Les pointes épileptiques intercritiques (IES) représentent une signature neuronale de l'activation transitoire hypersynchrone et excessive d'un grand ensemble de neurones corticaux hétérogènes. Elles sont considérées comme la signature de l’épileptogénicité du réseau neuronal sous-jacent. Dans cette étude, des changements sur la configuration neurale ont été observés chez des modèles animaux et humains au cours de l'IES. Pour la première fois, ces changements ont été détectés à l'aide de la spectroscopie optique rapide (FOS), qui correspond aux variations de la lumière diffusée par le tissu neural pendant l'activation. Ces chages [i.e. changements] sont associés à des mécanismes cellulaires plutôt qu'à des réponses hémodynamiques à haute résolution spatiale et temporelle. Pour étudier le mécanisme IES, une analyse simultanée multimodale des changements optiques rapides (FOS) et électriques (EEG/ECoG: temps et fréquence) a été développée chez des modèles animaux (15 rats) et humains (IES frontales,3 enfants). Pour évaluer de manière indépendante nos méthodes, un potentiel évoquant somatosensoriel et une réponse optique ont été conçus dans des modèles animaux et humains (5 volontaires sains).Les résultats suggèrent une relation entre la (dé)synchronisation et les changements optiques quel que soit le modèle épileptique. Nous avons démontré que cette approche multimodale non invasive multi-échelles (FOS, ECoG / EEG) permet d'étudier la physiopathologie de l'IES chez les patients et de mieux comprendre les mécanismes qui propulsent les neurones vers l'hypersynchronisation chez les modèles épileptiques humains et animaux / Interictal epileptic spikes (IES) represent a signature of the transient synchronous and excessive discharge of a large ensemble of cortical heterogeneous neurons and are widely accepted diagnostically as a signature of an epileptic underlying network. In this study, changes on neural configuration were observed in an animal and human models during the IES. For the first time, these changes were detected using Fast Optical Spectroscopy (FOS), which correspond to variations of scattered light from neural tissue during activation. These chages [i.e. changes] are associated with cellular mechanisms rather than hemodynamic responses with high spatial and temporal resolution. To investigate IES mechanism, a multimodal simultaneous analysis of the fast optical (FOS) and electrical (EEG/ECoG: time and frequency domain) changes was developed in both animal (15 rats) and human models (frontal IES, 3 children). To independently evaluate our methods, a control somatosensory evoked potential and optical response was designed in both animal and human models (5 healthy volunteers). The results suggest a relationship between (de)synchronization and optical changes whatever the epileptic model. This also proposed that changes in the fast optical signal which reflect changes in membrane configuration, are associated with the complex perturbations of the neuronal activation of the epileptic networks. We demonstrated that this non-invasive multiscale multimodal approach (FOS, ECoG/EEG) is suitable to study the pathophysiology of the IES in patients and shed new light on the mechanisms that propel neurons to the hypersynchronization in both animal and human epileptic models

