Monosynaptic Functional Connectivity in Cerebral Cortex During Wakefulness and Under Graded Levels of Anesthesia

The balance between excitation and inhibition is considered to be of significant importance for neural computation and cognitive function. Excitatory and inhibitory functional connectivity in intact cortical neuronal networks in wakefulness and graded levels of anesthesia has not been systematically investigated. We compared monosynaptic excitatory and inhibitory spike transmission probabilities using pairwise cross-correlogram (CCG) analysis. Spikes were measured at 64 sites in the visual cortex of rats with chronically implanted microelectrode arrays during wakefulness and three levels of anesthesia produced by desflurane. Anesthesia decreased the number of active units, the number of functional connections, and the strength of excitatory connections. Connection probability (number of connections per number of active unit pairs) was unaffected until the deepest anesthesia level, at which a significant increase in the excitatory to inhibitory ratio of connection probabilities was observed. The results suggest that the excitatory–inhibitory balance is altered at an anesthetic depth associated with unconsciousness

Fluorescence Imaging of Cortical Calcium Dynamics: A Tool for Visualizing Mouse Brain Functions, Connections, and Networks

Hemodynamic-based markers of cortical activity (e.g. functional magnetic resonance imaging (fMRI) and optical intrinsic signal imaging) are an indirect and relatively slow report of neural activity driven by electrical and metabolic activity through neurovascular coupling, which presents significant limiting factors in deducing underlying brain network dynamics. As application of resting state functional connectivity (FC) measures is extended further into topics such as brain development, aging, and disease, the importance of understanding the fundamental basis for FC will grow. In this dissertation, we extend functional analysis from hemodynamic- to calcium-based imaging. Transgenic mice expressing a fluorescent calcium indicator (GCaMP6) driven by the Thy1 promoter in glutamatergic neurons were imaged transcranially in both anesthetized (using ketaminze/xylazine) and awake states. Sequential LED illumination (λ=470, 530, 590, 625nm) enabled concurrent imaging of both GCaMP6 fluorescence emission (corrected for hemoglobin absorption) and hemodynamics. EEG measurements of the global cortical field potential were also simultaneously acquired. First, we validated the ability of our system to capture GCaMP6 fluorescence emission and hemodynamics by implementing an electrical somatosensory stimulation paradigm. The neural origins of the GCaMP6 fluorescent signal were further confirmed by histology and by comparing the spectral content of imaged GCaMP6 activity to concurrently-acquired EEG. We then constructed seed-based FC and coherence network maps for low (0.009-0.08Hz) and high, delta-band (0.4-4.0Hz) frequency bands using GCaMP6 and hemodynamic contrasts. Homotopic GCaMP6 FC maps using delta-band data in the anesthetized states show a striking correlated and anti-correlated structure along the anterior to posterior axis. We next used whole-brain delay analysis to characterize this correlative feature. This structure is potentially explained by the observed propagation of delta-band activity from frontal somatomotor regions to visuoparietal areas, likely corresponding to propagating delta waves associated with slow wave sleep. During wakefulness, this spatio-temporal structure is largely absent, and a more complex and detailed FC structure is observed. Collectively, functional neuroimaging of calcium dynamics in mice provides evidence that spatiotemporal coherence in cortical activity is not exclusive to hemodynamics and exists over a larger range of frequencies than hemoglobin-based contrasts. Concurrent calcium and hemodynamic imaging enables direct temporal and functional comparison of spontaneous calcium and hemoglobin activity, effectively spanning neurovascular coupling and functional hyperemia. The combined calcium/hemoglobin imaging technique described here will enable the dissociation of changes in ionic and hemodynamic functional structure and provide a framework for subsequent studies of sleep disorders and neurological disease

Functional integration in the cortical neuronal network of conscious and anesthetized animals

General anesthesia consists of amnesia, analgesia, areflexia and unconsciousness. How anesthetics suppress consciousness has been a mystery for more than one and a half centuries. The overall goal of my research has been to determine the neural correlates of anesthetic-induced loss of consciousness. I hypothesized that anesthetics induce unconsciousness by interfering with the functional connectivity of neuronal networks of the brain and consequently, reducing the brain\u27s capacity for information processing. To test this hypothesis, I performed experiments in which neuronal spiking activity was measured with chronically implanted microelectrode arrays in the visual cortex of freely-moving rats during wakefulness and at graded levels of anesthesia produced by the inhalational anesthetic agent desflurane. I then applied linear and non-parametric information-theoretic analyses to quantify the concentration-dependent effect of general anesthetics on spontaneous and visually evoked spike firing activity in rat primary visual cortex. Results suggest that desflurane anesthesia disrupts cortical neuronal integration as measured by monosynaptic connectivity, spike burst coherence and information capacity. This research furthers our understanding of the mechanisms involved with the anesthetic-induced LOC which may facilitate in the development of better anesthetic monitoring devices and the creation of effective anesthetic agents that will be free of unwanted side effects

A model of delta frequency neuronal network activity and theta-gamma interactions in rat sensorimotor cortex in vitro

