Migraine affects ~15% of the US population (nearly 40 million people), making it one of the most common neurological disorders; however currently available therapeutic options for migraine relief are often ineffective. Moreover, acute and prophylactic drugs are both commonly associated with contraindications and serious side effects, and routine use of acute treatments may result in medication overuse-headaches. Elevated levels of the neuropeptide calcitonin gene-related peptide (CGRP) are known to be a primary factor in migraine pathogenesis, although the mechanisms by which CGRP expression becomes errantly modulated are unclear. CGRP is a product of the trigeminal ganglion and can be released both peripherally onto the dura mater, leading to neurogenic inflammation, and centrally at the spinal trigeminal nucleus, leading to neuromodulation. A great deal of CGRP-relevant migraine research has focused on the trigeminovascular system, but whether the cerebral cortex may have a role in migraine pathophysiology been less well studied. A subset of migraineurs experience a premonitory aura, which often manifests as a disturbance in one visual hemifield. An aberration called cortical spreading depression (CSD) is the likely electrophysiological substrate of the migraine aura, but whether CSD and CGRP are functionally related is not known. CSD is characterized by an initial transient wave of neuronal and glial depolarization, followed by a prolonged period of quiescence that is largely refractory to subsequent stimulation. Converging evidence supports a facilitatory role for cortical spreading depression (CSD) in migraine with and without aura, and CSD propagation has been shown to be dependent on functional CGRP receptors. Moreover, reported effects of CSD overlap with those of CGRP-mediated neurogenic inflammation. The experiments described herein seek to test the hypothesis that induction of CSD in vivo will lead to increased CGRP expression in the rodent cerebral cortex. Preliminary data in rats suggests that 3M KCl-induced CSD can trigger increased CGRP expression in the ipsilateral cortex. Preliminary data in mice has been less conclusive. Presented here are the data obtained from mice and rats, as well as speculation on the cause(s) of the differences in CGRP expression between species and how these findings relate to human studies.

The studies in this thesis research were conducted to investigate if sensitivity to induced cortical spread depression (CSD) or the consequence of a CSD event is affected by sumatriptan induced latent sensitization. Previous studies in our lab showed persistent exposure of sumatripan to rats produced a latent state of sensitization. Using persistent sumatripan exposed rats as a model for medication overuse headache, behavior, electrical stimulation threshold to provoke a CSD event and the immunoreactivity of c-Fos in the trigeminal nucleus caudalis (TNC) were characterized. Current results showed no statistical difference of electrically induced CSD thresholds in anesthetized rats measured at day 20 in sumatripan exposed rats compared with saline treated rats. Topiramate (80 mg/kg, i.p.) used clinically for prophylaxis of migraine headache significantly increased CSD threshold in both saline and sumatriptan infused rats. CSD events appear to be associated with trigeminal vascular system activation in TNC because c-Fos expression significantly enhanced in rats with electrically stimulated CSD events. As compared to saline treated rats, sumatriptan-exposed rats demonstrated a significantly higher number of c-Fos positive cells following the electrically stimulated CSD event. Under environmental stress (bright light), sumatripan exposed rats demonstrated decreased response thresholds to periorbital and hindpaw tactile stimuli (i.e., allodynia) and enhanced c-Fos expression in TNC. A single dose of topiramate (80 mg/kg, i.p.) reversed environmental stress induced allodynia and c-Fos over-activity. Taken together, these results suggest that latent sensitization induced by persistent sumatripan exposure seems not correlated to the threshold of electrically stimulated CSD in current model. However, CSD enhanced the responses of trigeminal system in rats with sumatriptan-induced latent sensitization. The protective effects of topiramate shown in this model may be related to blocking the initiation of CSD events resulting from environmental stimulation as well as inhibiting the consequences of CSD events in primary afferents. These findings correlate with clinical observations of protective effects of topiramate for migraine prophylaxis.