Doctor of Philosophy / The general aim of this current work was to examine spinal cord injury (SCI), and in particular to examine the pathology of injury as it relates to changes in sensory transmission. Due to the limited possibilities for experimentation in humans, a range of animal models of SCI have been developed and are reviewed here. The weight drop SCI model is the most similar to the clinical presentation of SCI in humans and has been widely used in the rat. It was selected for the series of experiments reported in this thesis. Many of the functional deficits produced by SCI result from a cascade of biochemical events set into motion by the injury. Included amongst these is the activation of the enzyme nitric oxide synthase which produces the gaseous neuromodulator, nitric oxide (NO). NO is amongst the most widely distributed and widely utilised molecule in virtually all living organisms, and it is an important signalling molecule in the nervous system. One of the major functions performed by NO appears to relate to sensory transmission, and thus alterations in sensory transmission observed as a result of SCI may involve alterations to NO synthesis. One of the principal aims of this thesis was to examine the effect of SCI on the NO producing cells of the spinal cord and to consider what any changes in NO synthesis may suggest in regards to sensation. NO producing cells were examined using NADPH diaphorase (NADPH-d) histochemistry. As the symptoms of SCI such as motor loss and changes in sensory processing are functional changes, it was also useful to examine changes in neuronal function as a result of SCI. Widespread neuronal function was examined via immunohistochemical detection of the gene product of the immediate early gene, c-fos. It is not known how extensive the biochemical changes resulting from SCI may be, thus another of the aims of the present thesis was to examine the effects of SCI on NO synthesis not only at the level of injury, but also distant to the injury. Findings of the present thesis indicated that traumatic SCI resulted in a decrease in the number of NADPH-d positive cells from the superficial dorsal horn (SDH) of the spinal cord, while the number of these cells are increased in the ventral horn. These changes were restricted to spinal segments adjacent to the injury. Fos expression was also altered by injury and was found to decrease. The most profound changes were found to occur in lamina III, although the other laminae also demonstrated similar changes. Changes in fos expression however were notably more widespread than those for NADPH-d and were not restricted to the level of the injury, occurring at all levels of the spinal cord examined. It was interpreted that alterations in NO synthesis appear to be modulated by the local injury-induced environment while fos expression may be altered by widespread changes to the global level of activity within the central nervous system. Having observed that the number of NADPH-d positive cells of the SDH is reduced following injury, it was of interest to determine whether these cells were in fact killed, or whether they were still present but with reduced NADPH-d activity. Cell counts suggested that the NADPH-d positive cells, which were likely to represent a population of inhibitory interneurons, were not killed following injury, but rather are disrupted such that their normal biochemistry is altered. Since these cells were likely to be inhibitory and were located in laminae involved in sensory transmission, the question arose how disruption of these cells may relate to the neuropathic pain observed to develop following SCI. Thus both NADPH-d and fos expression were again examined, but this time in conjunction with the sensory function of the rats. Sensory thresholds to pain-like behaviour were determined prior to and after injury using Von Frey filaments. Rats that demonstrated a decrease in sensory threshold of at least two Von Frey filament gradations (>70%) were classed as allodynic, while those with a less than a 70% decrease in threshold were classed as non-allodynic. A subpopulation of each of the groups of rats (uninjured, non-allodynic and allodynic) underwent a somatic stimulation paradigm. It was found that stimulation resulted in an increase in the number of NO producing cells but only in the allodynic group of animals. Since this group of animals by definition would perceive this stimulation as noxious, it is likely that the noxious nature of the stimulation resulted in the increased number of NO producing cells observed. This effect occurred only in segments adjacent to the injury. When fos expression was examined in the uninjured animals it was noted that somatic stimulation resulted in a decrease in fos expression, almost exclusively in lamina III. Following injury, there was no change in fos expression in lamina III observed. Instead the only change observed was an increase in fos expression in the deep dorsal horn (DDH, lamina IV and V). This occurred most profoundly in the allodynic group. These results suggested that SCI may lead to misprocessing of sensory signals such that non-noxious somatic stimuli are processed in the DDH rather than lamina III following SCI. It is proposed here that this change in laminae processing may be responsible for the perception of pain towards a non-noxious stimulus, and that the reported injury-induced loss of NO producing inhibitory interneurons in the SDH may be responsible for this alteration in sensory processing following SCI. Sensation is also processed by a number of supraspinal structures and a number of these have been implicated in the development of neuropathic pain states. The effects of SCI on neuronal activity as well as NO synthesis were examined in the periaqueductal grey region of the mid brain (PAG). SCI was shown to result in reduced neuronal activity in the PAG. This reduction in activity did not follow the somatotopy of the lateral column of the PAG (lPAG). It was suggested the reduced activity may not be solely caused by reduced spinal input as a result of SCI. Reduced neuronal activity in the PAG may indicate reduced PAG function, which includes descending modulation of spinal sensory transmission. Injury was not found to alter NADPH-d expression in the PAG. The effect of traumatic lumbar SCI on the parietal (sensorimotor) cortex of the rat was also examined, as loss of inputs following SCI have been shown to result in a profound reorganisation of the cortex. Results indicated that SCI results in a virtual cessation of neuronal activity in areas 1 and 2 of the parietal cortex, likely as a result of lost afferent drive. Theories of cortical plasticity suggest that while the primary inputs via the lumbar spinal cord may be lost following SCI, other less dominants input will remain and become more dominant. It has been proposed previously that cortical reorganisation involves a rapid reorganisation of the entire sensory system. It was interpreted that a similar process may explain the system-wide reduction in neuronal activity observed in the present series of studies.

