The mammalian central nervous system maintains a delicate balance between neuronal excitation and inhibition. Conventional synaptic inhibition is mediated through the transient activity of postsynaptic γ-aminobutyric acid (GABA) at type A GABA (GABAA) receptors. A subset of GABAA receptors is also located outside of inhibitory synapses. These extrasynaptic receptors generate a tonic inhibitory conductance in response to low concentrations of extracellular GABA. Tonic inhibition broadly suppresses neuronal activity and regulates many vital processes such as sleep, consciousness and memory formation. This thesis examines the physiological effects of tonic inhibition at the cellular level and in the behaving animal. This thesis also explores whether gabapentin, a commonly used sedative, anxiolytic, and analgesic drug, enhances tonic GABAergic inhibition. I hypothesize that: (1) tonic GABAA receptor activity reduces the intrinsic excitability of neurons; (2) the activity of tonically active GABAA receptors in spinal pain pathways attenuates nociception; and (3) tonic inhibition can be upregulated by gabapentin. The results show that a tonic inhibitory current generated by α5 subunit-containing GABAA (α5GABAA) receptors reduces the excitability of hippocampal pyramidal neurons excitability by increasing the rheobase, but does not change the gain of action potential firing. A similar shunting inhibition is present in spinal cord lamina II neurons that is generated by δ subunit-containing GABAA receptors. The activity of these receptors in spinal nociceptive pathways reduces acute thermal nociception and may constrain central sensitization in a behavioural model of persistent pain. Finally, gabapentin increases a tonic inhibitory current in cultured hippocampal neurons independent from changes in the expression of α5GABAA receptors or in the concentration of GABAA receptor ligands. The results of this thesis demonstrate that tonically active GABAA receptors play an important role in the regulation of neuronal activity and nociception, and that tonic inhibition represents a novel target of therapeutic drugs.

This thesis describes experiments designed to investigate the effects of neuromuscular electrical stimulation (NMES) on corticospinal (CS) excitability in humans. NMES delivered at 100 Hz was more effective for increasing CS excitability than 10-, 50-, or 200-Hz NMES. CS excitability increases occurred after 24 min of 100-Hz NMES, were strongest in the stimulated muscle, and were mediated primarily at a supraspinal level. NMES of the common peroneal nerve of the leg increased CS excitability in multiple leg muscles, whereas NMES of the median nerve of the hand increased CS excitability in only the muscle innervated by that nerve. Additionally, CS excitability for the hand increased after 40 min of relatively high intensity and frequency NMES but not after 2 h of lower intensity and frequency NMES. These results have implications for identifying optimal NMES parameters to augment CS excitability for rehabilitation after central nervous system injury.

The plasma membrane electrical activities of neurons that secrete gonadotropin releasing hormone (GnRH), referred to as GnRH neurons hereafter, have been studied extensively. A couple of mathematical models have been developed previously to explain different aspects of these activities including spontaneous spiking and responses to stimuli such as current injections, GnRH, thapsigargin (Tg) and apamin. The goal of this paper is to develop one single, minimal model that accounts for the experimental results reproduced by previously existing models and results that were not accounted for by these models. The latter includes two types of membrane potential bursting mechanisms and the associated calcium oscillations in the cytosol. One of them has not been reported in experimental literatures on GnRH neurons and is thus regarded as a model prediction. Other improvements achieved in this model include the incorporation of a more detailed description of calcium dynamics in a three dimensional cell body with the ion channels evenly distributed on the cell surface. Although the model is mainly based on data collected in cultured GnRH cell lines, we show that it is capable of explaining some properties of GnRH neurons observed in several of other preparations including mature GnRH neurons in hypothalamic slices. One potential explanation is suggested. A phenomenological reduction of this model into a simplified form is presented. The simplified model will facilitate the study of the roles of plasma membrane electrical activities on the pulsatile release of GnRH by these neurons when it is coupled with a model of pulsatile GnRH release based on the autoregulation mechanism.

