Rozprawy doktorskie na temat „Migraine”

Migraine, comprised of migraine with aura (MA) and migraine without aura (MO), is a painful neurovascular disease, affecting approximately 16% of the general population. It is characterised by a wide variety of symptoms including headache, nausea and vomiting, and photo- and phonophobia. The disorder is complex involving not only multiple genes, but also specific environmental factors, which can induce attacks in genetically predisposed individuals. Hyperhomocysteinaemia is a known risk factor for cerebrovascular, peripheral vascular and coronary heart disease. The Methylenetetrahydrofolate Reductase (MTHFR) enzyme is involved in homocysteine metabolism. Furthermore, it has been reported that a homozygous mutation (677C to T; Ala to Val) in the 5,10-MTHFR gene is associated with an elevation in plasma homocysteine levels (Frosst et al., 1995). This common mutation in the MTHFR gene has recently been associated with migraine with aura in a Japanese cohort (Kowa et al., 2000). The present study was designed to determine the prevalence of the MTHFR C677T mutation in Australian patients with migraine and to determine whether this mutation is associated with the disease in Caucasians. A large case-control study, consisting of 270 patients with migraine (167 with aura and 103 without aura), and 270 normal matched controls was investigated. Genotypic results indicated that the prevalence of the homozygous (T/T) genotype in migraine sufferers (15%) was higher than that of controls (9%) (P = 0.084). Furthermore, the frequency of the mutant (T/T) genotype in individuals with MA (19%) was significantly higher than in controls (9%) (P = 0.006). Interestingly, the risk of MA was ~2.5-fold higher in suffers possessing the homozygous variant (OR = 2.52, CI: 1.42 - 4.47, P = 0.0012). To confirm the MTHFR allelic association with MA, family-based tests were performed in an independent pedigrees group, where only those with MA were considered affected. Results from both the Pedigree Disequilibrium Test (PDT) and Family-Based Association Test (FBAT) analysis revealed slight, although not significant (PDT test, P = 132; and FBAT test, P = 0.390), over-transmission of the mutant allele (T) from parents to affected offspring. However, despite the MTHFR variant having a high heterozygosity (0.48), there were a limited number of informative transmissions for the MTHFR variant in the pedigree group resulting in reduced power for these tests. In conclusion, our results support the trends reported in the Japanese migraine study and suggest that the homozygous 677T gene variant causing mild hyperhomocysteinaemia, is a genetic risk factor for migraine, and indicate that further studies investigating the role of this gene are warranted. Mutations in various ion channel genes are responsible for neurovascular and other neurological disorders. Inherited ion channel mutations or "channelopathies" are increasingly found to be the cause of various neurological disorders in humans. Wittekindt and colleagues (1998) reported that the calcium-activated potassium channel (hKCa3) gene is a good candidate for schizophrenia and bipolar disorder (BD), as well as for other neurological disorders such as migraine. The hKCa3 gene is a neuronal small conductance calcium-activated potassium channel, which contains a polyglutamine tract, encoded by a polymorphic CAG repeat in the gene. The hKCa3 gene encodes a protein of 731 amino acids containing two adjacent polyglutamine sequences in its N-terminal domain separated by 25 amino acids. The C-terminal polyglutamine sequence is highly polymorphic in length (Austin et al., 1999). hKCa3 plays a critical role in determining the firing pattern of neurons via the generation of slow after-polarization pulses and the regulation of intracellular calcium channels (Kohler et al., 1996). Three distinct mutations in the a1 calcium channel gene have been shown to cause SCA-6, episodic ataxia-2 and familial hemiplegic migraine (FHM) (Ophoff et al., 1996). The hKCa3 gene contains a highly polymorphic CAG repeat that was initially mapped (Chandy et al., 1997) to a schizophrenia locus on chromosome 22 (Pulver et al., 1994). Recently Austin et al (1999) re-mapped hKCa3 and found it to reside on chromosome 1q21, a region that has been linked to FHM (Austin et al., 1999), a rare subtype of MA (Ducros et al., 1997; Gardner et al., 1998), and a region recently showing genetic linkage to typical migraine (Lea et al., 2002). The hKCa3 polymorphism results in small variations in polyglutamine number, similar to those that occur in the calcium channel a1a subunit gene (CACNA1A), which is encoded by CAG expansions and thought to cause Spinocerebellar Ataxia Type 6 via loss of channel function (Austin et al., 1999). Given the recent linkage of FHM to the region of chromosome 1q21, to which hKCa3 resides, and also linkage of typical migraine to this region, a large case-control study investigating this hKCa3 CAG marker and consisting of 270 migraine and 270 stringently matched healthy controls was undertaken. Our results indicated that there was no statistically significant difference in allele distributions for this marker between migraine and non-migraine patients (P >0.05). No significant difference in the allelic distribution was observed in the MA or MO groups when compared to controls (P >0.05) and there was no significant difference in CAG repeat length distribution between the migraine group and controls (P = 0.92), or between the MA and MO groups (P = 0.72) collectively. Hence, the CAG repeat in this gene does not show expansion in migraine. Overall, our results provide no genetic evidence to suggest that the hKCa3 CAG repeat polymorphism is involved in migraine aetiology in Australian Caucasians. Thus the involvement of the hKCa3 gene in migraine is not likely, although the hKCa3 gene remains an important candidate for other neurological disorders that may be linked to the 1q21.3 chromosomal region.