Muscle fatigue is ubiquitous in every day life.Muscle fatigue might be considered as an altered state of motor behaviour, which impairs motor performance. By contrast, muscle fatigue could also be considered a positive phenomenon, which protects muscle tissue from damage that might be incurred to it by overuse. The principal aim of the thesis was to explore some of the mechanisms of muscle fatigue at the spinal level in animal models.The activation of multiple motor units of a single calf muscle may influence contractile properties of its neighbouring, otherwise inactive units, providing evidence for spatial spreading of fatigue between different muscle parts. The release of metabolites, their action on inactive muscle units and the effects of local hypoxia are the most likely causes. Fatigue-induced metabolite shift in the interstitium provokes excitation and/or sensitisation of high-threshold afferent fibers, with complex effects on the spinal premotoneuronal network involved in the modulation of motoneuronal output. This was examined by studing the intrasegmental lamellar distribution of the lumbar spinal interneurons following fatiguing contractions of the triceps surae muscle. Furthermore, fatigue of calf muscles enhanced the activity of fusimotor neurons to these muscles irrespective of the regime of muscle activity (isometric vs. lengthening) in conditions that simulate locomotion. Altered fusimotor activity, through increased or maintained muscle spindle afferent responsiveness may be advantageous, providing support to the skeletomotor activity and enhanced information about muscle periphery to higher nervous centres. The particular effects of interneuronal network at motor input (presynaptic inhibition system) and output (recurrent inhibition system) stages were then addressed. Fatigue of triceps surae muscle induced a suppression of the monosynaptic reflex. The intensity of presynaptic inhibition increased, while the intensity of recurrent inhibition decreased. Post fatigue-evoked changes in monosynaptic reflexes and presynaptic inhibition indicate the possibility that high-threshold afferents inhibit group Ia terminals pre-synaptically, which would allow fatigue-induced signals from the muscle to reduce the relevance of proprioceptive feedback. Besides intrasegmental, intersegmental spreading of nociceptive signals was explored. Activation of sensory afferents from dorsal neck muscles by capsaicin induces powerful activation of interneurons located in the cervical spinal cord, as well as a widespread activation of cells in lumbar spinal cord segments. The results confirm the pivotal role of small diameter muscle afferents in the orchestration of segmental responses to fatigue and show complex interactions that may lead to limited accuracy of motor output. They also depict processes that may be related to, and even become precursors of chronic muscle pain.

The supraspinal control of descending inhibition of genital reflexes (such as ejaculation) is poorly understood but is important in our global comprehension of how neural signals are integrated to produce sexual behavior, and in our understanding of sexual dysfunction. Sexual dysfunctions, such as premature ejaculation/delayed ejaculation in men, and involuntary vaginal spasms, dyspareunia, and anorgasmia in women, are common. An underlying dysregulation of genital reflexes may produce these dysfunctions, especially in those individuals being treated for depression and anxiety with serotonergic drugs. The nucleus paragigantocellularis (nPGi) of the rat medulla has been described as a descending inhibitory system for genital reflexes in rats, and a homologue is known in humans. Through retrograde tracing of nPGi afferents with the tracer Fluorogold in rats, we found that a number of brain regions implicated in sexual behavior, such as the medial preoptic area, paraventricular nucleus of the hypothalamus, and periaqueductal gray (PAG) provide sexually dimorphic projections to the nPGi, and that many of these regions contain receptors for gonadal steroids and are active during sexual behavior. We also found that excitotoxic lesions of the nPGi with N-methyl-D-aspartate facilitate male sexual behavior by reducing the number of intromissions required for ejaculation, and decreasing ejaculation latency. In females, such lesions attenuated sexual behavior by reducing the amount of time the female spent mating and reducing the reinforcement value of vaginocervical stimulation. Lastly, we found that by removing the source of serotonin to the nPGi (from the ventrolateral PAG) with the serotonergic neurotoxin 5,7-DHT in male rats, we were able to mimic the effects of nPGi lesions and facilitated male sexual behavior indicating that serotonin neurotransmission at the level of the nPGi is critical for genital reflex control. Taken together our results indicate that the nPGi is an important site of integration of internal signals for the regulation of sexual behavior that is sexually dimorphic and under serotonergic control. Our understanding of normal and dysfunction genital reflex control, and possible treatment options in people, is complemented by these results.

