Reading out a spatiotemporal population code by imaging neighbouring parallel fibre axons in vivo.

The spatiotemporal pattern of synaptic inputs to the dendritic tree is crucial for synaptic integration and plasticity. However, it is not known if input patterns driven by sensory stimuli are structured or random. Here we investigate the spatial patterning of synaptic inputs by directly monitoring presynaptic activity in the intact mouse brain on the micron scale. Using in vivo calcium imaging of multiple neighbouring cerebellar parallel fibre axons, we find evidence for clustered patterns of axonal activity during sensory processing. The clustered parallel fibre input we observe is ideally suited for driving dendritic spikes, postsynaptic calcium signalling, and synaptic plasticity in downstream Purkinje cells, and is thus likely to be a major feature of cerebellar function during sensory processing

VOLTAGE-SENSITIVE DYE IMAGING OF RAT PIRIFORM CORTEX BEFORE AND AFTER KINDLING

To determine the role of inhibitory cells in the propagation of activity in the rat piriform cortex (PC) before and after kindling, we used voltage-sensitive dye imaging technique to follow the membrane potential changes in three layers of the PC after stimulating the lateral olfactory tract (LOT) with beta and gamma frequencies. Stimulation of LOT was followed by propagation of excitatory (in layer II) and inhibitory responses (in layer III) through the PC. Decreasing the inhibition by applying gabazine, a GABAa- receptor antagonist, decreased the inhibitory responses and increased the excitatory responses in the control rats; however, it did not affect the excitatory and inhibitory responses in the kindled rats. Furthermore, cutting the slice below the layer II decreased the both responses. Thus, we concluded that disinhibition of layer III intemeurons is necessary for principle cells firing and kindling can result in seizures by increasing disinhibition in layer III of PC

Propofol suppresses synaptic responsiveness of somatosensory relay neurons to excitatory input by potentiating GABA(A )receptor chloride channels

Propofol is a widely used intravenous general anesthetic. Propofol-induced unconsciousness in humans is associated with inhibition of thalamic activity evoked by somatosensory stimuli. However, the cellular mechanisms underlying the effects of propofol in thalamic circuits are largely unknown. We investigated the influence of propofol on synaptic responsiveness of thalamocortical relay neurons in the ventrobasal complex (VB) to excitatory input in mouse brain slices, using both current- and voltage-clamp recording techniques. Excitatory responses including EPSP temporal summation and action potential firing were evoked in VB neurons by electrical stimulation of corticothalamic fibers or pharmacological activation of glutamate receptors. Propofol (0.6 – 3 μM) suppressed temporal summation and spike firing in a concentration-dependent manner. The thalamocortical suppression was accompanied by a marked decrease in both EPSP amplitude and input resistance, indicating that a shunting mechanism was involved. The propofol-mediated thalamocortical suppression could be blocked by a GABA(A )receptor antagonist or chloride channel blocker, suggesting that postsynaptic GABA(A )receptors in VB neurons were involved in the shunting inhibition. GABA(A )receptor-mediated inhibitory postsynaptic currents (IPSCs) were evoked in VB neurons by electrical stimulation of the reticular thalamic nucleus. Propofol markedly increased amplitude, decay time, and charge transfer of GABA(A )IPSCs. The results demonstrated that shunting inhibition of thalamic somatosensory relay neurons by propofol at clinically relevant concentrations is primarily mediated through the potentiation of the GABA(A )receptor chloride channel-mediated conductance, and such inhibition may contribute to the impaired thalamic responses to sensory stimuli seen during propofol-induced anesthesia

Characterization of the neocortical network excitability with multielectrode arrays: alterations in migraine and epilepsy mouse models.

missin

Cellular properties of the medial entorhinal cortex as possible mechanisms of spatial processing