Migraine, comprised of migraine with aura (MA) and migraine without aura (MO), is a painful neurovascular disease, affecting approximately 16% of the general population. It is characterised by a wide variety of symptoms including headache, nausea and vomiting, and photo- and phonophobia. The disorder is complex involving not only multiple genes, but also specific environmental factors, which can induce attacks in genetically predisposed individuals. Hyperhomocysteinaemia is a known risk factor for cerebrovascular, peripheral vascular and coronary heart disease. The Methylenetetrahydrofolate Reductase (MTHFR) enzyme is involved in homocysteine metabolism. Furthermore, it has been reported that a homozygous mutation (677C to T; Ala to Val) in the 5,10-MTHFR gene is associated with an elevation in plasma homocysteine levels (Frosst et al., 1995). This common mutation in the MTHFR gene has recently been associated with migraine with aura in a Japanese cohort (Kowa et al., 2000). The present study was designed to determine the prevalence of the MTHFR C677T mutation in Australian patients with migraine and to determine whether this mutation is associated with the disease in Caucasians. A large case-control study, consisting of 270 patients with migraine (167 with aura and 103 without aura), and 270 normal matched controls was investigated. Genotypic results indicated that the prevalence of the homozygous (T/T) genotype in migraine sufferers (15%) was higher than that of controls (9%) (P = 0.084). Furthermore, the frequency of the mutant (T/T) genotype in individuals with MA (19%) was significantly higher than in controls (9%) (P = 0.006). Interestingly, the risk of MA was ~2.5-fold higher in suffers possessing the homozygous variant (OR = 2.52, CI: 1.42 - 4.47, P = 0.0012). To confirm the MTHFR allelic association with MA, family-based tests were performed in an independent pedigrees group, where only those with MA were considered affected. Results from both the Pedigree Disequilibrium Test (PDT) and Family-Based Association Test (FBAT) analysis revealed slight, although not significant (PDT test, P = 132; and FBAT test, P = 0.390), over-transmission of the mutant allele (T) from parents to affected offspring. However, despite the MTHFR variant having a high heterozygosity (0.48), there were a limited number of informative transmissions for the MTHFR variant in the pedigree group resulting in reduced power for these tests. In conclusion, our results support the trends reported in the Japanese migraine study and suggest that the homozygous 677T gene variant causing mild hyperhomocysteinaemia, is a genetic risk factor for migraine, and indicate that further studies investigating the role of this gene are warranted. Mutations in various ion channel genes are responsible for neurovascular and other neurological disorders. Inherited ion channel mutations or "channelopathies" are increasingly found to be the cause of various neurological disorders in humans. Wittekindt and colleagues (1998) reported that the calcium-activated potassium channel (hKCa3) gene is a good candidate for schizophrenia and bipolar disorder (BD), as well as for other neurological disorders such as migraine. The hKCa3 gene is a neuronal small conductance calcium-activated potassium channel, which contains a polyglutamine tract, encoded by a polymorphic CAG repeat in the gene. The hKCa3 gene encodes a protein of 731 amino acids containing two adjacent polyglutamine sequences in its N-terminal domain separated by 25 amino acids. The C-terminal polyglutamine sequence is highly polymorphic in length (Austin et al., 1999). hKCa3 plays a critical role in determining the firing pattern of neurons via the generation of slow after-polarization pulses and the regulation of intracellular calcium channels (Kohler et al., 1996). Three distinct mutations in the a1 calcium channel gene have been shown to cause SCA-6, episodic ataxia-2 and familial hemiplegic migraine (FHM) (Ophoff et al., 1996). The hKCa3 gene contains a highly polymorphic CAG repeat that was initially mapped (Chandy et al., 1997) to a schizophrenia locus on chromosome 22 (Pulver et al., 1994). Recently Austin et al (1999) re-mapped hKCa3 and found it to reside on chromosome 1q21, a region that has been linked to FHM (Austin et al., 1999), a rare subtype of MA (Ducros et al., 1997; Gardner et al., 1998), and a region recently showing genetic linkage to typical migraine (Lea et al., 2002). The hKCa3 polymorphism results in small variations in polyglutamine number, similar to those that occur in the calcium channel a1a subunit gene (CACNA1A), which is encoded by CAG expansions and thought to cause Spinocerebellar Ataxia Type 6 via loss of channel function (Austin et al., 1999). Given the recent linkage of FHM to the region of chromosome 1q21, to which hKCa3 resides, and also linkage of typical migraine to this region, a large case-control study investigating this hKCa3 CAG marker and consisting of 270 migraine and 270 stringently matched healthy controls was undertaken. Our results indicated that there was no statistically significant difference in allele distributions for this marker between migraine and non-migraine patients (P >0.05). No significant difference in the allelic distribution was observed in the MA or MO groups when compared to controls (P >0.05) and there was no significant difference in CAG repeat length distribution between the migraine group and controls (P = 0.92), or between the MA and MO groups (P = 0.72) collectively. Hence, the CAG repeat in this gene does not show expansion in migraine. Overall, our results provide no genetic evidence to suggest that the hKCa3 CAG repeat polymorphism is involved in migraine aetiology in Australian Caucasians. Thus the involvement of the hKCa3 gene in migraine is not likely, although the hKCa3 gene remains an important candidate for other neurological disorders that may be linked to the 1q21.3 chromosomal region.
Thesis (Masters)
Master of Philosophy (MPhil)
School of Health Sciences
Full Text

Better characterisation of migraine is critical to enhancing its diagnosis, assessment and, ultimately, effective treatments. The aim of this thesis was to better characterise migraine through more detailed investigation of selected headache-related factors and to compare these factors with those seen in other commonly occurring recurrent headaches. The factors investigated in this thesis were neurochemical profile, cervical musculoskeletal impairments, and patient experience, represented by pain and disability characteristics, emotional state and other personal factors. This thesis provides deeper information regarding the nature and characteristics of migraine compared with non-migraine headaches (tension-type and cervicogenic headaches). This thesis has established the potential of GABA as a biomarker for migraine and implies the possible role of GABA in the disease process. This thesis has also characterised migraine according to cervical musculoskeletal impairments and patient experience embodying disability, pain, central sensitisation, and other personal factors. The implications for clinical practice are to assess cervical musculoskeletal impairments and patient experience to facilitate differential diagnosis and prognostication, and to educate patients on the nature of their headaches. Findings from the thesis may also be used by guideline developers, providing stimulus for further discussions on the definition of migraine and reporting of participant selection criteria, with reference to this definition, in clinical trials. Future research directions are identified in validating GABA as a migraine biomarker and elucidating its pathophysiology. By characterising migraine more fully, findings from this thesis will inform the development of effective treatments that could possibly target GABA or clinical characteristics found to be present in migraine. Ultimately this should achieve better health outcomes for people with migraine.