No description available.

Vocal plasticity is presumed to be a key element underlying the evolution of human speech and language, but the mechanisms and neuroanatomical basis for this plasticity remain largely unknown. The Mexican free-tailed bat, Tadarida brasiliensis, presents a unique opportunity to advance our understanding of the evolution and neurobiology of mammalian vocal communication because this animal displays elements of vocal complexity and plasticity that are more sophisticated than any mammal other than humans, including non-human primates. Current models of vocal control in mammals do not account for the vocal complexity of free-tailed bats. The purpose of this dissertation is to fill that gap in knowledge by identifying a possible neuronal basis for vocal complexity in free-tailed bats. This will be achieved by 1) providing a detailed analysis of the free-tailed bat’s vocal behaviors, 2) mapping the distribution of neurotransmitter receptor types suspected of involvement in vocal control, 3) identifying brain regions that exhibit increased neuronal activity during vocalizing, and 4) pharmacologically manipulating putative vocal control regions to confirm and characterize their function in vocalizing. Analysis of Tadarida’s vocal behavior indicated that they have a vast vocal repertoire, including many different call types, context-dependent sensory-feedback driven vocal plasticity, and syntactically-organized stereotyped songs. Their vocal behavior changed seasonally, so I mapped the distribution of melatonin binding sites in the brain, finding high densities in the striatum, similar to dopamine receptor distribution. I then used immunohistochemical labeling of the immediate early gene cfos to map neuronal activation in brains of highly vocal bats to find ROIs activated by vocal production. This technique not only identified all previously known regions of the mammalian vocal motor pathway but also revealed activity in novel brain regions that could potentially account for vocal plasticity, including a localized region of the basal ganglia, the dorsolateral caudate nucleus, and the anterior cingulate region of the frontal cortex. Pharmacological excitation of these regions evoked complex vocal sequences similar to the songs recorded in the field and lab. These results support the hypothesis that the mammalian basal ganglia may play a crucial role in the plasticity and complexity of mammalian vocal behaviors.

Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 3 uppsatser.

Few transcription factors (TFs) have been studied in the context of an integrative analysis incorporating genomic datasets from diverse genome regulatory mechanisms. Such an analysis allows the testing of specific regulatory associations in an unbiased and comprehensive manner. The promoter binding TF complex NF-Y regulates a diverse set of constitutive, inducible, developmental, oncogenic and tissue-specific genes. Using cancer models, ChIP-Seq, shRNA, and genomics, I have undertaken a genome-wide study of NF-Y. NF-Y binds to not only promoters but also extensively to enhancers, select classes of repetitive elements, inactive chromatin domains and insulators. NF-Y is a “pioneer”-like factor able to access its motif within closed, transcriptionally inactive chromatin domains. NF-Y pervasively associates with FOS, usually in the absence of JUN and the AP-1 motif, and with a group of growth controlling oncogenic TFs. I also show that NF-Y asymmetrically binds to its motif and stereo-aligns with specific TFs and their motifs. My results indicate that NF-Y is not merely a commonly-used, proximal promoter TF, but rather functions at a more diverse set of genomic elements. The dynamics of TF occupancy, cis-regulatory element (CRE) usage and their linkage to gene expression during a differentiation process, from a genome-wide perspective, is poorly understood and is critical to the understanding of fundamental aspects of development and disease. I utilize a model of inflammation-mediated oncogenic transformation, siRNA, ChIP-Seq, FAIRE-Seq, and microarrays to study the genomic aspects of transformation driven by Src-mediated activation of the inflammatory TF STAT3. I show that CRE usage is static, even in the presence of induced STAT3 activity, and large-scale transcriptional and phenotypic changes. STAT3 induced occupancy is tightly associated with FOS, pre-existing CREs, and does not create CREs de novo. I also highlight a putative role of TSC22D3 in inhibiting an epigenetic switch and in STAT3 and AP-1 factors driving the embryonic-like and bone-like phenotypes of breast cancer. The research presented here suggests that phenotypic alterations occurring during disease are not driven by large-scale perturbations of CRE usage. Overall, this dissertation provides an invaluable resource of genome-scale datasets within cancer models that will assist in future endeavors of scientific discovery.

