Migraine is a complex neurological disorder that affects approximately 38 million Americans. For over 25 years, the neuropeptide calcitonin gene-related peptide (CGRP) has been implicated in the pathogenesis of migraine. In fact, several pharmaceutical companies are tailoring treatments to antagonize CGRP actions. However, due to the complexity of migraine, exactly how and where CGRP acts to contribute to migraine have remained controversial: whereas several studies suggest that CGRP acts in the central nervous system (CNS) in this context, others have indicated a role in the periphery. Central nervous system sites of action include the trigeminal nucleus and several higher brain regions, and peripheral sites include the vasculature and dural mast cells in the meninges. Among the sites of CGRP action, the trigeminal nerve, which is the major somatosensory structure of the face, is of particular interest because it bridges the CNS and the periphery. Migraine is generally thought to involve abnormal signaling in the trigeminovascular system, and about 50% of trigeminal neurons have CGRP immunoreactivity. Although the notion that CGRP has a central site of action in relation to migraine had gained ground over the past decade, the recent discovery that monoclonal antibodies against CGRP can prevent migraine attacks has resurrected the possibility that a peripheral site of action is involved as well. Clarification of the sites of CGRP action in migraine will be crucial to developing an understanding of mechanisms that underlie migraine so that future treatments can be rationally designed. One diagnostic criterion for migraine is photophobia, a painful and often debilitating response to non-noxious levels of light. Our laboratory previously developed a preclinical model of migraine in which the light-aversive behavior of mice is used as a surrogate of photophobia. Specifically, mice were sensitized to CGRP by introducing a nestin/hRAMP1 transgene. In these mice versus control littermates, light aversion in response to central (intracerebroventricular, ICV) injection of CGRP was enhanced in dim light. In wild-type mice, CGRP (ICV) also elicited aversion to very bright light; this did not occur in vehicle-treated mice. Additionally, I have shown that CGRP injected peripherally (intraperitoneal, IP) can induce significant light aversion in wild-type mice. I have begun to identify the sites of action outside of the central nervous system, using four lines of transgenic mice with different patterns of overexpression of CGRP receptors: global hRAMP1 mice (expression in all tissues), nestin/hRAMP1 mice (expression only in nervous tissue), tagln/hRAMP1 (expression only in smooth muscle cells), and tek2/hRAMP1 (expression in endothelial cells). As predicted, in the global hRAMP mice light aversion, in response, to IP-injected CGRP was enhanced. However, in nestin/hRAMP1 mice, only ICV-injected, and not IP-injected, CGRP induced enhanced light aversion. This finding suggests that peripheral CGRP activates neural pathways involved in light aversion, but by an indirect mechanism. To determine where in the periphery CGRP is acting, a pharmacological and genetic approach was taken. Since CGRP is one of the most potent vasodilators in the body, it is well positioned to have vascular effects that induce light aversive behavior. This hypothesis was based on findings that 1) intravenous administration of CGRP in human subjects can cause migraine pain, and 2) perivascular CGRP can sensitize the trigeminal nerve, which could alter synaptic transmission to the central nervous system and 3) CGRP monoclonal antibodies are effective in clinical trial and likely do not cross the blood brain barrier. Thus, there is a mechanism by which CGRP in the periphery can sensitize the trigeminal nerve and alter sensory perception, leading to photophobia. The role of the vasculature in migraine, specifically vasodilation, has been controversial and now the consensus is that it is neither necessary nor sufficient. First, I wanted to test the role of vasodilation in this model. I pharmacologically inhibited CGRP-induced vasodilation using two vasoconstrictors, phenylephrine and endothelin-1. Blocking CGRP-induced vasodilation partially attenuates the light aversive response. Moreover, mice that overexpress the CGRP receptor in smooth muscle, but not endothelial, cells exhibit enhanced light aversion indicating a role for vascular actions of CGRP in this preclinical model of migraine. These results present clear evidence that CGRP has actions on the vasculature to induce light aversion. Additionally, the inability of blocking vasodilation to completely rescue the light aversion suggests that the vasculature may not be the only peripheral target of CGRP in migraine pathophysiology. This work improves the understanding of peripheral CGRP actions in migraine and raises awareness that contribution of the vasculature in migraine should not be ignored. The identification of sites of CGRP action in regions inside and outside of the CNS could lead to improved and more successful therapeutics for migraine.