Migraine is a common, highly disabling episodic neurological disorder affecting more than 10% of the population and arises from a primary brain dysfunction that leads to episodic activation and sensitization of the trigeminovascular pain pathway. Familial hemiplegic migraine (FHM) is a rare and very severe monogenic autosomal dominant subtype of migraine with aura. Apart from the motor aura and the possible longer duration of the aura, typical FHM attacks resemble migraine with aura attacks (Pietrobon and Moskowitz, 2013); thus, FHM can be considered as a model of the common forms of migraine. Gain-of-function missense mutations in CACNA1A gene, encoding the pore-forming subunit of the neuronal voltage-gated Ca2+ channel CaV2.1 (also known as P/Q-type Ca2+ channel), cause FHM type 1 (FHM1) and loss-of-function mutations in ATP1A2, the gene encoding the astrocytic Na,K-ATPase α2 subunit, cause FHM type 2 (FHM2) (Ophoff et al., 1996; De Fusco et al., 2003). FHM knock-in (KI) mice carrying mutations causing either FHM1 or FHM2 show facilitation of induction and propagation of experimental CSD (van den Maagdenberg et al., 2004, 2010; Leo et al., 2011), the phenomenon underlying migraine aura and a key triggering event for trigeminovascular activation. FHM1 KI mice, carrying the R192Q mutation, show an increased influx of Ca2+ through P/Q-type Ca2+ channels in neurons, including cortical pyramidal cells, and an increased probability of glutamate release at cortical pyramidal cells synapses (Pietrobon, 2010; Tottene et al., 2009), that may explain the facilitation of experimental CSD. Recent findings in our laboratory (Fabbro, Sessolo, Vecchia and Pietrobon, manuscript in preparation) show that the frequency of the up-states recorded in acute cortical slices, that resemble slow oscillation in vivo (Steriade et al., 1993), is larger in FHM1 KI than WT mice, suggesting that the gain-of-function of P/Q-type Ca2+ channels facilitates the mechanisms of up-states generation and/or decreases the refractory period after an up-state. The first aim of my work was to further investigate the role of P/Q-type Ca2+ channels in the recurrent network activity underlying the up-states in WT mice. I studied the effect of pharmacological inhibition of P/Q-type Ca2+ channels on the up-state activity recorded from layer 2/3 pyramidal neurons in acute slices of mouse somatosensory cortex, by performing single and double patch clamp experiments. I found that the block of P/Q-type Ca2+ channels transforms the up-states into events resembling interictal epileptiform discharges. I evaluated the mean excitatory and inhibitory synaptic conductances (Ge and Gi) during the control up-states, during the simil-interictal epileptiform events after P/Q-type Ca2+ channels block as well as immediately after the application of the P/Q-type Ca2+ channels blocker when the channels were not completely blocked. I showed that 1) P/Q-type Ca2+ channels have a dominant role in controlling both excitatory and inhibitory synaptic transmission onto pyramidal cells during the spontaneous recurrent network activity that underlies the up-states. However, the block of P/Q-type Ca2+ channels reduces recurrent inhibition more than recurrent excitation and shifts the cortical excitation-inhibition balance towards excitation. 2) When, as a consequence of the block of P/Q-type Ca2+ channels, Ge/Gi increases above a critical value, the spontaneous network activity changes and the up-states are transformed into events resembling simil-interictal epileptiform discharges. These data suggest that, in the cerebral cortex, P/Q-type Ca2+ channels play a more prominent role in controlling inhibitory compared to excitatory synaptic transmission. Given that at many cortical synapses P/Q- and N-type (also known as CaV2.2) Ca2+ channels cooperate in controlling synaptic transmission, I also investigated the effect of blocking N-type Ca2+ channels on up-state activity. Pharmacological inhibition of this channel strongly reduces the up-states frequency, indicating a role for N-type Ca2+ channel in controlling up-states frequency. After the block of N-type Ca2+ channels, Ge/Gi increases but not sufficiently to transform up-states in simil-interictal epileptiform events. The aim of my second project was to investigate the unknown mechanisms underlying facilitation of experimental CSD in FHM2 KI mice. After setting the conditions in which facilitation of CSD was observed in vitro, I studied three possible mechanisms that may underlie the facilitation of CSD in heterozygous FHM2 KI mice, in acute slices of mouse somatosensory cortex. Given the specific localization and functional coupling of the α2 Na,K-ATPase to glutamate transporters in astrocyte processes surrounding cortical glutamatergic synapses (Cholet et al., 2002), I first investigated whether the loss-of-function of α2 Na,K-ATPase results in an impaired astrocyte-mediated clearance of glutamate from the synaptic cleft during cortical neuronal activity. I monitored the rate of glutamate clearance electrophysiologically, by measuring the synaptically-activated glutamate transporter-mediated current (STC) evoked in astrocytes of layer 1 by extracellular stimulation of neuronal afferents in the same layer. Either single pulse stimulation or trains of stimuli at high frequencies (50 and 100 Hz) were delivered. I isolated the STC pharmacologically in order to measure the STC decay time course that provides a relative measure of the glutamate clearance by astrocytes (Bergles and Jahr, 1997; Diamond and Jahr, 2000). I found that the clearance of glutamate release is slower in FHM2 KI compared to WT mice. The slowing of glutamate clearance was more pronounced after a train stimulation than a single stimulus and increased with increasing frequency of the train. My data show that the loss-of-function of α2 Na,K-ATPase results in an impairment of glutamate clearance and suggest that the impairment increases with increasing frequency of cortical activity. Surprisingly, the STC amplitude after a single pulse stimulation was higher in FHM2 KI than in WT mice. Given that the STC amplitude is proportional to the glutamate release evoked at the synapses by the extracellular stimulation (Bergles and Jahr, 1997; Diamond and Jahr, 2000), this result would suggest that the extracellular stimulation elicits a larger glutamate release in FHM2 KI than in WT mice. Indeed, during repetitive stimulation the STC depressed more in FHM2 KI than WT mice, as expected if the probability of glutamate release is increased in the mutant mice. Given the key role of NMDA receptors in the positive feedback cycle, that ignites CSD (Tottene et al., 2011; Pietrobon and Moskowitz, 2014), both the reduced clearance of glutamate and the increased glutamate release may be implicated in the facilitation of CSD in FHM2 KI mice. Given that most models of CSD include local increase of extracellular K+ concentration above a critical value, as a triggering event in the initiation of CSD (Pietrobon and Moskowitz, 2014), and that pharmacological evidence indicates that α2 and/or α3 Na,K-ATPase participate in the clearance of K+ from the extracellular space during intense neuronal activity (D’Ambrosio et al., 2002; Kofuji and Newman, 2004), I investigated whether K+ clearance by astrocytes is impaired in FHM2 KI mice. I evaluated the rate of K+ clearance from the interstitial space, by recording the slowly decaying current, which is mainly due to K+ influx through Kir channels, evoked in layer 1 astrocytes by extracellular stimulation. I measured the decay time course of this current, which provides an indirect measure of the K+ clearance rate by astrocytes. Preliminary experiments show that the decay time course of the K+ current evoked by train stimulation is similar in WT and FHM2 KI mice. If confirmed, this result would indicate that there are no changes in the rate of K+ clearance in FHM2 KI mice compared to WT. Given that α2 Na,K-ATPase is tightly coupled to the Na+/Ca2+ exchanger at plasma membrane microdomains that overlay the endoplasmic reticulum (Lencesova et al., 2004; Golovina et al., 2003) and hence its loss-of-function could influence Ca2+ homeostasis, we investigated whether the Ca2+ content in the intracellular Ca2+ stores of astrocytes in FHM2 KI mice is increased. We obtained an indirect measure of the amount of Ca2+ in the stores, by measuring in cultured cortical astrocytes the Ca2+ transient induced by ionomycin in Ca2+-free medium. This transient was larger in FHM2 KI mice compared to WT mice, indicating that the Ca2+ content is increased in the intracellular Ca2+ stores of astrocytes in FHM2 KI mice. I measured CSD threshold and velocity after depletion of intracellular Ca2+ stores by CPA, a SERCA inhibitor, and I observed that depletion of Ca2+ stores reduces the facilitation of CSD in FHM2 KI mice, without affecting CSD in WT mice. This result suggests a role of increased Ca2+ concentration within the astrocytes intracellular stores in the facilitation of experimental CSD in FHM2 KI mice.
L’emicrania è un disturbo neurologico comune e altamente invalidante, che colpisce più del 10% della popolazione, dovuto ad una disfunzione primaria del cervello che porta all’attivazione e alla sensibilizzazione episodica delle vie nocicettive trigeminovascolari. L’emicrania emiplegica familiare (FHM) è un rara forma di emicrania con aura considerata un buon modello per lo studio dell’emicrania; infatti gli attacchi tipici di FHM sono simili a quelli della normale emicrania con aura, eccetto per il sintomo dell’emiparesi (Pietrobon and Moskowitz, 2013). Mutazioni missenso con guadagno di funzione nel gene CACNA1A, codificante la subunità formante il poro dei canali del Ca2+ voltaggio dipendenti CaV2.1 (denominati anche canali del Ca2+ di tipo P/Q), causano FHM di tipo 1 (FHM1) e mutazioni con perdita di funzione nel gene ATP1A2, codificante la subunità astrocitaria α2 della Na,K-ATPasi, causano FHM di tipo 2 (FHM2) (Ophoff et al., 1996; De Fusco et al., 2003). Topi knock-in (KI) per le mutazioni che causano FHM1 e FHM2 presentano una facilitazione nell’induzione e nella propagazione della cortical spreading depression (CSD) (van den Maagdenberg et al., 2004, 2010; Leo et al., 2011), il fenomeno neurologico alla base dell’aura emicranica e un evento chiave innescante l’attivazione del sistema trigeminovascolare. Topi FHM1 KI per la mutazione R192Q, mostrano un aumentato influsso di Ca2+ attraverso i canali del Ca2+ di tipo P/Q e un aumento nella probabilità di rilascio di glutammato alle sinapsi piramidali della corteccia (Pietrobon, 2010; Tottene et al., 2009), che potrebbe spiegare la facilitazione della CSD sperimentale osservata in questi topi. Recentemente, nel nostro laboratorio, è stato dimostrato che la frequenza degli up-state registrati in fettine acute di corteccia, simili alle oscillazioni lente riportate in vivo (Steriade et al., 1993), è maggiore nel topo FHM1 KI che nel WT. Questo dato suggerisce che il guadagno di funzione dei canali del Ca2+ di tipo P/Q faciliti i meccanismi di generazione degli up-state e/o riduca il periodo refrattario dopo un up-state (Fabbro, Sessolo, Vecchia and Pietrobon, dati non pubblicati). Lo scopo della prima parte del mio lavoro è stato quello di approfondire il ruolo dei canali del Ca2+ di tipo P/Q nell’attività ricorrente di circuito alla base degli up-state nei topi WT. Ho studiato l’effetto dell’inibizione farmacologica dei canali del Ca2+ di tipo P/Q sulla attività ad up-state registrata da neuroni piramidali dello strato 2/3 in fettine acute di corteccia somatosensoriale di topo. Per questo scopo ho eseguito esperimenti di singolo e doppio patch clamp. Ho trovato che il blocco di questi canali del Ca2+ trasforma gli up-state in eventi che ricordano le scariche epilettiformi interictali. Ho analizzato le conduttanze medie eccitatorie ed inibitorie (Ge and Gi) durante gli up-state in controllo, durante gli eventi epilettiformi simil-interictali dopo il blocco dei canali del Ca2+ di tipo P/Q e nel periodo iniziale di applicazione dell’inibitore di questi canali, ovvero quando solo una parte dei canali era stata bloccata. Ho trovato che 1) i canali del Ca2+ di tipo P/Q svolgono un ruolo fondamentale nel controllare sia la trasmissione sinaptica eccitatoria sia quella inibitoria sulle cellule piramidali durante l’attività ricorrente di circuito sottostante gli up-state. Tuttavia, il blocco di questi canali riduce maggiormente l’inibizione ricorrente rispetto all’eccitazione, spostando di conseguenza l’equilibrio eccitazione-inibizione a favore dell’eccitazione. 2) Quando, come risultato del blocco dei canali del Ca2+ di tipo P/Q, il rapporto Ge/Gi supera un valore critico, l’attività spontanea di circuito cambia e gli up-state vengono trasformati in eventi simili alle scariche epilettiformi interictali. Questi dati suggeriscono che, nella corteccia cerebrale, i canali del Ca2+ di tipo P/Q svolgono un ruolo predominante nel controllo della trasmissione sinaptica inibitoria rispetto a quella eccitatoria. Dal momento che in molte sinapsi corticali, i canali del Ca2+ di tipo P/Q e di tipo N (denominati anche CaV2.2) cooperano nel controllare la trasmissione sinaptica, ho studiato anche l’effetto del blocco dei canali del Ca2+ di tipo N sull’attività ad up-state. L’inibizione farmacologica di questi canali causa una riduzione della frequenza degli up-state, suggerendo che i canali del Ca2+ di tipo N sono coinvolti nel regolarne la frequenza. Dopo il blocco dei canali del Ca2+ di tipo N, il rapporto Ge/Gi aumenta ma non sufficientemente a trasformare gli up-state in eventi epilettiformi simil-interictali. L’obiettivo della seconda parte del mio progetto è stato quello di studiare i meccanismi, ancora non noti, alla base della facilitazione della CSD sperimentale nel topo FHM2 KI. Dopo aver determinato le condizioni sperimentali in cui poter osservare la facilitazione della CSD in vitro, ho indagato tre possibili meccanismi che potrebbero spiegare la facilitazione della CSD nel topo eterozigote FHM2 KI, in fettine acute di corteccia somatosensoriale di topo. Dato lo specifico accoppiamento sia funzionale che strutturale negli astrociti tra la α2 Na,K-ATPasi e i trasportatori del glutammato a livello delle sinapsi corticali glutammatergiche (Cholet et al., 2002), ho verificato se la perdita di funzione della α2 Na,K-ATPasi compromettesse la rimozione, mediata dagli astrociti, del glutammato dalla fessura sinaptica durante l’attività neuronale. Ho pertanto valutato il tasso di rimozione del glutammato misurando elettrofisiologicamente la corrente attivata sinapticamente mediata dai trasportatori del glutammato (STC), indotta negli astrociti dello strato 1 attraverso la stimolazione extracellulare delle afferenze neuronali nello stesso strato sia con singoli impulsi che con treni di impulsi ad alta frequenza (50 and 100 Hz). Ho isolato farmacologicamente la STC, al fine di misurarne il tempo di decadimento che fornisce una misura relativa della rimozione del glutammato mediata dagli astrociti (Bergles and Jahr, 1997; Diamond and Jahr, 2000). Ho trovato che la rimozione del glutammato rilasciato è effettivamente più lenta nei topi FHM2 KI rispetto ai topi WT. Il rallentamento della rimozione del glutammato era più pronunciato dopo un treno di impulsi rispetto a dopo un singolo stimolo e aumentava all’aumentare della frequenza del treno. I miei dati dimostrano che la perdita di funzione della α2 Na,K-ATPasi compromette la rimozione del glutammato e suggeriscono che la rimozione del glutammato diventa più inefficiente all’aumentare della frequenza dell’attività corticale. Sorprendentemente, l’ampiezza della STC dopo un singolo stimolo era più grande nel topo FHM2 KI che nel topo WT. Dal momento che l’ampiezza della STC è proporzionale al rilascio di glutammato evocato alle sinapsi dalla stimolazione extracellulare (Bergles and Jahr, 1997; Diamond and Jahr, 2000), questo risultato potrebbe suggerire che la stimolazione extracellulare provoca un rilascio di glutammato maggiore nel topo FHM2 KI che nel topo WT. Infatti, durante stimolazioni ripetute, la STC deprime di più nel topo FHM2 KI che nel topo WT, come previsto nel caso di probabilità di rilascio di glutammato aumentata nel topo FHM2 KI. Considerato il ruolo chiave dei recettori NMDA nel ciclo di feedback positivo che innesca la CSD (Tottene et al., 2011; Pietrobon and Moskowitz, 2014), sia la ridotta rimozione del glutammato sia l’aumentato rilascio del neurotrasmettitore potrebbero essere implicati nella facilitazione osservata nel topo FHM2 KI. Visto che molti modelli della CSD includono un aumento della concentrazione extracellulare di K+ al di sopra di un valore critico, come un evento innescante la CSD (Pietrobon and Moskowitz, 2014), e alla luce delle evidenze farmacologiche che indicano che l’α2 e/o l’α3 Na,K-ATPasi partecipano alla rimozione del K+ dallo spazio extracellulare durante l’attività neuronale intensa (D’Ambrosio et al., 2002; Kofuji and Newman, 2004), ho indagato se la rimozione del K+ mediata dagli astrociti fosse compromessa nel topo FHM2 KI. Ho registrato elettrofisiologicamente la corrente sostenuta indotta negli astrociti dello strato 1 da stimolazione extracellulare; questa corrente è per lo più dovuta all’influsso di K+ attraverso i canali Kir e il suo tempo di decadimento fornisce una misura indiretta della velocità di rimozione del K+ mediata dagli astrociti. Questi esperimenti preliminari evidenziano che il tempo di decadimento della corrente di K+ evocata da treni di impulsi è simile nel topo FHM2 KI e nel topo WT. Se tale risultato venisse confermato, indicherebbe che non ci sono variazioni nel tasso di rimozione del K+ nel topo FHM2 KI rispetto al topo WT. Dal momento che l’α2 Na,K-ATPasi è strettamente accoppiata allo scambiatore Na+/Ca2+ a livello di microdomini di membrana sovrapposti al reticolo endoplasmatico (Lencesova et al., 2004; Golovina et al., 2003), abbiamo valutato se il contenuto di Ca2+ nei depositi intracellulari degli astrociti nel topo FHM2 KI fosse aumentata. Abbiamo ottenuto una misura indiretta della quantità di Ca2+ nei depositi andando a misurare negli astrociti corticali in coltura i transienti di Ca2+ indotti da ionomicina in un mezzo senza Ca2+. Il transiente di Ca2+ nel topo FHM2 KI era maggiore che nel topo WT, indicando un aumentato contenuto di Ca2+ nei depositi degli astrociti del topo FHM2 KI. Misurando la soglia e la velocita della CSD dopo aver svuotato i depositi di Ca2+ usando acido ciclopiazonico (CPA), un inibitore della SERCA, ho osservato che lo svuotamento delle riserve di Ca2+ riduce la facilitazione della CSD nel topo FHM2 KI, senza influenzare la CSD nel topo WT. Questo risultato suggerisce che l’aumentata concentrazione di Ca2+ all’interno dei depositi degli astrociti è coinvolta nella facilitazione della CSD sperimentale nel topo FHM2 KI.

