Spinal cord injury (SCI) causes life-long neurological impairment, with loss of sensory and motor function distal to the point of injury. There are approximately 300,000 patients living with SCI in the United States, and currently no effective treatment, reducing their quality of life. Amongst other things, proprioception, which has been determined essential for normal locomotion, can be lost with SCI. Epidural Electric Stimulation (EES), that is thought to excite large diameter afferent fibers (LDAF), has been found to improve recovery from spinal cord injury in conjunction with movement rehabilitation in animal models and humans. This represents an exciting new approach to help these patients. However, many open questions remain about how and why EES works. Chief among them are 1) which of the afferent fibers are necessary and sufficient to promote better recovery, and 2) what are the mechanisms of plasticity in the spinal cord that underly improvement. Here, we sought to address the first question by using viral and genetic tools to begin to target specific subsets of LDAF. First, we use a viral vector that preferably transduces only in the large diameter afferent fibers (LDAF) in the Dorsal Root Ganglia (DRG), and then specifically only the proprioceptors within the LDAF, by using a transgenic rat line that expresses Cre recombinase in Parvalbumin, a marker for proprioceptive neurons in the DRG. This approach consists of using the chemogenetic modulator of neuronal activity Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), which are activated by a putatively inert drug, clozapine-N-oxide (CNO), that crosses the blood brain barrier. While we were able to specifically target LDAF with excitatory DREADDs in L3-L5 DRGs in wild type rats, we were unsuccessful at specifically targeting proprioceptors by using the Pvalb-iCre rat line. Additionally, we studied the effect of exciting LDAF on rats with a 200KDyn SCI. CNO withdrawal on the week 7 stage of the recovery was associated with worse ladder performance than the previous and following weeks, as well as worse kinematic behavior of the same week on lower speeds in ankle movement. These results suggest that DREADDs activation is necessary for changes in movement at longer times post injury. It does not rule out that plasticity in neural circuitry has occurred but suggests that plasticity may rely on afferent activation. Finally, we sought to develop new methods to overcome skin motion artifact in rat kinematics by tattooing the knee area under the skin and recording infrared high-speed videos of moving rats which would correct joint calculations beyond just triangulation methods, as well as a novel MATLAB application that can accurately and reliably perform automated H-Reflex measurements, test the stimulating electrodes, and carry out typical instantaneous analyses, which in return allows for faster data collection with reduced human error, and subsequently result in higher research quality. / Bioengineering

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23923号 / 工博第5010号 / 新制||工||1782(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 浜地 格, 教授 森 泰生, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

<div><div><div><p>Increased meal size on a western diet is a major contributor to development and maintenance of obesity. This also leads to decreased sensitivity to the satiating effects of the western diet. Excitation of cells during consumption of a meal in the caudal two-thirds of the nucleus solitary tract (cNTS) in the brainstem are thought to produce satiation and inhibit feeding. Currently, it is unknown how excitation of these cells inhibits feeding. A major obstacle has been the inability to selectively manipulate these cells without affecting intermixed cells that mediate other autonomic functions. We propose a novel approach using inducible, activity-dependent chemogenetics or optogenetics to test whether artificial excitation of cells in the caudal two-thirds of the nucleus solitary tract (cNTS) activated during satiation can reduce food intake and could contribute to preventing or reversing obesity in humans.</p><p>We tested four different mouse models with potential for answering this question: double transgenic mice with cFos-tTA & Tet-O-hM3Dq genes, a single transgenic cFos-tTA mouse with a virally delivered hM3Dq gene injected into the cNTS, a double transgenic mice with the TRAP2- tdTomato genes and double transgenic mice with c-Fos-tTA and ChEF genes. Evidence suggested that clozapine-N-oxide might activate satiation-related cells in the absence of the hM3Dq receptor and this should be taken into consideration for future experiments. All four models had promising aspects for studying feeding as well as serious limitations. These limitations will need to be considered when deciding to use any of these models to study any feeding behaviors, especially satiation.</p></div></div></div>