Orientador: Antonio de Queiroz Neto / Banca: Renata Navarro Cassu / Banca: Luís Carlos Rêgo Oliveira / Banca: Luciane Helena Gargaglianoni Batalhão / Banca: Guilherme de Camargo Ferraz / Resumo: Os efeitos antinociceptivos, comportamentais (atividade locomotora espontânea - ALE, altura de cabeça - AC) e sobre as variáveis fisiológicas de seis equinos tratados com tramadol, como agente analgésico preventivo, nas doses intravenosas de 2 (TT2), 3 (TT3) e 5 mg/kg (TT5), assim como da associação tramadol (3 mg/kg) e xilazina (0,5 mg/kg) (TTX) ou ainda da xilazina (0,5 mg/kg) isolada (TX) foram avaliados. Para ALE, diferenças (P<0,05) foram observadas entre os grupos TT2, TT3 e TT5, porém estas não foram significativas (P>0,05) entre esses e os grupos TTX e TX. Para a AC os grupos TTX e TX foram semelhantes sendo esses diferentes dos grupos tratados com tramadol isolado (P<0,05). Diferenças não foram observadas (P>0,05) quanto à ação antinociceptiva. No grupo TTX as variações nas frequências cardíaca e respiratória, pressão arterial sistólica e motilidade intestinal foram significativas (P<0,05). Pode-se concluir pelo exposto que, embora o tramadol isoladamente não promova alteração significativa no estado comportamental de equinos, não constitui um fármaco analgésico somático ao menos para o estímulo usado, e que a associação tramadol/xilazina, não constitui uma opção como associação, visando à sedação e à analgesia, principalmente quando for desejado incrementar, nas técnicas de anestesia, a antinocicepção somática preventiva. / Abstract: Antinociceptive and behavioral effects (spontaneous locomotor activity [SLA] and head height [HH]) and effects on physiological parameters in six horses treated with tramadol as a preventive analgesic agent were assessed. Tramadol was administered at intravenous doses of 2 (TT2), 3 (TT3) and 5 mg/kg (TT5), as well as a combination of tramadol (3 mg/kg) and xylazine (0.5 mg/kg) (TTX) or xylazine alone (0.5 mg/kg) (TX). Differences in SLA (P<0.05) were seen in TT2, TT3, and TT5 groups but they were not statistically significant (P>0.05) between these groups and TTX and TX groups. TTX and TX groups showed similar HHs but there were differences of HH between TTX and TX and those groups treated with tramadol alone (P<0.05). However, no differences (P>0.05) were found regarding antinociceptive action. Significant changes (P<0.05) of heart and respiratory rates, systolic blood pressure, and intestinal motility were seen in TTX group. Although tramadol alone does not have a significant effect on horse behavior, it failed to produce analgesia and it has no somatic analgesic action to the stimulus studied. In conclusion, the combination of tramadol plus xylazine should be carefully prescribed to patients with prior cardiovascular and gastrointestinal conditions but it is not an adequate drug combination for sedation and analgesia, especially when anesthesia is intended to increase preventive somatic antinociception. / Doutor