Migraine is a common disabling condition likely to be associated with dysfunction of brain pathways involved in pain and other sensory modalities. Functional brain imaging in acute migraine has proved challenging due to the logistic problems associated with an episodic condition although it has provided us with important insights into the pathophysiology of the condition. Since the seminal observation of brainstem activation in migraine there has only been a single case substantiating this finding. Our objectives were, first, to test the hypothesis that brainstem activation could be detected in migraine and to refine the anatomical localisation with higher resolution positron emission tomography (PET) and more advanced analysis techniques. Secondly, we wanted to explore the glyceryl trinitrate (GTN) model of migraine further to determine reliability and reproducibility. Finally, using this model we wished to explore the issue of laterality in migraine and, in particular, how it relates to brainstem activation. H2150-labelled PET was used to study acute migraine attacks occurring spontaneously in five migraineurs. Using GTN-triggered migraine twenty-four migraineurs (divided into three groups according to the site of their headache: right/ left/ bilateral) and eight controls were scanned. The data was analysed using statistical parametric mapping (SPM99). Significant brainstem activation was seen in the dorsolateral pons (P < 0.05 after small volume correction) during the migraine state versus the pain-free state in both spontaneous and induced migraine groups. When the induced group was analysed separately, to investigate laterality, it was found that the dorsal pontine activation was ipsilateral in the right-sided and left-sided groups and bilateral in the bilateral headache group with a left-sided preponderance. Looking at the GTN model, thirty-three of the forty-four patients administered GTN had a migraine attack fulfilling International Headache Society criteria. Twelve patients described typical premonitory symptoms, which have not been previously documented with GTN-induced migraine. A repeat attack was triggered in all subjects but one and laterality was also remarkably reproducible. In one case a visual aura was also triggered both times. Our study shows that GTN-induced triggering is common in our patients, and remarkably reproducible. Overall, our findings provide clear evidence for dorsal pontine activation in migraine, and reinforce the view that migraine is a subcortical disorder involving modulation of afferent neural traffic. The results also suggest that lateralisation of pain in migraine is due to lateralised brain dysfunction.