Addiction is a complex process in which behavioral sensitization may be an important component. While the behavioral effects of sensitization are well established, the intricate neurobiology of the phenomenon is still largely unknown. Dopamine systems mediate the induction of behavioral sensitization in adult rats, but there is a large amount of evidence showing that other neurotransmitter systems also modulate the induction process. For example, the α1b-adrenergic and 5-HT2A receptor systems are known to modulate the sensitized responding of adult rats, but the roles that these receptor systems play in the induction and expression of behavioral sensitization during the preweanling period has yet to be investigated. Therefore, the purpose of this thesis was to determine whether the serotonergic and adrenergic receptor systems mediate the induction and/or expression of cocaine-induced one-trial behavioral sensitization in preweanling rats. I used a novel approach to address this question, as the receptors of interest were “protected” from the alkylating effects of EEDQ (an irreversible nonselective receptor antagonist) by prior treatment with selective antagonist drugs. More specifically, rats were given ritanserin (a serotonergic receptor antagonist), prazosin (an adrenergic receptor antagonist), or a combination of the two drugs prior to an injection of EEDQ. To study the induction of behavioral sensitization, this series of injections was administered on PD 18 (24 h before the pretreatment injection of cocaine). To study the expression of behavioral sensitization, the injections were administered on PD 20, which was the day between the drug pretreatment day and the test day. In all experiments, the test day (i.e., the day on which the challenge dose of cocaine was given) was on PD 21. Control experiments were performed for both the induction and expression paradigms in order to determine whether prazosin and ritanserin independently affected sensitization. Results showed that the receptor inactivation caused by EEDQ blocked both the induction and expression of cocaine-induced one-trial behavioral sensitization. Importantly, administering prazosin and ritanserin did not protect the induction of the sensitized locomotor response, which suggests that serotonergic and adrenergic receptors do not mediate cocaine-induced one-trial behavioral sensitization in preweanling rats. This conclusion should be tempered, however, because co-administration of prazosin and ritanserin affected the locomotor activity and sensitized responding of cocaine-treated rats independent of the actions of EEDQ. Considering both past and present results, the most harmonious conclusion is that multiple receptor systems (i.e., dopaminergic, serotonergic, adrenergic, etc.) work in unison to produce the complex phenomenon of behavioral sensitization.