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-04-27T14:10:38Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Dout_Lima_DSCL_2016.pdf: 2341451 bytes, checksum: e4310955dbc4f0c3af7730606a09c7f3 (MD5) / Made available in DSpace on 2017-04-27T14:10:39Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese_Dout_Lima_DSCL_2016.pdf: 2341451 bytes, checksum: e4310955dbc4f0c3af7730606a09c7f3 (MD5) Previous issue date: 2016-07-29 / O aminoácido glutamina (Gln) é precursor dos neurotransmissores cerebrais glutamato e GABA. O aumento de sua disponibilidade pode modular a excitabilidade cerebral. O objetivo deste trabalho foi descrever os efeitos do tratamento com diferentes doses de Gln, durante o desenvolvimento cerebral, sobre o comportamento de ansiedade, a depressão alastrante cortical (DAC) e a ativação da microglia no córtex de ratos recém-desmamados (D) e adultos (A). Os animais foram amamentados em ninhadas com 9 (L9; lactação normal) e com 15 filhotes (L15; lactação desfavorável). Do 7º ao 27º dia de vida pós-natal (P7-P27), os filhotes machos receberam por gavagem 250, 500 ou 750 mg/kg/dia de Gln (grupos Gln250, Gln500 e Gln750, respectivamente). Os grupos controles foram formados por animais que receberam o veículo (água destilada) no qual a Gln foi dissolvida por gavagem e por animais que não receberam gavagem (grupo ingênuo). Aos P28-P30 (D) e P88-P90 (A), os animais foram submetidos aos testes para comportamentos sugestivos de ansiedade no labirinto em cruz elevado (LCE) e no campo aberto. Dos P30-35 (D) ou P90-120 (A), registrou-se a DAC, obtendo-se dados de sua velocidade de propagação, duração e amplitude. Em seguida, os cérebros de alguns animais foram processados para imunomarcação com anticorpos anti-Iba1, específicos para microglia. No grupo D, os ratos tratados com Gln apresentaram um comportamento menos ansioso, tanto no LCE quanto no campo aberto. Este efeito ansiolítico da Gln foi mais evidente nos animais da condição L15. Na idade adulta (A), os grupos Gln500 e Gln750 da condição L15 percorreram uma maior distância e apresentaram menor tempo de imobilidade no LCE. Em relação à DAC, os animais da condição L15 apresentaram maior velocidade de propagação do que os correspondentes L9. Com exceção do grupo Gln250 da condição L15 na idade adulta, todos os grupos tratados com Gln apresentaram maior velocidade de propagação quando comparados aos respectivos controles. Além disso, esse efeito acelerador foi dependente da dose, uma vez que os grupos Gln500 e Gln750 apresentaram maiores velocidades de propagação do que os correspondentes Gln250. Quanto à reação da microglia, os animais tratados com Gln apresentaram maior imunorreatividade, tanto no córtex parietal quanto no hipocampo dos grupos D e A. Nos animais A da condição L9, a imunorreatividade da microglia e o percentual de área marcada foram maiores no grupo Gln500 do que no grupo Gln250. A partir desses resultados, sugere-se que o tratamento com Gln durante o período neonatal module a excitabilidade cerebral, resultando nas alterações eletrofisiológicas, comportamentais e imunohistoquímicas descritas neste estudo. Essas alterações persistem até a idade adulta e são dependentes da dose e da condição nutricional do animal. / The amino acid glutamine (Gln) is precursor of the brain neurotransmitters glutamate and GABA. Therefore, the increase of its availability can modulate brain excitability. The aim of this study was to describe the effects of treatment with different doses of Gln during brain development on anxiety-like behavior, cortical spreading depression (CSD) and microglial reaction in the cortex of developing (D) and adults (A) rats. Wistar rats were suckled in litters with 9 (L9; normal condition) or 15 (L15; unfavorable condition) pups. From 7th to 27th postnatal day (P7-P27), male rats received Gln by gavage at the doses of 250 mg/kg/day or 500 mg/kg/day or 750 mg/kg/day (respectively Gln250, Gln500 and Gln750 groups). The control groups were formed by animals that received vehicle which Gln was dissolved (distilled water) and animals that were not submitted to the gavage procedure (naive group). At P28-P30 (D) and P88-P90 (A), animals were tested in elevated plus maze (EPM) and open field. At P30-35 (D) and P90-120 (A), we recorded the CSD, obtaining data from its velocity of propagation, duration and amplitude. The brains of some animals were processed for microglial immunolabeling with anti-Iba-1 antibodies to analyze cortical microglia. In the D group, Gln treated rats showed less anxious behavior, both in EPM and open field. This anxiolytic effect of Gln was more evident in L15 condition. In adult rats (A), Gln500 and Gln750 groups of L15 condition traveled a greater distance and displayed shorter immobility time in the LCE when compared to controls. Regarding CSD, L15 animals presented with higher propagation velocity than the corresponding L9. Except for the Gln250 group of L15 condition in adulthood, all groups treated with Gln showed higher CSD velocity when compared to their respective controls. Moreover, the accelerating effect was dose dependent, since Gln500 and Gln750 groups displayed higher CSD velocity than the corresponding Gln250. Gln treated groups had greater immunoreactivity in both the parietal cortex and hippocampus. In adult rats of L9 condition, Gln500 group had greater immunoreactivity and higher percentage of labeled area when compared Gln250 group. Our findings suggest that neonatal treatment with Gln modulates brain excitability, resulting in the electrophysiological, behavioral and microglial alterations here described. These alterations persist into adulthood and are modulated by dose and lactation conditions.