Typical migraine, comprised of migraine with aura (MA) and migraine without aura (MO), is a chronic, painful and debilitating neurovascular disease which is generally characterised by recurrent attacks of severe headache usually accompanied by nausea, vomiting, photo and phonophobia. Migraine has been shown to affect a large proportion of Caucasian populations with a recent comprehensive study indicating that around 25% of women and 8% of men suffer from the disease. Strong familial aggregation of typical migraine and an increased concordance for the disease in MZ twins over DZ twins, suggests that it has a significant genetic component. Heritability estimates are calculated to be between 40% and 60%, indicating that disease variation, in part, is explained by environmental determinants. The mode of transmission of typical migraine is not clear but is most likely multifactorial. Although the MA and MO subtypes exhibit some clinical heterogeneity, segregation analysis has suggested that there may be a common genetic aetiology for MA and MO, and a major gene contributing to typical migraine pathogenesis. This idea is substantiated by the fact that both subtypes of migraine can occur within the same family and even within the same individual, with up to 33% of sufferers experiencing both types of the disease. In addition, migraine prophylactics have been shown to result in similar effects in patients treated for both types of migraine. However, whether the two subtypes are truly separate entities or not remains unclear. At present, the type and number of genes involved in typical migraine is not known. Despite this, several studies into Familial Hemiplegic Migraine (FHM), a very severe subtype of MA, have led to the discovery that mutations in a brain specific calcium channel subunit gene (CACNA1A) located on chromosome 19, cause FHM in about 50% of affected families. FHM is a rare disease and is distinguished from typical migraine by its association with hemiparesis and clear autosomal dominant mode of inheritance. However, certain clinical features are common to both FHM and typical migraine including similarities in headache characteristics and triggers. Hence, FHM genetic studies provide a valuable model for investigating the genes involved in the more prevalent types of migraine with and without aura. For this reason the Genomics Research Centre has been conducting linkage studies utilising large Australian migraine pedigrees with a focus on the known FHM (CACNA1A) gene region on chromosome 19p13. Our results to date have indicated suggestive linkage to the FHM region on 19p13 in a large multigenerational pedigree (MF1) affected with typical migraine, with a maximum parametric LOD score of 1.92 (P = 0.001) obtained for a triplet repeat polymorphism situated in exon 47 of the CACNA1A gene. Expansion of this repeat was not observed, but is possible that mutations elsewhere in the CACNA1A gene may be responsible for migraine in this pedigree. To investigate this possibility, the current research involved sequencing two patients carrying the critical susceptibility haplotype surrounding the CACNA1A gene. The results of this mutation screen revealed no disease causing mutations or polymorphisms in any of the 47 exons screened. To determine whether the CACNA1A genomic region was implicated in typical migraine susceptibility in the general Caucasian population, 82 independent pedigrees and a large case-control group were also analysed using highly polymorphic microsatellite markers. There was no linkage or association detected in these groups and thus, it was concluded that if CACNA1A plays a role in typical migraine it does not confer a major effect on the disease. However, subsequent case-control studies of SNPs in the INSR gene, which is located ~15cM telomeric from CACNA1A, provided evidence of association to typical migraine. Thus, the INSR gene may now emerge as the new migraine susceptibility gene in this genomic region on chromosome 19. Family linkage studies conducted by Gardner et al have implicated an additional FHM susceptibility region on chromsome 1q31. Furthermore, independent research carried out by Ducros et al. has indicated a second FHM locus at 1q21-23, which is ~ 30cM centromeric to the region reported by Gardner et al. At this stage it is not clear whether there is a single locus, or two distinct loci, on the chromosome 1q region. This research also involved a family-based linkage and association approach to investigating the FHM susceptibility region on chromosome 1q31 for involvement in typical migraine susceptibility in affected Australian pedigrees. Initial multipoint ALLEGRO analysis provided strong evidence for linkage of Chr1q31 markers to typical migraine in a large multigenerational pedigree. The 1-LOD* unit support interval for suggestive linkage spanned ~18cM with a maximum allele sharing LOD* score of 3.36 obtained for marker D1S2782, P = 0.00004. Subsequent analysis of an independent sample of 82 affected pedigrees added support to the initial findings with a maximum LOD* of 1.24 (P = 0.008). Utilising the independent sample of 82 pedigrees we also performed a family-based association test. Results of this analysis indicated distortion of allele transmission at marker D1S249 (global c2(5) of 15.00, P = 0.010) in these pedigrees. These positive linkage and association results will need further confirmation by independent researchers, but overall they provide good evidence for the existence of a typical migraine locus near these markers on Chr1q31, and reinforce the idea that an FHM gene in this genomic region may also contribute to susceptibility to the more common forms of migraine. The serotonergic system has long been implicated in the pathophysiology of migraine. Researchers have therefore focused on the serotonin receptors and the genes that code for them when investigating this disease. Although serotonin receptor agonists have proven to be effective in the treatment of migraine, there has been little evidence of a serotonin receptor gene being associated with the disorder. However, in 1998, Ogilvie et al reported that a VNTR in the serotonin transporter gene (SERT) showed altered allelic distributions in a Danish migraine population. In addition to serotonin, there has been renewed interest in the involvement of the dopaminergic pathways in migraine. This interest has gained impetus since the study of Peroutka et al who reported an allelic association between the dopamine receptor gene DRD2 and migraine with aura. Another dopamine related gene, the dopamine beta-hydroxylase gene (DBH), has been localised to Chr 9q34 and codes for the enzyme that catalyses the conversion of dopamine to norepinephrine. It therefore plays an important role in dopaminergic and noradrenergic neurotransmission. Serum levels of DbH enzyme have been reported to be elevated in migrainous patients during the headache phase of an attack. Also, significantly increased DbH enzyme activity has been observed in migraine patients during the headache-free interval. Thus, the DBH gene is another good candidate for involvement in migraine pathophysiology and, to our knowledge, has not been previously implicated in this disease. Candidate gene studies may be useful strategies for identifying genes involved in complex diseases such as migraine, especially if the gene being examined contributes only a minor effect to the overall phenotype. This research also involved a linkage and association approach to investigating neurotransmitter related migraine candidate genes. Specifically, polymorphisms within the serotonin transporter gene (SERT), the dopamine receptor gene (DRD2) and the dopamine beta-hydroxylase (DBH) gene were tested in unrelated Caucasian migraineurs and non-migraine control individuals. In addition, an independent sample of 82 families affected with migraine were examined. Unrelated case-control association analysis of a DBH intragenic dinucleotide polymorphism indicated altered allelic distribution between migraine and control groups (c2 = 16.53, P = 0.019). Furthermore, the transmission/disequilibrium test (TDT) which was implemented on the family data also indicated distortion of allele transmission for the same DBH marker (c2 = 4.44, P = 0.035). Together, these results provide evidence for allelic association of the DBH gene with typical migraine susceptibility (Fisher's Combined P-value = 0.006) and indicate that further research into the role of the DBH gene in migraine aetiology is warranted. Nitric oxide (NO) is emerging as a key molecule affecting the pain associated with migraine. Since nitric oxide synthase (NOS) enzymes catalyse the synthesis of NO, the genes that code for these enzymes are good candidates for migraine molecular genetic analysis. This research involved investigating the role of a functionally relevant bi-allelic tetranucleotide polymorphism located in the promoter region of the human inducible nitric oxide synthase (iNOS) gene in migraine aetiology. A large group of migraine affected individuals were genotyped and compared to an age and sex matched group of unaffected controls. Results of a chi-squared analysis indicated that allele distributions for both migraine cases and controls were not significantly different (c2 = 1.93, P = 0.16). These findings offer no evidence for an allelic association of the tested iNOS polymorphism with the common forms of the disease and therefore do not support a role for this gene in migraine pathogenesis. In summary, this research involved linkage and association analysis of migraine candidate genes and genomic susceptibility regions. Whilst, the known FHM gene (CACNA1A) was excluded for significant involvement in typical migraine the adjacent INSR gene has been associated. Migraine is genetically heterogeneous and the results of this research also provide good evidence that the DBH gene is involved in disease predisposition, whilst the DRD2, SERT and INOS gene were not shown to be implicated. An additional susceptibility region for typical migraine is also likely to localise to chromosome 1q31. Overall, the results presented in this thesis have contributed valuable data to the understanding of the molecular genetics of migraine with and without aura. Future research into the molecular pathophysiological mechanisms of migraine will greatly facilitate the development of more effective diagnosis and treatment strategies.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Health Sciences
Full Text