During development, intrinsic genetic programs give rise to distinct cellular lineages through the establishment of cell type specific chromatin states. These distinct chromatin states instruct gene expression primarily through the genome-wide demarcation of enhancers. In addition to maintaining cellular identity, the chromatin state of a cell provides a platform for transcriptional responses to environmental signals. However, relatively little is known about the influence of extracellular stimuli on chromatin state at enhancers, and it is not clear which enhancers among the tens of thousands that have been recently identified function to drive stimulus-responsive transcription. In the nervous system, the chromatin state of terminally differentiated neurons not only maintains neuronal identity but also provides a platform for sensory experience-dependent gene expression, which plays a critical role in the development and refinement of neural circuits and in long-lasting changes in neuronal function that underlie learning, memory, and behavior. Using chromatin-immunoprecipitation followed by high through put sequencing (ChIP-Seq), we determined the effects of neuronal stimuli on the active chromatin landscape of mouse cortical neurons. We discover that stimulation with neuronal activity and brain derived neurotrophic factor (BDNF) cause rapid, widespread, and distinct changes in the acetylation of histone H3 lysine 27 (H3K27Ac) at thousands of enhancers throughout the neuronal genome. We find that functional stimulus-responsive enhancers can be identified by stimulus- inducible H3K27Ac, and we use this dynamic chromatin signature to discover neuronal enhancers that respond to neuronal activity, BDNF, or both stimuli. Finally, we investigate the transcriptional mechanisms underlying the function of stimulus responsive enhancers. We show that a subset of stimulus-responsive enhancers in the nervous system require the coordinated action of the stimulus-general transcription factor activator protein 1 (AP1) with additional stimulus-specific factors. Our studies reveal the genome-wide basis for transcriptional specificity in response to distinct neuronal stimuli. Furthermore, the comprehensive identification of neuronal activity and BDNF-dependent enhancers in cortical neurons provides a critical resource for elucidating the role of stimulus-responsive transcription in synaptic plasticity, learning and memory, behavior, and disease. Finally, the epigenetic signature of stimulus-inducible H3K27Ac may aid in the identification and study of stimulus- regulated enhancers in other tissues.

Terrestrial salamanders of the genus Plethodon are among many vertebrates possessing both main olfactory and vomeronasal systems, which the Volatility Theory posits are for detection of volatile and soluble olfactory cues, respectively. Further recent work showing a high amount of convergence between the two olfactory subsystems at the level of the central nervous system suggests complementary or overlapping roles for them. This study examined the use of the olfactory subsystems in prey detection from the perspectives of behaviour and neurobiology. Red-backed salamanders, Plethodon cinereus, were observed in standardized behavioural assays with both volatile and soluble prey olfactory cues. Naïve salamanders showed an increase in nosetapping as well as a side preference in the presence of soluble and volatile prey cues when tested in a 22°C day/20°C night room. In a 15°C day /12°C night room, salamanders increased nosetapping in the presence of soluble prey cues. Salamanders showed a pattern of responses that differed based on their previous experience with the assay, as well as the temperature of the testing room. Attempts to study the neurobiology of olfactory function in Plethodon shermani were inconclusive up to this point, but future directions are discussed. This study shows the importance of olfaction in prey detection by salamanders and that prey searching behaviour is exhibited in the exclusive presence of olfactory cues.