Migraine is a debilitating neurological disorder, which affects over 10% of the general population. In addition to headache, migraine includes a host of associated symptoms, such as nausea and hypersensitivity to light, noise, and touch. While great strides have been made in migraine treatment in recent decades, the basic biological and pathophysiological mechanisms underlying migraine are still not well understood. Pain signals travel via a polysynaptic pathway from the periphery to the cortex, where conscious perception of pain occurs. This multi-neuron pathway produces a message that can be modified at any step of its transit. One peptide that may modify this pathway is calcitonin gene-related peptide (CGRP). CGRP is a potent vasodilator and neuromodulator, and mounting evidence suggests CGRP may play a causative role in migraine. CGRP levels are increased during migraine, but can be reduced upon successful treatment with drugs in the triptan class. Importantly, injection of CGRP into migraine patients can elicit a delayed, migraine-like headache. Finally, CGRP receptor antagonists are clinically effective in providing relief to migraine patients. In addition to CGRP, the CGRP gene (CALCA) expresses another peptide that may also be relevant to migraine. Procalcitonin (proCT) is a recognized biomarker for sepsis, but emerging evidence suggests it may have actions similar to CGRP in migraine. First, proCT has biological activity at the CGRP receptor. Second, proCT is reported to be increased during migraine. We hypothesized that regulation of CGRP and proCT may be altered in migraineurs, and that migraineurs may also be sensitized to the effects of these peptides. To study the role of these peptides in migraine pathways, a number of methods have been employed. Studies exploring regulation of gene expression were performed in cultured trigeminal ganglia, as well as primary cultures of trigeminal and cortical glia. These studies show that the Calca gene can be regulated by a number of stimuli, including hypoxia and reactive oxygen species. These insults have the ability to induce CALCA gene and peptide expression to varying degrees on different cell types. In addition to in vitro experiments on Calca gene regulation, the in vivo effects of CGRP on mouse behavior were also investigated. Animals were genetically sensitized to CGRP via overexpression of the rate-limiting CGRP receptor subunit. In these animals, injection of CGRP is sufficient to induce light aversion, which is used to model photophobia. Physiological and biochemical triggers of migraine were tested using this behavioral paradigm. While stress and mast cell degranulation are sufficient to induce light aversion, the role of CGRP in these events remains unclear, as both CGRP sensitized and control animals displayed a light aversion phenotype. Together, these studies show the dynamic regulation of the Calca gene in migraine pathways as well as highlight some of the challenges of modeling a complex disease in an animal model.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

The purpose of this study was to examine the effects of cortical spreading depression on neuronal activity in the rat motor (M1) cortex. It is known that cortical spreading depression causes widespread neuronal and glial activity in the cortex, but the degree to which it exerts its effects is unclear. Cortical spreading depression was induced in eight Sprague-Dawley male rats. After two hours, animals were euthanized and immunohistochemistry was performed on the brain to stain for the presence of c-Fos, an immediate early gene that is a well-known marker of neuronal activity. Sections were counterstained for Nissl substance to reveal two populations of cells: Nissl-stained neurons that were c-Fos positive, activated cells and Nissl-stained neurons that were c-Fos negative, non-activated cells. Three sections for each animal were examined and 20-30% of the total M1 cortex was analyzed. Cells were counted using systematic random sampling for each of the six cortical layers. Our results show that the cortical spreading depression did not produce an activation of all neurons. When layers were individually examined, there was a main effect of layer on neuronal activation. This confirmed previous findings that cortical spreading depression had the strongest effect on superficial layers of the cortex