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Typical migraine, comprised of migraine with aura (MA) and migraine without aura (MO), is a chronic, painful and debilitating neurovascular disease which is generally characterised by recurrent attacks of severe headache usually accompanied by nausea, vomiting, photo and phonophobia. Migraine has been shown to affect a large proportion of Caucasian populations with a recent comprehensive study indicating that around 25% of women and 8% of men suffer from the disease. Strong familial aggregation of typical migraine and an increased concordance for the disease in MZ twins over DZ twins, suggests that it has a significant genetic component. Heritability estimates are calculated to be between 40% and 60%, indicating that disease variation, in part, is explained by environmental determinants. The mode of transmission of typical migraine is not clear but is most likely multifactorial. Although the MA and MO subtypes exhibit some clinical heterogeneity, segregation analysis has suggested that there may be a common genetic aetiology for MA and MO, and a major gene contributing to typical migraine pathogenesis. This idea is substantiated by the fact that both subtypes of migraine can occur within the same family and even within the same individual, with up to 33% of sufferers experiencing both types of the disease. In addition, migraine prophylactics have been shown to result in similar effects in patients treated for both types of migraine. However, whether the two subtypes are truly separate entities or not remains unclear. At present, the type and number of genes involved in typical migraine is not known. Despite this, several studies into Familial Hemiplegic Migraine (FHM), a very severe subtype of MA, have led to the discovery that mutations in a brain specific calcium channel subunit gene (CACNA1A) located on chromosome 19, cause FHM in about 50% of affected families. FHM is a rare disease and is distinguished from typical migraine by its association with hemiparesis and clear autosomal dominant mode of inheritance. However, certain clinical features are common to both FHM and typical migraine including similarities in headache characteristics and triggers. Hence, FHM genetic studies provide a valuable model for investigating the genes involved in the more prevalent types of migraine with and without aura. For this reason the Genomics Research Centre has been conducting linkage studies utilising large Australian migraine pedigrees with a focus on the known FHM (CACNA1A) gene region on chromosome 19p13. Our results to date have indicated suggestive linkage to the FHM region on 19p13 in a large multigenerational pedigree (MF1) affected with typical migraine, with a maximum parametric LOD score of 1.92 (P = 0.001) obtained for a triplet repeat polymorphism situated in exon 47 of the CACNA1A gene. Expansion of this repeat was not observed, but is possible that mutations elsewhere in the CACNA1A gene may be responsible for migraine in this pedigree. To investigate this possibility, the current research involved sequencing two patients carrying the critical susceptibility haplotype surrounding the CACNA1A gene. The results of this mutation screen revealed no disease causing mutations or polymorphisms in any of the 47 exons screened. To determine whether the CACNA1A genomic region was implicated in typical migraine susceptibility in the general Caucasian population, 82 independent pedigrees and a large case-control group were also analysed using highly polymorphic microsatellite markers. There was no linkage or association detected in these groups and thus, it was concluded that if CACNA1A plays a role in typical migraine it does not confer a major effect on the disease. However, subsequent case-control studies of SNPs in the INSR gene, which is located ~15cM telomeric from CACNA1A, provided evidence of association to typical migraine. Thus, the INSR gene may now emerge as the new migraine susceptibility gene in this genomic region on chromosome 19. Family linkage studies conducted by Gardner et al have implicated an additional FHM susceptibility region on chromsome 1q31. Furthermore, independent research carried out by Ducros et al. has indicated a second FHM locus at 1q21-23, which is ~ 30cM centromeric to the region reported by Gardner et al. At this stage it is not clear whether there is a single locus, or two distinct loci, on the chromosome 1q region. This research also involved a family-based linkage and association approach to investigating the FHM susceptibility region on chromosome 1q31 for involvement in typical migraine susceptibility in affected Australian pedigrees. Initial multipoint ALLEGRO analysis provided strong evidence for linkage of Chr1q31 markers to typical migraine in a large multigenerational pedigree. The 1-LOD* unit support interval for suggestive linkage spanned ~18cM with a maximum allele sharing LOD* score of 3.36 obtained for marker D1S2782, P = 0.00004. Subsequent analysis of an independent sample of 82 affected pedigrees added support to the initial findings with a maximum LOD* of 1.24 (P = 0.008). Utilising the independent sample of 82 pedigrees we also performed a family-based association test. Results of this analysis indicated distortion of allele transmission at marker D1S249 (global c2(5) of 15.00, P = 0.010) in these pedigrees. These positive linkage and association results will need further confirmation by independent researchers, but overall they provide good evidence for the existence of a typical migraine locus near these markers on Chr1q31, and reinforce the idea that an FHM gene in this genomic region may also contribute to susceptibility to the more common forms of migraine. The serotonergic system has long been implicated in the pathophysiology of migraine. Researchers have therefore focused on the serotonin receptors and the genes that code for them when investigating this disease. Although serotonin receptor agonists have proven to be effective in the treatment of migraine, there has been little evidence of a serotonin receptor gene being associated with the disorder. However, in 1998, Ogilvie et al reported that a VNTR in the serotonin transporter gene (SERT) showed altered allelic distributions in a Danish migraine population. In addition to serotonin, there has been renewed interest in the involvement of the dopaminergic pathways in migraine. This interest has gained impetus since the study of Peroutka et al who reported an allelic association between the dopamine receptor gene DRD2 and migraine with aura. Another dopamine related gene, the dopamine beta-hydroxylase gene (DBH), has been localised to Chr 9q34 and codes for the enzyme that catalyses the conversion of dopamine to norepinephrine. It therefore plays an important role in dopaminergic and noradrenergic neurotransmission. Serum levels of DbH enzyme have been reported to be elevated in migrainous patients during the headache phase of an attack. Also, significantly increased DbH enzyme activity has been observed in migraine patients during the headache-free interval. Thus, the DBH gene is another good candidate for involvement in migraine pathophysiology and, to our knowledge, has not been previously implicated in this disease. Candidate gene studies may be useful strategies for identifying genes involved in complex diseases such as migraine, especially if the gene being examined contributes only a minor effect to the overall phenotype. This research also involved a linkage and association approach to investigating neurotransmitter related migraine candidate genes. Specifically, polymorphisms within the serotonin transporter gene (SERT), the dopamine receptor gene (DRD2) and the dopamine beta-hydroxylase (DBH) gene were tested in unrelated Caucasian migraineurs and non-migraine control individuals. In addition, an independent sample of 82 families affected with migraine were examined. Unrelated case-control association analysis of a DBH intragenic dinucleotide polymorphism indicated altered allelic distribution between migraine and control groups (c2 = 16.53, P = 0.019). Furthermore, the transmission/disequilibrium test (TDT) which was implemented on the family data also indicated distortion of allele transmission for the same DBH marker (c2 = 4.44, P = 0.035). Together, these results provide evidence for allelic association of the DBH gene with typical migraine susceptibility (Fisher's Combined P-value = 0.006) and indicate that further research into the role of the DBH gene in migraine aetiology is warranted. Nitric oxide (NO) is emerging as a key molecule affecting the pain associated with migraine. Since nitric oxide synthase (NOS) enzymes catalyse the synthesis of NO, the genes that code for these enzymes are good candidates for migraine molecular genetic analysis. This research involved investigating the role of a functionally relevant bi-allelic tetranucleotide polymorphism located in the promoter region of the human inducible nitric oxide synthase (iNOS) gene in migraine aetiology. A large group of migraine affected individuals were genotyped and compared to an age and sex matched group of unaffected controls. Results of a chi-squared analysis indicated that allele distributions for both migraine cases and controls were not significantly different (c2 = 1.93, P = 0.16). These findings offer no evidence for an allelic association of the tested iNOS polymorphism with the common forms of the disease and therefore do not support a role for this gene in migraine pathogenesis. In summary, this research involved linkage and association analysis of migraine candidate genes and genomic susceptibility regions. Whilst, the known FHM gene (CACNA1A) was excluded for significant involvement in typical migraine the adjacent INSR gene has been associated. Migraine is genetically heterogeneous and the results of this research also provide good evidence that the DBH gene is involved in disease predisposition, whilst the DRD2, SERT and INOS gene were not shown to be implicated. An additional susceptibility region for typical migraine is also likely to localise to chromosome 1q31. Overall, the results presented in this thesis have contributed valuable data to the understanding of the molecular genetics of migraine with and without aura. Future research into the molecular pathophysiological mechanisms of migraine will greatly facilitate the development of more effective diagnosis and treatment strategies.