Le cerveau génère spontanément des activités électriques enregistrables à toutes les échelles spatiales, de l'électroencéphalogramme (EEG) jusqu'à la membrane neuronale. La fréquence et l'amplitude de ces activités varient en fonction des états de vigilance. Afin de comprendre comment cette activité synaptique endogène sculpte à chaque instant l'intégration des événements exogènes, j'ai mis au point une nouvelle stratégie expérimentale in vivo visant à comparer les réponses neuronales à divers stimuli, en présence et en absence d'activité endogène. J'ai généré, chez le rat, une activité de type éveil ou sommeil puis, j'ai induit un état isoélectrique durant lequel toute activité spontanée était supprimée. J'ai montré que la suppression de l'activité synaptique dans les neurones du cortex somatosensoriel induisait une diminution de sensibilité neuronale et un accroissement de la régularité des réponses. La persistance d'une excitabilité neuronale dans cet état de coma profond m'a conduit à poursuivre mes recherches avec le service de réanimation neurologique afin d'explorer, chez des patients placés dans un tel coma, la réactivité corticale à des stimulations sensorielles. J'ai démontré chez l'homme et l'animal la persistance de potentiels évoqués sensoriels dans l'EEG et les neurones corticaux. Ces réponses apparaissaient plus tardivement, avec une amplitude plus importante et une plus grande fiabilité d'un essai à l'autre. Ainsi, il apparaît que l'activité synaptique spontanée, qui caractérise le fonctionnement " normal " du cerveau, a essentiellement comme effet d'augmenter la sensibilité des neurones ainsi que la variabilité statistique des réponses à l'environnement. / The brain spontaneously generates electrical activities, which can be recorded at all the spatial level, from the electroencephalogram (EEG) to the neuronal membrane. The frequency and the amplitude of these activities vary with the states of vigilance. To understand how this endogenous synaptic activity sculpts the integration of exogenous events, I developed a new in vivo experimental strategy to compare the cortical neuronal responses to various stimuli in the presence and absence of endogenous activity. I generated a waking-like or sleep-like synaptic activity in the rat. Then, I induced an isoelectric state in which spontaneous activity was completely suppressed. I showed that the suppression of synaptic activity in somatosensory cortex neurons resulted in a decrease in neuronal sensitivity and an increase in the regularity of responses to repeated identical stimuli. The persistence of neuronal excitability while the animal was immersed in a deep comatose led me to continue my research in collaboration with the neurological intensive care unit to explore the sensory-evoked cortical responses in patients exhibiting an isoelectric EEG. I demonstrated in humans and animals the persistence of sensory-evoked potentials in the EEG and individual cortical neurons. These cortical responses occurring in the absence of spontaneous brain activity had an augmented latency, a larger amplitude and a higher trial-to-trial reliability. It thus seems that the primary effect of the sustained background synaptic activity, the hallmark of a "normal" functioning of the brain, is to increase the sensitivity of neurons and the statistical variability of responses to the environment.

The plasma membrane electrical activities of neurons that secrete gonadotropin releasing hormone (GnRH), referred to as GnRH neurons hereafter, have been studied extensively. A couple of mathematical models have been developed previously to explain different aspects of these activities including spontaneous spiking and responses to stimuli such as current injections, GnRH, thapsigargin (Tg) and apamin. The goal of this paper is to develop one single, minimal model that accounts for the experimental results reproduced by previously existing models and results that were not accounted for by these models. The latter includes two types of membrane potential bursting mechanisms and the associated calcium oscillations in the cytosol. One of them has not been reported in experimental literatures on GnRH neurons and is thus regarded as a model prediction. Other improvements achieved in this model include the incorporation of a more detailed description of calcium dynamics in a three dimensional cell body with the ion channels evenly distributed on the cell surface. Although the model is mainly based on data collected in cultured GnRH cell lines, we show that it is capable of explaining some properties of GnRH neurons observed in several of other preparations including mature GnRH neurons in hypothalamic slices. One potential explanation is suggested. A phenomenological reduction of this model into a simplified form is presented. The simplified model will facilitate the study of the roles of plasma membrane electrical activities on the pulsatile release of GnRH by these neurons when it is coupled with a model of pulsatile GnRH release based on the autoregulation mechanism. / Science, Faculty of / Mathematics, Department of / Graduate

The process of movement execution can be separated into two sections; the foreperiod and the response time. The foreperiod represents the time between the warning signal (WS) and the presentation of the imperative “go” signal, and the response time incorporates both the reaction time (RT) and the movement time (Schmidt & Lee, 2011). Transcranial magnetic stimulation (TMS) was used to probe corticospinal excitability (CE) which has been measured in a variety of RT tasks during both the foreperiod and the response time periods. The purpose of the two studies in this thesis was to measure when and at what rate changes in CE occur in both simple and complex tasks. The results of the first experiment indicated that CE levels quickly increased from baseline with the presentation of the WS. This was followed by a holding period in which CE was held constant until a decline in CE occurred prior to the presentation of the IS. This decline was followed by a rapid increase in CE as the movement was initiated and released. Importantly, even though levels of CE were decreasing relative to the start of the decline, participants were still in a heightened state as they prepared to release their movements. Furthermore, it is suggested that selective inhibitory control mechanisms were at least partly responsible for the decline prior to the IS. The results of the second experiment indicated that MEP amplitudes in a simple task were significantly larger compared to those in a complex task relative to both the IS and the onset of electromyography. These findings suggest that simple and complex tasks achieve differing levels of corticospinal excitability, and it is suggested that the complex requires the use of the cerebellum, which suppresses excitatory projections to the thalamus, and consequently to the motor cortex.

