In mammals, spatial learning and memory depend on neural processing carried out in the hippocampal formation. Interestingly, extracellular recordings from behaving animals have shown that cells in this region exhibit spatially modulated activity patterns, thus providing insights into the neural activity underlying spatial behaviour. One area within the hippocampal formation, layer II of the medial entorhinal cortex, houses cells that encode a grid-like map of space using a firing rate code. At the same time, oscillatory signals at distinct theta (4–12 Hz) and gamma (30–120 Hz) frequencies are also present in layer II, providing a substrate for a timing code. To understand how layer II of the medial entorhinal cortex produces these outputs I sought to characterise the electrical properties and functional computational architecture of its microcircuitry. The functionality of any neural circuit depends on the electrical properties of its constituent cells. Because the grid cells in layer II are likely to be stellate cells, I used the perforated patch-clamp technique to accurately assess the intrinsic excitable properties of this cell type. Compared to whole-cell recordings, these recordings indicate that some intrinsic properties of stellate cells, such as spike clustering, which is revealed to be robust, are more likely to play a functional role in circuit computation. Conversely, other intrinsic properties, such as spontaneous membrane potential fluctuations, which are confirmed to be insufficiently stable to support reliable interference patterns, are revealed to be less likely than other, more robust electrical properties to play a direct role in circuit function. The characteristic connectivity profiles of different cell types are also critical for circuit function. To investigate cell type-specific connectivity in layer II I used optogenetic stimulation in combination with in vitro electrophysiology to record synaptic activity in different cell types while selectively activating distinct subpopulations of cells with light. Using this method I found that connections between stellate cells are absent or very rare and that communication between stellate cells is instead mediated by strong feedback inhibition from fast-spiking interneurons. Dissecting oscillatory activity in neural circuits may be important for establishing functionally relevant circuit architecture and dynamics but is difficult in vivo. I accomplished this in vitro by recapitulating the interacting theta and gamma rhythms that are observed in vivo with an optogenetic method. I found that locally driving a subset of neurons in the layer II microcircuit at theta frequency with a light stimiulus produced a nested field rhythm at gamma frequency that was also evident as rhythmic inhibition onto stellate cells. Critically, these interacting rhythms closely resembled those recorded from behaving animals. In addition, I found that this thetanested gamma is sufficiently regular to act as a clock-like reference signal, indicating its potential role in implementing a timing code. To functionally dissect the circuit I performed multiple simultaneous whole-cell patch-clamp recordings during circuit activation. These recordings revealed how feedback interactions between stellate cells and fast-spiking interneurons underpin the theta-nested gamma rhythm. Together, these results suggest that feedback inhibition in layer II acts as a common substrate for theta-nested gamma oscillations and possibly also grid firing fields, thereby providing a framework for understanding how computations are carried out in layer II of the medial entorhinal cortex.