This research was conducted to expand the understanding of the role of the X chromosome in common and familial typical migraine. The primary objective of this study was to identify new X chromosomal genetic targets that cause migraine. Overall this research has identified nine genetic targets of interest. Various obstacles were encountered throughout this study, but the knowledge gained for overcoming these obstacles are invaluable for implementation and improvement of future genetic studies investigating the X chromosome.

Introduction: Bipolar Disorder (BD) is a chronic mental illness associated with functional decline, mortality, and significant health care costs; furthermore, specific general medical conditions have been found to occur disproportionately within BD patient populations, among them, migraine is one of the most studied. Migraine has a global prevalence of 10%, and it is a disorder with elevated direct and indirect costs, the later mostly derived from its association with mood and anxiety disorders. Specifically, the reported prevalence of migraine in the BD population ranges from 24.8% to 39.8%, rates that are considerable higher than those found in the general population.
Objective: To explore the prevalence and clinical characteristics of BD patients with and without migraine (Study 1), and to examine the psychiatric comorbidity in patients suffering from migraine (Study 2).
Methods: 323 BD patients were studied, using SADS-L and SCID as diagnostic interviews, and ill-Migraine questionnaire to assess the presence of migraine. Statistical analyses were conducted using parametric analysis and the development of log-linear models. Additionally, 102 migraine patients were interviewed using SADS-L, and the descriptive characteristics of the sample were analyzed.
Results: For Study 1, we found that 24.5% of BD patients suffer from migraine, and it is significantly associated with BD 2, suicidal behaviour, and a variety of anxiety disorders. As well, over 70% of migraine patients showed a lifetime psychiatric diagnosis, mainly within the spheres of mood and anxiety disorders; specifically, the prevalence of BD among migraine patients was 12.7%.
Conclusions: Our study highlights the high prevalence of migraine among BD patients, and the elevated prevalence of psychiatric comorbidity among migraine sufferers. The study of this comorbidity will deepen our understanding of the mechanisms that underlie both disorders and provide a better framework for the developing of molecular techniques to further analyze the molecular physiopathology of Bipolar Disorder.

Migraine can be conceptualised as a disorder of sensory processing, manifest by such symptoms as headache (pain), phonophobia and photophobia. Current models of migraine pathophysiology incorporate a significant role for the brainstem. Vestibular migraine (VM) is a subtype of the disorder in which significant brainstem dysfunction has been documented. The condition is known to have a significant effect on mental health. This study was designed to investigate disturbances in audiovestibular brainstem function in vestibular migraine in a four part study: 1. Otoacoustic emission suppression by contralateral noise, a test of auditory efferent pathway function, was measured in a group of 33 VM patients and compared with 31 healthy controls. Regression analysis showed a higher rate of abnormality amongst the VM group (p=0.03). 2. Vestibular evoked myogenic potentials were recorded in a group of 30 VM patients and compared with 35 healthy controls. Recordings showed a higher rate of abnormal responses in the VM group than amongst controls (p=0.008). 3. The potential for vestibular stimuli to act as migraine triggers was investigated by observing the effect of vestibular testing or a control condition on 148 individuals. Vestibular stimulation was associated with a significant increase in the probability of developing a migraine attack over the following 24 hour period (p=0.01). 4. Psychological symptoms of depression and anxiety were assessed using questionnaires 39 patients with VM and compared with a control group of 44 patients with dizziness of other causes. Although the VM group had a significantly higher load of symptoms of depression and anxiety, regression modelling showed that this effect was largely accounted for by an excess of dizziness symptoms. In conclusion, this study documents a number of audiovestibular sensory processing abnormalities using a variety of techniques. Vestibular migraine has a significant effect on psychological wellbeing, largely via the associated balance symptoms.