In recent decades, advances in electrophysiological techniques have enabled understanding of neuronal network activity, with in vitro brain slices providing insights into the mechanisms underlying oscillations at various frequency ranges. Understanding the electrical and neuro-pharmacological properties of brain networks using selective receptor modulators in native tissue allows to compare such properties with those in disease models (e.g. epilepsy and Parkinson’s). In vivo and in vitro studies have implicated M1 in execution of voluntary movements and, from both local network in vitro and whole brain in vivo perspectives. M1 has been shown to generate oscillatory activity at various frequencies, including beta frequency and nested theta and gamma oscillations similar to those of rat hippocampus. In vivo studies also confirmed slow wave oscillations in somatosensory cortex including delta and theta band activity. However, despite these findings, non-thalamic mechanisms underlying cortical delta oscillations remain almost unexplored. Therefore, we determined to explore these oscillations in vitro in M1 and S1. Using a modified sagittal plane slice preparation with aCSF containing neuroprotectants, we have greatly improved brain slice viability, enabling the generation and study of dual rhythms (theta and gamma oscillations) in deep layers (LV) of the in vitro sensorimotor slice (M1 and S1) in the presence of KA and CCh. We found that theta-gamma activity in M1 is led by S1 and that the amplitude of gamma oscillations was (phase-amplitude) coupled to theta phase in both regions. Oscillations were dependent on GABAAR, AMPAR and NMDAR and were augmented by DAR activation. Experiments using cut/reduced slices showed both M1 and S1 could be intrinsic generators of oscillatory activity. Delta oscillations were induced in M1 and S1 by maintaining a neuromodulatory state mimicking deep sleep, characterised by low dopaminergic and low cholinergic tone, achieved using DAR blockade and low CCh. Delta activity depends on GABAAR, GABABR and AMPAR but not NMDAR, and once induced was not reversible. Unlike theta-gamma activity, delta was led by M1, and activity took >20mins to develop in S1 after establishement of peak power in M1. Unlike M1, S1 alone was unable to support delta activity. Dopamine modulates network activity in M1 and it is known that fast-spiking interneurons are the pacemakers of network rhythmogenesis. Recent studies reported that dopamine (DA) controled Itonic in medium spiny, ventrobasal thalamus and nucleus accumbens neurons by modulation of GABARs or cation channels. In the current study, voltage-clamp whole cell recordings were performed in fast spiking interneurons (FS cells) in Layer V of M1. These recordings revealed tonic and phasic GABAAR inhibition and when DA was bath applied, a slow inward current (IDA) was induced. IDA was mediated by non-specific cationic TRPC channels following D2R-like receptor activation. Overall, my studies show the strong interdependence of theta-gamma rhythmogenesis between M1 and S1, dominanace of M1 at delta frequency and the crucial role of dopamine in controlling FS cell activity. Further exploration of these rhythms in models of pathological conditions such as Parkinson`s disease and Epilepsy may provide insights into network changes underlying these disease conditions

THE THALAMIC RETICULAR NUCLEUS: A MULTIFACETED GUARDIAN

Interactions between the cortex and the thalamus are essential for major brain functions such as sensory information processing and integration, sleep and wake regulation and cognitive processes. The thalamic reticular nucleus (TRN) is strategically positioned within the thalamocortical circuit and has a strong inhibitory control over the thalamus. It can act on a global scale, such as suppressing the flow of sensory information from the thalamus to the cortex during sleep. The TRN also acts locally on the activity of single cells or small cell groups. To reconcile both of these global and local aspects of TRN functions, we studied the cellular, synaptic and functional heterogeneity of the TRN, with a focus on the comparison between the classical sensory TRN and the less well-described limbic TRN. In study 1, using anatomical tracing and cellular electrophysiology, we identified the dorsal presubiculum (dPreS), the retrosplenial cortex (RSC) and the anterior thalamic nuclei (ATN) as part of a novel thalamo-cortical circuit involving the limbic TRN in mice. The dPreS, RSC and ATN are three key structures for spatial navigation. dPreS/RSC excitatory glutamatergic synapses formed on TRN and ATN are part of a feedforward circuit through which TRN-mediated inhibition generates large burst-mediated inhibitory synaptic currents. The PreS/RSC afferents to the TRN showed driver-like characteristics, which is unprecedented for corticoreticular synapses and expands the scope of the TRN heterogeneity to the nature of its synaptic afferents. We further investigated the role of the limbic TRN in the control of head-direction neurons that were previously described to be located in the anterodorsal thalamus. The width of the tuning curve of head-direction neurons in the thalamus was broadened upon chemogenetic silencing of the TRN, revealing a novel form of internal sensory gating by the TRN. About half of the head-direction neurons showed action potential discharge patterns consistent with feedforward inhibitory responses upon light activation of dPreS/RSC. These data suggest that the limbic TRN sharpens the tuning of thalamic head-direction neurons under dPreS/RSC control. Finally, we investigated the potential function of the limbic TRN in the hidden version of the Morris watermaze. We discovered that chemogenetic silencing of the limbic TRN biased the search patterns towards allocentric strategies and generated perseverance to previously learned escape positions, suggesting an impairment of the egocentric system in which the head-direction system plays a critical role. In study 2, we combined opto-tagging of TRN sectors with in vitro electrophysiological recordings and discovered that the limbic TRN neurons produced less repetitive burst firing than their sensory counterpart. The burst discharge of sensory TRN neurons is known to generate sleep spindles that propagate to the cortex, that are a marker of sleep quality and that correlate with memory consolidation. Consistently, local field potential recordings in the prefrontal cortex that is related to the less bursty limbic TRN revealed smaller amplitude and slower sleep spindles compared to sensory ones, making the heterogeneity of the TRN a critical player in local sleep rhythms. This thesis summarizes elements supporting the heterogeneity of the TRN, in particular between the sensory and the limbic TRN. It also provides a novel function for the limbic TRN in the spatial navigation system