Transcranial direct current stimulation (TDCS) allows for the noninvasive modulation of cortical activity. In this study, the effects of cathodal and anodal TDCS treatment on baseline activity in the motor cortex of rats were investigated via translaminar electroencephalogram (EEG) recording and power spectral density analysis. Treatment with low intensity anodal TDCS for five minutes was found to increase delta and theta frequency cortical activity during and for up to five minutes following treatment. This study also assessed the interaction of TDCS with the phenomenon of cortical spreading depression (CoSD), which has been implicated in numerous disease states, including migraine and stroke. TDCS treatment was given concurrently with induction of CoSD via administration of potassium chloride to the surface of the dura. The presence of the spreading depression event, a characteristic low frequency wave observed to travel outwards from the point of CoSD induction and downwards through the cortex, was used as a proxy measure for the occurrence of CoSD. It was observed that animals treated with cathodal TDCS exhibited fewer spreading depression events relative to those treated with anodal TDCS or those receiving sham treatment. In this study, animals were segregated into groups that exhibited stimulus artifact during TDCS treatment and those that did not. Stimulus artifact was defined as a characteristic alpha and/or beta frequency activity spike lasting throughout and not longer than the period of stimulation. Those animals receiving TDCS without exhibiting stimulus artifact were considered for the purposes of this study to not have received proper TDCS treatment, and acted as a sham treatment group. Because salient differences emerged between the stimulus artifact positive and stimulus artifact negative groups, this study suggests that the presence of stimulus artifact could be used as a proxy measure for successful TDCS dosage.

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.

No description available.

No / Cortical spreading depression (CSD) is a temporary disruption of local ionic homeostasis that propagates slowly across the cerebral cortex, and may contribute to the pathophysiology of stroke and migraine. Previous studies demonstrated that nitric oxide (NO) formation promotes the repolarisation phase of CSD, and this effect may be cyclic GMP (cGMP)-mediated. Here, we have examined how phosphodiesterase (PDE) inhibition, either alone or superimposed on NO synthase (NOS) inhibition, alters CSD and the associated changes in extracellular cGMP. Microdialysis probes incorporating an electrode were implanted into the frontoparietal cortex of anaesthetised rats for quantitative recording of CSD, pharmacological manipulations, and dialysate sampling for cGMP measurements. CSD was induced by cathodal electrical stimulation in the region under study by microdialysis. Extracellular cGMP increased, but only slightly, during CSD. Perfusion of either zaprinast or sildenafil through the microdialysis probe, at concentrations that inhibited both PDE5 and PDE9 (and possibly other PDE), increased significantly extracellular cGMP. Unexpectedly, these levels remained high when NOS was subsequently inhibited with N¿-nitro- -arginine methyl ester hydrochloride ( -NAME, 1 mM). The most interesting pharmacological effect on CSD was obtained with sildenafil. This drug altered neither CSD nor the subsequent characteristic effect of NOS inhibition, i.e. a marked widening of CSD. The fact that NOS inhibition still widened CSD in the presence of the high extracellular levels of cGMP associated with PDE inhibition, suggests that NO may promote CSD recovery, independently of cGMP formation.

No / Preconditioning of the cerebral cortex was induced in mice by repeated cortical spreading depression (CSD), and the major ionotropic glutamate (GluRs) and nicotinic acetylcholine receptor (nAChRs) subunits were compared by quantitative immunoblotting between sham- and preconditioned cortex, 24 h after treatment. A 30% reduction in ¿-amino-3-hydroxy-5-methyl-4-iso- xazolepropionate (AMPA) GluR1 and 2 subunit immunoreactivities was observed in the preconditioned cortex (p < 0.03), but there was no significant change in the NMDA receptor subunits, NR1, NR2A and NR2B. A 12¿15-fold increase in ¿7 nAChR subunit expression following in vivo CSD (p < 0.001) was by far the most remarkable change associated with preconditioning. In contrast, the ¿4 nAChR subunit was not altered. These data point to the ¿7 nAChR as a potential new target for neuroprotection because preconditioning increases consistently the tolerance of the brain to acute insults such as ischaemia. These data complement recent studies implicating ¿7 nAChR overexpression in the amelioration of chronic neuropathologies, notably Alzheimer's disease (AD).