Rheumatoid arthritis (RA) is caused by aberrant attack of the joints by native inflammatory system. This can lead to joint destruction and pain that can be debilitating. Increased angiogenesis and innervation by nociceptive afferent fibers are characteristic features of RA joints, which in addition to the elevated levels of a wide variety of inflammatory mediators, are thought to play an important role in the pathogenesis of chronic inflammatory pain associated with RA. Interestingly, a recent report indicates that C–type natriuretic peptide (CNP) is increased in the blood serum of RA patients. Natriuretic peptides (NPs) control natriuresis and normalize changes in blood pressure. Many biological effects of NPs are mediated by guanylate cyclase (GC)–coupled NP receptors, NPR–A and NPR–B, whereas the third NP receptor, NPR–C, lacks the GC kinase domain and acts as the NP clearance receptor. In addition, NPR–C can couple to specific Gái–βã–mediated intracellular signaling cascades in numerous cell types. Recent studies suggest that NPs are also involved in the regulation of pain sensitivity, although the underlying mechanisms remain largely unknown. In Aim 1, I show that CNP acutely sensitized the excitation of mouse dorsal root ganglia (DRG) sensory neurons that is dependent on the transient receptor potential vanilloid–1 (TRPV1). CNP potentiated capsaicin– and proton–activated TRPV1 currents in cultured mouse DRG neurons and increased neuronal firing frequency, an effect that was absent in DRG neurons from TRPV1−/−mice. Further, CNP injection into mouse hind paw led to the development of thermal hyperalgesia, which was absent in TRPV1−/−mice. In Aim 2, I dissected the signaling mechanism underlying TRPV1 sensitization by CNP. My results show that all 3 functional NPRs are expressed in mouse DRG neurons; however NPR–A/B–cGMP signaling is not involved in CNP–mediated sensitization of TRPV1. Interestingly, I observed that sensitization of TRPV1 by CNP is dependent on protein kinase C (PKC) activity. Furthermore, I found that NPR–C is co–expressed in TRPV1–expressing mouse DRG neurons and can be co–immunoprecipitated with Gαi, but not with Gαq/11 or Gαs subunits. CNP treatment induced translocation of PKCå to the plasma membrane of these neurons, which was attenuated by pertussis toxin pre–treatment. Accordingly, CNP–induced sensitization of TRPV1 was attenuated by pre–treatment of DRG neurons with the specific inhibitors of Gβã, phospholipase–Cβ (PLCβ) or PKC, but not of protein kinase A (PKA), and by mutations at two PKC phosphorylation sites, S502 and S800, in the TRPV1 protein. Furthermore, the development of thermal hyperalgesia in CNP–injected hindpaw was attenuated by administration of specific inhibitors of Gβã or PKC. Thus, my work identifies the Gβã–PLCâ–PKC–dependent potentiation of TRPV1 as a novel signaling cascade recruited by CNP in mouse DRG neurons that can lead to enhanced nociceptor excitability and thermal hypersensitivity. Such signaling cascade could presumably constitute one of the mechanisms underlying chronic inflammatory joint pain associated with RA.

<p>Smoking is a worldwide problem and it is the second major cause of death. People often try to quit, but few succeed mainly because of withdrawal symptoms such as irritability, anxiety, increased appetite, hyperventilation and difficulty concentrating.</p><p>The overall aim of this project was to study neurochemical changes in the brain following sensitization to nicotine which could give more information about what causes an individual to go from using drugs to abusing the drugs. Therefore, we investigated messenger ribonucleic acid (mRNA) expression of several genes known to be involved in the mesolimbic dopamine pathway in the nucleus accumbens, caudate putamen, prefrontal cortex and medial prefrontal cortex using real-time polymerase chain reaction (real-time PCR).</p><p>The results showed that in the nucleus accumbens, mRNA expression of gamma-aminobutyric acid (GABA) Aα1 subunit receptor and GABA transporter 3 (GAT-3) were significantly increased following nicotine administration, while in the caudate putamen no difference in expression was observed. In prefrontal cortex, the expression of adrenergic subunit receptor α2A was significantly increased following hexamethonium administration. In medial prefrontal cortex a significant decrease of expression of GAT-1 was shown following nicotine and hexamethonium administration, while a decrease of CART expression only was shown following nicotine administration.</p><p>Overall, these changes in the GABA system may help to explain the mechanism of nicotine sensitization.</p>