The pedunculopontine tegmental (PPT) nucleus is a brainstem structure thought to be important in the regulation of sleep/wake states. The PPT is comprised of three distinct types of neurons (cholinergic, GABAergic, and glutamatergic), and each may serve different functions. It remains unknown how PPT neurons affect specific sleep/wake states and which of their axonal projections mediate their effect. Therefore, we used optogenetics to selectively activate glutamatergic PPT (PPTglut) neurons at both the cell soma and axon terminals in a temporally and spatially precise manner. The purpose of these experiments was to determine the role of PPTglut neurons during wake, non-rapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep, and to identify the key projections through which PPTglut neurons produce their effects. Using transgenic mice, we transfected PPTglut neurons with an adeno-associated viral vector to induce expression of a light-dependent ion channel, channelrhodopsin-2 (ChR2). While recording electroencephalography (EEG), electromyography (EMG), and video feed, we photostimulated the transfected PPTglut neurons and measured the effects on sleep/wake states of the mice. Stimulation of the PPTglut soma during NREM sleep produced a frequency-dependent wake response. With increasing frequency of stimulation, we observed an increase in the speed of the wake response, as well as the amplitude and duration of the wake response. Stimulating the PPTglut soma increased time spent in wake, decreased NREM sleep, and slightly decreased REM sleep. We also noticed that mice did not exhibit spontaneous body movements during stimulation of the PPTglut soma. Stimulating individual PPTglut axon terminal fields partially recapitulated the phenotype observed with stimulation of the cell soma. Photostimulation of axon terminals in the basal forebrain, lateral hypothalamus, and thalamus elicited a fast wake response, stimulation of both the basal forebrain and lateral hypothalamus terminal fields produced a strong wake response, but long-lasting wakefulness was observed only with high-frequency stimulation of axon terminals in the lateral hypothalamus. In summary, photostimulation of PPTglut neurons promotes wake, and slightly decreases REM sleep. Our experiments strongly support the role of PPTglut neurons in promoting wakefulness from NREM sleep, and this wake response is carried out through several axonal projections which, in sum, recapitulate the wake phenotype observed with stimulation of the cell soma in the PPT itself. Further exploration of the axonal projections of PPTglut neurons is warranted to elucidate the neuronal targets through which this response is carried out. / 2019-07-03T00:00:00Z

The remapping of cortical networks after stroke is hypothesized to be one of the mechanisms subserving functional recovery. Our understanding of cortical remapping remains limited due to the inability to resolve which cells are active while performing motor tasks with high temporal and spatial specificity. The experiments presented in the first chapter of this thesis evaluate the ability of the inducible Arc-CreERT2:Rosa-YFPf/f model to label cells in the motor cortex activated by a motor-related behaviour. Through the modification of previously published 4-hydroxytamoxifen treatment paradigms, this model can differentiate between animals that performed the rotarod task at two time points and home cage controls. In addition, 65% of cells active at the first behavioural time point are reactivated. Taken together, these data suggest that the Arc-CreERT2:Rosa-YFPf/f model is able to reliably label networks used to perform the same behavioural task at two time points. The second chapter of this thesis details a pilot study in which the Arc-CreERT2:Rosa-ChR2:YFPf/f model was used to test the effect of daily optogenetic stimulation of the contralateral cortex on functional recovery. The results of this chapter suggest that stimulating the contralesional motor cortex may impair functional recovery. Overall, the results of this thesis lay the foundation to use this model to investigate motor networks in both naïve and pathological conditions, such as stroke.

No description available.

Perinatal stroke, which occurs before or shortly after birth, may result in both beneficial and maladaptive plasticity in surviving tissue. However, current preclinical and clinical work have an unclear understanding on the relationship between functional outcome and neurophysiology. This thesis aims to dually characterize and correlate behaviour with cortical motor representations in a mouse model of perinatal stroke. On postnatal day 7, a unilateral photothrombotic stroke was produced in the primary motor cortex of Thy1-ChR2 mice. Sensorimotor function was evaluated in adulthood with a battery of behavioural tests. Subsequently, a transcranial window was implanted, and motor maps were created through optogenetic point stimulation. To evaluate the impact of skilled motor training on cortical reorganization, mapping was conducted before and after training on the single pellet reaching task. P7 stroke caused functional impairments across a battery of motor tasks, while both motor map size and movement latency were bilaterally impacted. Spontaneous limb use was positively correlated with map size of both hemispheres, but single pellet performance was only positively correlated with map size in the injured hemisphere. Following skilled motor training, both map size reductions and delayed latency was partially restored. Additionally, significant correlations between map size expansion and movement latency reduction following skilled motor training not only demonstrate that training-induced plasticity was beneficial, but also primarily mediated by the uninjured hemisphere. As the first study to conduct within-animal optogenetic motor mapping following perinatal stroke, we show that 1) perinatal stroke bilaterally impacts both cortical and descending aspects of the motor system, 2) the remaining movement sites in both the uninjured and injured hemispheres have a positive impact on functional outcome, and 3) skilled forelimb training can partially restore cortical and descending motor neurophysiology.