Denervation leads to skeletal muscle atrophy, which is a decrease in muscle mass and force; the latter exceeding expectation from mass loss. In some cases, nerve regeneration following an injury takes several months. During this time, muscle mass and force loss become severe as fibers are replaced by connective and fat tissue, which can further prolongs normal muscle function recovery once reinnervation occurs. The objectives of this study were 1) document the angiotensin 1-7 (Ang 1-7) hypertrophic effect in innervated mouse skeletal muscle; 2) test the hypothesis that Ang 1-7 prevents muscle atrophy and maintain force following short 2 and 4 week denervation; 3) as well as following long 16 week denervation. Innervated and denervated mice were treated with Ang 1-7 or diminazene aceturate (DIZE), an ACE2 activator, to increase plasma Ang 1-7 level. In normal innervated extensor digitorum longus (EDL) and soleus muscle, Ang 1-7 increased muscle weight, cross sectional area (CSA) and tetanic force, which represents the muscle maximum force. During the short denervation period (2-4 weeks), Ang 1-7 did not prevent muscle mass and CSA loss, but fully abolished the loss of normalized tetanic force to CSA while accentuating twitch force. Normalized tetanic force was maintained as Ang 1-7 partially reduced the extent of membrane depolarization which normally observed with denervation, and it fully prevented the loss of membrane excitability. The protective effect of Ang 1-7 on maximum tetanic force was also observed after 16 weeks of denervation, but only in EDL not in soleus. About 35-40% of denervated EDL and soleus muscle fibers became reinnervated over the 16 week period and Ang 1-7 enhanced the recovery of muscle mass and tetanic force in both EDL and soleus. All Ang 1-7 effects on twitch and tetanic force were completely blocked by A779, a Mas receptor (MasR) antagonist, and were not observed in MasR deficient (MasR / ) muscles. Ang 1-7 did not affect how denervation modulates changes in the protein content MuRF-1 atrogin-1, two atrophic proteins, total and phosphorylated Akt, S6K and 4EPB, three hypertrophic proteins. So, the Ang 1-7 effect involves an activation of its MasR, but it is not clear which intracellular pathway it then affects. This is the first study providing evidence that Ang 1-7 maintains normal muscle function in terms of tetanic force and membrane excitability during 2, 4 and 16 week denervation periods.

Although inhibitory cortical interneurons play a critical role in regulating brain excitability and function, the effects of stroke on these neurons is poorly understood. In particular, interneurons expressing vasoactive intestinal peptide (VIP) specialize in inhibiting other classes of inhibitory neurons, and thus serve to modulate cortical sensory processing. To understand how stroke affects this circuit, we imaged VIP neuron responses (using GCaMP6s) to low and high intensity forepaw stimulation, both before and after focal stroke in somatosensory cortex. Our data show that the fraction of forelimb responsive VIP interneurons and their response fidelity (defined as a cell’s number of responsive trials out of eight trials at a certain imaging week) was significantly reduced in the first week after stroke, especially when lower intensity forepaw stimulation was employed. The loss of responsiveness was most evident in highly active VIP neurons (defined by their level of responsiveness before stroke), whereas less active neurons were minimally affected. Of note, a small fraction of VIP neurons that were minimally active before stroke, became responsive afterwards suggesting that stroke may unmask sensory responses in some neurons. Although VIP responses to forepaw stimulation generally improved from 2-5 weeks recovery, the variance in response fidelity after stroke was comparatively high and therefore less predictable than that observed before stroke. Lastly, stroke related changes in response properties were restricted to within 400μm of the infarct border. These findings reveal the dynamic and resilient nature of VIP neurons and suggest that a sub-population of these cells are more apt to lose sensory responsiveness during the initial phase of stroke, whereas some minimally responsive cells are progressively recruited into the forelimb sensory circuit. Furthermore, stroke appears to disrupt the predictability of sensory-evoked responses in these cortical interneurons which could have important consequences for sensory perception. / Graduate / 2021-01-13