Background In the past half-century, the prevalence of asthma among children and adolescents has risen and asthma has become an important public health challenge in Sweden as well as in many other countries, necessitating further studies on this complex disease and its risk factor pattern. The studies included in this thesis aimed to investigate the clinical expression of childhood asthma over time, to describe the determinants of new-onset and remission of asthma, and to evaluate possible environmental risk factors in northern Sweden. Methods As the result of a repeated questionnaire survey among primary school children aged 7-8 years in three municipalities in the north of Sweden, two pediatric cohorts were formed, one in 1996 (n=3430) and one in 2006 (n=2585). The cohort created in 1996 was followed annually until the age of 19 years. Skin prick testing was performed on children in both cohorts. Lung function and bronchial hyperreactivity testing were carried out in children with asthma in the first cohort. The study participation and retention rates were very high in both cohorts. Among children in the second cohort living in Luleå, the home addresses were assigned to coordinates in a geographical information system (GIS) to evaluate the impact on respiratory health of living near roads with much traffic, which was measured as the number of vehicles daily. We used a validated reported diagnosis of asthma and International Study of Asthma and Allergies in Childhood (ISAAC) questions were incorporated into the questionnaire. A cross-sectional study of children of the same age ten years apart, longitudinal studies on asthma incidence and remission as well as a cross-sectional study on vehicle traffic were performed. Results While children aged 7-8 years in 2006 more often had a physician-diagnosed asthma compared to children of the same age in 1996 (7.4% vs 5.7%, p&lt;0.001), they had less asthma symptoms, especially severe symptoms. In parallel, a more beneficial environment and a more intense treatment with inhaled corticosteroids (ICS) were observed. The explanation for this change in clinical expression probably includes also an increased awareness and diagnosing of asthma. From age 12 years to age 19 years, the cumulative incidence of physician-diagnosed asthma was 7.2% and of current wheeze 22.0%. The risk of new-onset asthma in adolescence was increased among girls, sensitized and those with heredity for asthma. Smoking and home dampness increased the risk for incident wheeze. The risk for both incident asthma and wheeze was inversely related to number of siblings. Among children with current asthma at age 7-8 years, 21% were in remission, 38% had periodic asthma and 41% had persistent asthma at a follow-up at age 19 years. Subjects in remission and with periodic asthma had significantly less airway obstruction and showed less bronchial hyperreactivity compared to subjects with persistent asthma. The probability of asthma remission from childhood to early adulthood was significantly increased by absence of allergic sensitization, male gender and a low asthma severity scoring at age 7-8 years. Sensitization to furred animals was more important as a determinant of both incidence and remission than sensitization to pollen. Living close to roads with high traffic flows, especially with heavy vehicles, was associated with an increased risk for current wheeze. Stratified analyses showed that the effect of traffic on asthma and wheeze was restricted to non-sensitized subjects. Conclusion Asthma onset in adolescence was more common among girls and remission was more common among boys. Children sensitized to furred animals and children with a more severe asthma were risk groups for persistence of asthma. Environmental factors such as smoking and dampness were associated to onset of asthma symptoms during adolescence, and vehicle traffic was associated with asthma symptoms among children also in a small city with relatively low traffic flows. Preventive measures like smoking reduction programs, improvement of damp housing conditions and separation of areas where many children live from heavily trafficked roads could prove to be beneficial. / OLIN-studierna

Inflammatory and neuropathic pains arise in part from sensitization at nociceptive synapses in the spinal cord. Activity-dependent signaling cascades converge onto the tyrosine kinase Src, which participates in augmenting the function of N-methyl-D-aspartate receptors (NMDARs) and thus potentiates the nociceptive system. Src is capable of these effects because it is anchored to the NMDAR complex via an adaptor protein called NADH dehydrogenase subunit 2 (ND2). There is evidence that this interaction occurs between amino acids 40-49 of Src and amino acids 310-321 of ND2. I have determined that a peptide consisting of amino acids 310-321 of ND2, and affixed to the HIV Tat domain for cell permeability, is capable of alleviating tactile allodynia induced by Complete Freund's Adjuvant (CFA) in rats. Src40-49Tat was not effective in two models of inflammatory pain. This work further implicates the Src-ND2 interaction in pain hypersensitivity and suggests that Tat ND2 310-321 may alleviate it.

Inflammatory and neuropathic pains arise in part from sensitization at nociceptive synapses in the spinal cord. Activity-dependent signaling cascades converge onto the tyrosine kinase Src, which participates in augmenting the function of N-methyl-D-aspartate receptors (NMDARs) and thus potentiates the nociceptive system. Src is capable of these effects because it is anchored to the NMDAR complex via an adaptor protein called NADH dehydrogenase subunit 2 (ND2). There is evidence that this interaction occurs between amino acids 40-49 of Src and amino acids 310-321 of ND2. I have determined that a peptide consisting of amino acids 310-321 of ND2, and affixed to the HIV Tat domain for cell permeability, is capable of alleviating tactile allodynia induced by Complete Freund's Adjuvant (CFA) in rats. Src40-49Tat was not effective in two models of inflammatory pain. This work further implicates the Src-ND2 interaction in pain hypersensitivity and suggests that Tat ND2 310-321 may alleviate it.