The striatopallidal (indirect) pathway is considered as the main modulatory locus for the basal ganglia control of motor functions. According to the classic basal ganglia model, the striatopallidal pathway inhibits motor activity mainly via its projection to globus pallidus (GPe). However, striatopallidal medium spiny neurons (MSNs) form extensive feedback and lateral inhibitory networks via their collaterals. Thus, the striatopallidal pathway may control motor activity either through its projections onto GPe or through the striatal collaterals. To further define the circuit mechanism whereby the striatopallidal pathway controls motor activity, we have developed two new optogenetic transgenic mouse lines expressing channelrhodospin-2 (ChR2) or archaerhodopsin-3 (Arch) selectively in the striatopallidal neurons under the Adora2a gene promoter. Consistent with previous optogenetic studies, we found that ChR2 activation and Arch silencing of the striatopallidal neurons in dorsolateral striatum (DLS) suppressed and increased motor activity, respectively. However, contrary to the prediction from the classical model, we found that selective activation of the striatopallidal axon projections in GPe increased locomotor activity. Thus, light stimulation of MSN cell bodies and collaterals in DLS, versus stimulation in GPe axon projections, produced opposite motor responses. This led us to reassess the function of the striatopallidal collaterals and to test the hypothesis that the profuse projections and collaterization within the striatum may contribute to striatopallidal pathway control of motor activity. We found that ChR2-mediated activation of the striatopallidal neurons in DLS induced c-Fos expression in ChR2/GFP-positive MSNs. Conversely, Arch-mediated silencing of the striatopallidal neurons induced c-Fos expression and MAPK phosphorylation in Arch/GFP-negative MSNs surrounding the Arch/GFP-positive MSNs. This c-Fos/pMAPK expression pattern in MSNs is consistent with the suppression of GABA release in GFP-positive cells, resulting in the induction of c-Fos in GFP-negative cells having collateral connections with the GFP-positive cells. Together, our findings revealed a previously unrecognized complexity and novel motor control mechanism of the striatopallidal pathway: activation of striatopallidal projections to GPe increases motor activity while activation of striatopallidal neurons and collaterals in the DLS may contribute to motor suppression. These findings call for a revisit of GPe as a potential locus for deep brain stimulation in Parkinson’s disease.

Large numbers of people suffer from nervous system injuries and neurodegenerative diseases each year, with little success in regaining lost neural functions. Attempts to successfully regenerate nervous tissue in the mammalian Central Nervous System have meet with limited success. Simpler models have thus been useful in determining conserved mechanisms in the enhancement of neural regeneration. One such mechanism is intracellular calcium signaling. We used <italic>Caenorhabditis elegans</italic> as a model system to study the effects of optogenetic stimulation on regeneration. Using a femtosecond laser we cut individual <italic>C. elegans</italic> axons <italic>in vivo</italic> and then periodically stimulated the neurons by activating the genetically encoded light activated channel, Channelrodopsin-2. We found that periodic photo-activation could increase regeneration over 24h by at least 31%. We repeated these experiments with dantrolene treatment and in <italic>unc-68(e540)</italic> mutants to assess the effects from a lack of internal calcium ion signaling in these worms. In both cases, we found a complete suppression of stimulated regeneration when calcium signaling was blocked. This indicates that intracellular calcium ion signaling is crucial in the initiation of neural regeneration in the first 24 hours and mediates the enhanced outgrowth we observe with periodic photo-activation. The importance of intracellular calcium ion signaling can lead to further studies to enhance the stimulation of neural regeneration, and improve therapies for patients with neural damage and loss of neural functions.

No description available.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第24452号 / 理博第4951号 / 新制||理||1707(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 高橋 淑子, 准教授 佐藤 ゆたか, 教授 中務 真人 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

No description available.