortex. Moreover, our findings offer one explanation for how the responses of spared and deprived cortex to sensory deprivation can be dissociated in developed animals.