<p>Adolescence is a critical period for drug addiction in humans. Most lifelong drug addiction is initiated during adolescence and the progression from initial drug use to the expression of addictive behaviors occurs more rapidly during adolescence than in adulthood. The purpose of this work was to examine if the adolescent brain uniquely responds to the addictive stimulant cocaine. This was accomplished by comparing the following measures in adolescent and adult male rats: locomotor responses to cocaine across a range of doses in two acute cocaine binge models, plasma cocaine and brain concentrations, locomotor responses to apomorphine, the relative magnitude of locomotor sensitization induced by a single high dose of cocaine (40 mg/kg), and cocaine-induced c-fos and zif268 expression. We determined that young adolescent (PN 28) rats had greater stereotypy responses to all doses of a repeated dose cocaine binge (15 mg/kg), the highest dose of an escalating dose binge (25 mg/kg), and low dose apomorphine. In addition to showing exaggerated acute locomotor responses to cocaine, young adolescents demonstrated a form of intrabinge sensitization that was absent in adults. Exaggerated adolescent locomotor responses could not be attributed to cocaine metabolism as we did not observe greater cocaine plasma or brain concentrations in adolescents compared to adults. A single high dose of cocaine (40 mg/kg) induced more ambulatory and stereotypy sensitization in young adolescents than adults. Further, the magnitude of the acute locomotor response to cocaine predicted the magnitude of locomotor sensitization in individual adolescents. We also showed that cocaine dose-dependently caused age-specific increases in the expression of the plasticity-associated immediate early genes c-fos and zif268: low dose (10 mg/kg) cocaine caused greater increases in striatal c-fos expression in adolescents whereas high dose (40 mg/kg) cocaine caused greater increases in striatal c-fos and zif268 expression in adults. Both doses of cocaine stimulated bigger increases in cortical zif268 expression in adults compared to adolescents. Finally, we demonstrated that the coordinated expression of striatal c-fos and zif268 develops during adolescence: there was no correlation between striatal c-fos and zif268 expression in individual adolescents but a strong correlation was seen in adults. The results of these experiments demonstrate that adolescents have unique molecular responses to acute cocaine and may help explain how adolescents show unique adaptive changes following continued cocaine use.</p> / Dissertation

No description available.

Smoking is a worldwide problem and it is the second major cause of death. People often try to quit, but few succeed mainly because of withdrawal symptoms such as irritability, anxiety, increased appetite, hyperventilation and difficulty concentrating. The overall aim of this project was to study neurochemical changes in the brain following sensitization to nicotine which could give more information about what causes an individual to go from using drugs to abusing the drugs. Therefore, we investigated messenger ribonucleic acid (mRNA) expression of several genes known to be involved in the mesolimbic dopamine pathway in the nucleus accumbens, caudate putamen, prefrontal cortex and medial prefrontal cortex using real-time polymerase chain reaction (real-time PCR). The results showed that in the nucleus accumbens, mRNA expression of gamma-aminobutyric acid (GABA) Aα1 subunit receptor and GABA transporter 3 (GAT-3) were significantly increased following nicotine administration, while in the caudate putamen no difference in expression was observed. In prefrontal cortex, the expression of adrenergic subunit receptor α2A was significantly increased following hexamethonium administration. In medial prefrontal cortex a significant decrease of expression of GAT-1 was shown following nicotine and hexamethonium administration, while a decrease of CART expression only was shown following nicotine administration. Overall, these changes in the GABA system may help to explain the mechanism of nicotine sensitization.