Cells of the rodent medial entorhinal cortex (EC) possess cellular properties hypothesized to underlie the spatially periodic firing behaviors of 'grid cells' (GC) observed in vivo. Computational models have simulated experimental GC data, but a consensus as to what mechanism(s) generate GC properties has not been reached. Using whole cell patch-clamp and computational modeling techniques this thesis investigates resonance, rebound spiking and persistent spiking properties of medial EC cells to test potential mechanisms generating GC firing. The first experiment tested the voltage dependence of resonance frequency in layer II medial EC stellate cells. Some GC models use interference between velocity-controlled oscillators to generate GCs. These interference mechanisms work best with a linear relationship between voltage and resonance frequency. Experimental results showed resonance frequency decreased linearly with membrane potential depolarization, suggesting resonance properties could support the generation of GCs. Resonance appeared in medial EC but not lateral EC consistent with location of GCs. The second experiment tested predictions of a recent network model that generates GCs using medial EC stellate cell resonance and rebound spiking properties. Sinusoidal oscillations superimposed with hyperpolarizing currents were delivered to layer II stellate cells. Results showed that relative to the sinusoid, a limited phase range of hyperpolarizing inputs elicited rebound spikes, and the phase range of rebound spikes was even narrower. Tuning model parameters of the stellate cell population to match experimental rebound spiking properties resulted in GC spatial periodicity, suggesting resonance and rebound spiking are viable mechanisms for GC generation. The third experiment tested whether short duration current inputs can induce persistent firing and afterdepolarization in layer V pyramidal cells. During muscarinic acetylcholine receptor activation 1-2 second long current injections have been shown to induce persistent firing in EC principal cells. Persistent firing may underlie working memory performance and has been used to model GCs. However, input stimuli during working memory and navigation may be much shorter than 1-2 seconds. Data showed that input durations of 10, 50 and 100 ms could elicit persistent firing, and revealed time courses and amplitude of afterdepolarization that could contribute to GC firing or maintenance of working memory

An Introduction to In Vitro Slice Approaches for the Study of Neuronal Circuitry

Abstract The acute slice preparation can be a powerful tool to study brain networks that would otherwise be difficult to manipulate at the synaptic and cellular levels. In the first part of this chapter, we discuss the specific challenges of preparing brain slices to study neural networks, and we review solutions to overcome problems that can be faced during slice preparation and maintenance. In addition, we describe slice preparations that preserve the connectivity between multiple brain areas, such as hippocampal and thalamocortical slices. In the second part, we introduce several techniques that can be used to stimulate specific cells or networks in acute slices. We begin by reviewing methods for electrical stimulation, glutamate-based stimulation, and optogenetic stimulation. An additional procedure that combines the use of laser photostimulation with flavoprotein autofluorescence is also presented. We discuss advantages and disadvantages of these methods for neural network investigation in the acute slice preparation

An absence of dystrophin in cerebellar Purkinje cells impairs inhibitory synaptic function in mature dystrophic mice