Spinal cord injury (SCI) results in persistent neurological deficits and significant long-term disability. Stimulation of peripheral afferents by epidural electrical stimulation (EES) has been reported to reduce spasticity by reorganizing spared and disrupted descending pathways and local circuits. However, a current barrier to the field is that the plasticity mechanisms that underly improved recovery is unknown. Using the power of hM3Dq Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), we aim to accelerate the dissection of the mechanisms underlying enhanced recovery. In these studies, we identified the effect of clozapine-N-oxide (CNO) on the H-reflex of naïve animals; investigated the baseline influence of hM3Dq DREADDs in peripheral afferents in the intact animal using a novel behavioral tool, an addition of angled rungs to the horizontal ladder walking task; and began to uncover the neural and behavioral changes that accompany hM3Dq DREADDs activation in peripheral afferents after SCI. We observed no significant differences in the H-reflex with 4 mg/kg dosage of CNO administration (pre-CNO vs. CNO-active: p=0.82; CNO-active vs. CNO wash-out: p=0.98; n=6). On our novel ladder, we found significant differences in correct hind paw placement (p=0.0002, n=7) and incorrect placement (p=0.01) when DREADDs were activated with CNO (4 mg/kg). In our SCI study, we report that acute and chronic DREADDs activation may activate extensor muscles about the hip (32 cm/s: p=0.047; controls: n=6; DREADDs: n=8 and hereafter unless otherwise stated) as well as induce sprouting and synaptogenesis within motor pools and Clarke’s column in the lumbar spinal cord (motor pool: p=0.00053; Clarke’s column: p=0.021; controls: n=4; DREADDs: n=6). This muscle recruitment may have long-term effects such as increased hindquarter heights (e.g., 16 cm/s: p=0.017) and more frequent hindlimb coordination (p=0.002). Results from this study suggest hM3Dq DREADDs may have the potential to recapitulate EES-activation of afferents as well as provide a platform with which to functionally map changes that occur both within targeted afferents and second order neurons they effect. Future work, such as using C-Fos to examine and map changes in interneuronal networks, could seek to more directly tie changes in kinematics to observed changes in plasticity. / Bioengineering

No description available.

Decision-making is necessary to adapt to the variable environment in everyday life. During this process, our goal is to select the most beneficial course of action in order to obtain the best outcome, to develop efficient choice strategies. That is, estimating the probability to obtain any of the available outcomes as well as their value. Moreover, poor decision-making ability is a common symptom to several psychiatric disorders, such as pathological gambling, depression, schizophrenia and bipolar disorder.The cognitive and emotional mechanisms controlling decision-making processes depend, among others, on the striatum, Basal Ganglia’s main input nucleus. The striatum is divided into the dorsal striatum, responsible for motor and cognitive control that initiate actions (Dorsomedial Striatum, DMS) and generate habits (Dorsolateral Striatum, DLS), and Nucleus Accumbens (NAc) which manages reward and the influence of motivation on motor behavior. A2A-expressing and D1-expressing medium spiny neurons (iMSNs and dMSNs, respectively), accounting for 95% of striatal neurons act in coordination to generate adaptive behavioral responses. It has been shown that imbalanced activity between these two populations leads to abnormal behaviors: overactivation of striatonigral neurons promotes an increased locomotion as well as a higher sensitivity for reward, whereas overactivation of striatopallidal neurons produces the exact opposite effects. However, the specific contributions to decision-making of these two populations in each striatal territory remains unclear. Here, we made use of a chemogenetic (DREADD) tool to manipulate striatal projection neurons’ activity within each specific striatal area and tested their role in a decision-making operant protocol. To do so, we used two different mouse models that allowed us to target specifically iMSNs (A2A-Cre mice) or dMSNs (D1-Cre mice) and induce neuronal-specific expression of the hM3Dq DREADD receptor. CNO-mediated activation of these receptors led to neuronal activation. Then, we tested DREADD-dependent activation of MSNs during the Iowa Gambling Task (IGT), a test used to assess the influence of different rewards on choice and to evaluate the ability of mice to develop advantageous choice strategies. We found an exclusive role of DMS’ dMSNs in controlling choice preference, as DREADD-induced activation of these neurons produced a loss of preference. Manipulations of MSNs in other striatal areas led to altered task performance without affecting choice preference.These results contribute to a better understanding of the role of the striatum on decision-making and moreover, suggest the existence of a high level of functional specialization in this area, a fact that could be explained by the local circuits in which each MSN population is involved. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Aging and Alzheimer’s disease (AD), are associated with disabling sleep and cognitive deficits. Specifically, aging and Alzheimer’s disease is associated with reduced quantity and quality of the deepest stage of sleep, called slow-wave-sleep (SWS). Interestingly, SWS has been implicated in hippocampus-dependent memory in mice. More importantly, sleep deprivation, aging, and AD are all associated with deficits in memory. Therefore, I hypothesize that, in aging and AD, the sleep deficits are, at least in part, responsible for memory impairments and increasing the quantity and quality of SWS will reverse these memory deficits. I first developed mouse models of SWS enhancement in aging and AD. Chemogenetic activation of the parafacial zone GABAergic neurons enhances SWS in aged mice as previously described in adult mice. Similarly, in AD mice, SWS enhancement is as effective as in littermate wild-type controls. Then, I used these mouse models to characterize the role of SWS in memory using novel gain-of-sleep experiments. I found that acute SWS enhancement: 1) reduce spatial memory in adult mice and 2) failed to improve spatial memory in aged mice. In a preliminary study, acute SWS enhancement seems to improve contextual memory in AD mice. Collectively, my work provides a novel mouse model of SWS enhancement in aging and AD, offering a pivotal tool to study the role of SWS in physiological functions and neurodegenerative diseases. Furthermore, my results suggest that acute SWS enhancement does not benefit the behavioral manifestation of memory consolidation in adult mice and aged mice.