Duchenne muscular dystrophy (DMD) is a rapidly progressive X-linked recessive disease affecting about 1 in 3500 live male births. It is caused by mutations in the dystrophin gene, which result in the loss of dystrophin or expression of a non-functional truncated protein product. Full-length dystrophin is mainly expressed in muscles and the central nervous system. In addition to the degeneration of skeletal musculature, about one-third of patients with DMD display various degrees of intellectual impairment, commonly found with intelligence quotient (IQ) scores of one standard deviation below (IQ of 85) the normal population mean (IQ of 100). However, the mechanism underlying the cognitive deficits in DMD remains unclear and no effective treatment is available to reverse this condition in the affected individual. Recent studies showed that the life span of DMD patients today has increased from teens to their fourth decades. With longer survival, the quality of life becomes increasing important. Therefore, research on the cognitive aspect of DMD is as important as research on the muscular aspects because improvements in cognitive function will enhance the quality of life for the growing population of adult DMD patients. The aim of this thesis was to investigate the role of dystrophin in the central nervous system of the mdx mouse, a widely accepted murine model for DMD. This study employed the use of animal with different age groups, corresponding to young (3-4 months), adult (11-12 months), and aged (23-26 months). Adult and aged mdx mice are the focus in this study with findings from the older mouse especially valuable as, disease progression in aged mice closely resembling that of DMD. As numerous evidence has shown a high similarity between the specific cognitive dysfunctions seen in DMD (i.e. impaired verbal intelligence) and in patients with cerebellar lesions (i.e. language disorders), this study examined the function of cerebellar Purkinje cells in mdx mice using electrophysiological recording and calcium imaging. Overall, the data presented in this thesis provides new insights into the role of dystrophin in cerebellar Purkinje neurons. The findings suggest that dystrophin is important for normal inhibitory synaptic function, intrinsic electrophysiological properties, and calcium handling of the mature cerebellar Purkinje cells. The consequences of the absence of dystrophin including the altered GABAA receptor clustering and reduced peak amplitude of mIPSCs could be ameliorated when dystrophin was successfully rescued with Pip6f-PMO in an organotypic mdx cerebellar culture. If mdx mice and DMD patients share similar neuropathogenesis, the development of drugs targeting the altered functions in mdx Purkinje cells may serve as a potential therapy in alleviating the cognitive impairments seen in DMD

Characterization of the neocortical network excitability with multielectrode arrays: alterations in migraine and epilepsy mouse models.

missin

INTRINSIC AND SYNAPTIC MECHANISMS CONTROLLING THE EXCITABILITY OF LAYER 5 CORTICOCALLOSAL AND CORTICOCOLLICULAR NEURONS IN AUDITORY CORTEX

Auditory cortex (AC) layer (L) 5B contains both corticocollicular neurons, a type of pyramidal-tract neuron projecting to the inferior colliculus, and corticocallosal neurons, a type of intratelencephalic neuron projecting to contralateral AC. It is known that these neuronal types display dichotomous in vivo responses to sound. While corticocollicular neurons display robust evoked responses to wide range of sound frequencies, corticocallosal neurons are responsive to a limited range of sound frequencies. However, the intrinsic and synaptic mechanisms shaping these dichotomous responses remain unexplored. It is also known that corticocollicular neurons are critical for learning-induced plasticity involved in relearning sound localization after monaural occlusion. This learning induced-plasticity also requires the release of acetylcholine (ACh) in the AC. However, the effect of ACh release on the excitability of corticocollicular neurons is unknown. Therefore, we recorded in brain slices of mouse AC from retrogradely labeled corticocollicular and neighboring corticocallosal neurons in L5B to identify the intrinsic and synaptic mechanisms that contribute to the in vivo responses of these neurons to sound, and to examine the effect of ACh release on corticocollicular and corticocallosal neurons to identify cell-specific mechanisms that enable corticocollicular neurons to participate in relearning sound localization. In comparison to corticocallosal neurons, corticocollicular neurons display a more depolarized resting membrane potential, faster action potentials and less spike frequency adaptation. In paired recordings between single L2/3 and labeled L5B neurons, trains of EPSCs showed no synaptic depression in L2/3→corticocollicular connections, but substantial depression in L2/3→corticocallosal connections. We propose that these differences in intrinsic and synaptic properties contribute to the dichotomous in vivo responses of corticocallosal and corticocollicular neurons to sound. Additionally, ACh release generates nicotinic acetylcholine receptor (nAChR)-mediated depolarizing potentials in both corticocallosal and corticocollicular neurons, but muscarinic acetylcholine receptor (mAChR)-mediated hyperpolarizing potentials in corticocallosal neurons and mAChR-mediated prolonged depolarizing potentials in corticocollicular neurons. This prolonged mAChR-mediated depolarizing potential leads to persistent firing in corticocollicular neurons, whereas corticocallosal neurons lacking this prolonged mAChR-mediated depolarizing potential do not fire persistently. We propose that this mAChR-mediated persistent firing in corticocollicular neurons may be a mechanism required for relearning sound localization after monaural occlusion