Das deklarative Gedächtnis beruht auf Wechselwirkungen zwischen dem medialen Temporallappens (MTL) und Neokortex. Aufgrund der verteilten Natur neokortikaler Netzwerke bleiben zelluläre Ziele und Mechanismen der Gedächtnisbildung im Neokortex jedoch schwer fassbar. Im sechsschichtigen Säugetier-Neokortex konvergieren die Top-Down-Inputs auf Schicht 1 (L1). Wir untersuchten, wie Top-Down-Inputs von MTL die neokortikale Aktivität während der Gedächtnisbildung modulieren. Wir haben zunächst ein Kortex- und Hippocampus-abhängiges Lernparadigma angepasst, in dem Tiere gelernt haben, direkte kortikale Mikrostimulation und Belohnung zu assoziieren. Neuronen in den tiefen Schichten des perirhinalen Kortex lieferten monosynaptische Eingaben in L1 des primären somatosensorischen Kortex (S1), wo die Mikrostimulation vorgestellt wurde. Die chemogenetische Unterdrückung der perirhinalen Inputs in L1 von S1 störte die Gedächtnisbildung, hatte jedoch keinen Einfluss auf die Leistung der Tiere nach abgeschlossenem Lernen. Dem Lernen folgte das Auftreten einer klaren Subpopulation von Pyramidenneuronen der Schicht 5 (L5), die durch hochfrequentes Burst-Feuern gekennzeichnet war und durch Blockieren der perirhinalen Inputs zu L1 reduziert werden konnte. Interessanterweise zeigte ein ähnlicher Anteil an apikalen Dendriten von L5-Pyramidenneuronen ebenfalls eine signifikant erhöhte Ca2+-Aktivität während des Gedächtnisabrufs bei Expertentieren. Wichtig ist, dass die Störung der dendritischen Ca2+-Aktivität das Lernen beeinträchtigte, was darauf hindeutet, dass apikale Dendriten von L5-Pyramidenneuronen eine entscheidende Rolle bei der Bildung des neokortikalen Gedächtnisses spielen. Wir schließen daraus, dass MTL-Eingaben das Lernen über einen perirhinalen vermittelten Gating-Prozess in L1 steuern, der sich in einer erhöhten dendritischen Ca2+-Aktivität und einem Burst-Firing in pyramidalen L5-Neuronen manifestiert. / Declarative memory relies on interactions between the medial temporal lobe (MTL) and neocortex. However, due the distributed nature of neocortical networks, cellular targets and mechanisms of memory formation in the neocortex remain elusive. In the six-layered mammalian neocortex, top-down inputs converge on its outermost layer, layer 1 (L1). We examined how layer-specific top-down inputs from MTL modulate neocortical activity during memory formation. We first adapted a cortical- and hippocampal-dependent learning paradigm, in which animals learned to associate direct cortical microstimulation and reward, and characterized the learning behavior of rats and mice. We next showed that neurons in the deep layers of the perirhinal cortex not only provide monosynaptic inputs to L1 of the primary somatosensory cortex (S1), where microstimulation was presented, but also actively reflect the behavioral outcome. Chemogenetic suppression of perirhinal inputs to L1 of S1 disrupted early memory formation but did not affect animals’ performance after learning. The learning was followed by an emergence of a distinct subpopulation of layer 5 (L5) pyramidal neurons characterized by high-frequency burst firing, which could be reduced by blocking perirhinal inputs to L1. Interestingly, a similar proportion of apical dendrites (~10%) of L5 pyramidal neurons also displayed significantly enhanced calcium (Ca2+) activity during memory retrieval in expert animals. Importantly, disrupting dendritic Ca2+ activity impaired learning, suggesting that apical dendrites of L5 pyramidal neurons have a critical role in neocortical memory formation. Taken together, these results suggest that MTL inputs control learning via a perirhinal-mediated gating process in L1, manifested by elevated dendritic Ca2+ activity and burst firing in L5 pyramidal neurons. The present study provides insights into cellular mechanisms of learning and memory representations in the neocortex.