Migraine is a common yet debilitating condition worldwide, affecting individuals, particularly women, of working age, causing significant impacts on quality of life and productivity. In this study, we set out to evaluate a group of individuals with sporadic migraine. We aimed to evaluate the effect of botulinum toxin or the novel calcitonin gene-related peptide (CGRP) erenumab. We were also able to consider studies on patients with familial hemiplegic migraine, where we were particularly looking to see if there were alterations in axonal excitability related to the gene mutations. A study has identified differences in episodic ataxia type 2, and patients with familial hemiplegic migraine type 1 (FHM1) carry mutations of the same gene CACNA1A. We sought to see if FHM1 patients had similar changes to those of episodic ataxia type 2, and whether axonal excitability allowed identification of genetically affected migraine individuals. We collected data on clinical outcomes, thermal threshold perception and sensory and motor axonal excitability parameters from the median nerve for our cohort of sporadic chronic migraine. No significant excitability changes were identified when compared to healthy controls at baseline, or after treatment. No alterations were seen when patients were divided into subgroups of erenumab or botulinum toxin treatment. We did note significant clinical improvements in patients treated with either botulinum toxin or erenumab, with some patients experiencing a large treatment response. We did not identify any significant change in thermal perception or thermal pain thresholds, before and after treatment. We were unable to recruit sufficient numbers of hemiplegic migraine patients for this study, and future work will involve further recruitment, before definite conclusions can be drawn about excitability changes in hemiplegic migraine.

Migraine is a common, episodic neurological disorder that includes headache, nausea and hypersensitivity to sensory stimuli. During the headache phase of migraine, migraine patients can be especially hypersensitive to thermal stimuli. The unpredictable and episodic nature of migraine makes it difficult to treat and much of the mechanism of migraine has yet to be elucidated. A T44A substitution in casein kinase 1δ is inherited with migraine with aura. A transgenic mouse model suggests that animals with this mutation exhibit increased sensitivity to thermal stimuli after injection with nitroglycerin (NTG). We performed behavior assays that measure animal responses to thermal stimuli, after injection with NTG, a known migraine-inducer in human migraine patients. Female animals with the CK1δ-T44A mutation are more sensitive than wildtype littermates, suggesting a sex difference emerges in pain sensitivity in animals that express the CK1δ-T44A but not in wildtype siblings. Female CK1δ-T44A animals are more sensitive to the effects of NTG on pain than male CK1δ-T44A mice. This indicates a potential sex hormone related pain response. Since estrogen is implicated in both migraine and pain response, we test the thermal sensitivity of heterozygous ERβKO/+ and CK1δ-T44A; ERβKO/+ mice compared to wildtype and CK1δ-T44A mice. Overall thermal sensitivity is decreased before stress of injection in both male and female ERβKO/+ and CK1δ-T44A: ERβKO/+ mice. This demonstrates that ERβ is necessary for thermal nociception in untreated mice. However, after injection with saline or NTG, animals of all genotypes responded to thermal stimuli similarly. This suggests that estrogen signaling through ERβ is likely not part of the pathway of NTG-induced thermal sensitivity or that one copy of ERβ is sufficient for NTG-induced thermal sensitivity. Since ERβ is fully functional in CK1δ-T44A mice and CK1δ-T44A mice have wildtype thermal sensitivity at baseline, we can conclude that CK1δ-T44A does not modulate ERβ to affect thermal sensitivity in untreated animals.

Migraine is a serious neurological disorder that affects the central nervous system causing painful attacks of headache. Attacks of head pain vary widely in intensity, frequency and duration lasting from anywhere between 4-72 hours and are often accompanied by further symptoms of nausea, vomiting, photo- and phonophobia. In 1988, a group of world leaders in the diagnosis of migraine formed the International Headache Society (IHS) and compiled and published a consensus set of diagnostic criteria known as International Classification of Headache Disorders, ICHD-I 1988. This was the first classification system and was subsequently updated in 2004, ICHD-II 2004 and more recently a 3rd Edition beta version has been released (ICHD-3rd Ed Beta Version) and is the gold standard for diagnosing headache disorders. Migraine displays two main subtypes termed migraine with or without aura (MA and MO respectively). The two forms are distinguished from each other based on the development of aura, a period of variable and diverse neurological symptoms that precede the headache phase.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Science, Environment, Engineering and Technology
Full Text

Nella tesi è descritto un modello matematico per l'aura emicranica e, più precisamente, per lo scotoma scintillante (schema di fortificazione) e per la Cortical Spreading Depression, il fenomeno neuropatofisiologico alla base dell'aura. In particolare è spiegato un modello cinematico per l'evoluzione della CSD nella corteccia visiva primaria, considerata un mezzo debolmente eccitabile, la mappa retino-corticale e il modello, tramite fibrato, dei Pinwheels di V1.

Research into the association between migraine and menstruation has been hampered by lack of an agreed definition for 'menstrual' migraine. This thesis presents evidence for increased risk of migraine without aura on or between the two days before menstruation and the first three days of bleeding. Within individual women menstrual attacks differ from attacks at other times of the cycle, being longer and more severe. These findings led to the development of definitions for 'pure menstrual migraine' and 'menstrually-related migraine', which have subsequently been adopted by the International Headache Society. Further research identified an inverse relationship between oestrogen and migraine incidence. The follicular phase oestrogen rise was associated with reduced risk of migraine late luteal oestrogen 'withdrawal' at menstruation was associated with increased risk of migraine. In order to counteract the luteal phase oestrogen drop and prevent menstrual attacks, oestrogen supplements were used from the luteal phase oestrogen peak (day-6) through to the early follicular rise of endogenous oestrogen (day +2). Identification of ovulation using a fertility monitor enabled prediction of menstruation and accurate timing of oestrogen supplements, despite a wide inter- and intra-individual range in cycle length. The results showed that use of oestrogen supplements was associated with a significant reduction in migraine days compared to placebo. However, the benefits were offset by delayed oestrogen 'withdrawal' migraine. In women with migraine in the pill-free interval of combined hormonal contraceptives, there was a trend for oestrogen supplements to prevent 'menstrual' attacks, although the dose used was suboptimal. These findings support the hypotheses that menstrual migraine is a discrete clinical entity and is associated with oestrogen 'withdrawal'. Further, oestrogen 'withdrawal' migraine can be independent of menstruation and independent of ovulation. Oestrogen 'withdrawal' migraine can be prevented with oestrogen supplements, although the optimal regime has yet to be established.

The exact neural mechanisms underlying migraine have been strongly debated for decades. It is well accepted the trigeminovascular system plays a role in migraine, however the precise neurobiological mechanism by which migraine attacks are initiated is presently unknown. Existing literature has demonstrated that even between attacks, the migraine brain differs from that of controls and has led to the current theory of migraine as a “cycling” brain disorder. This suggests that the changes observed in migraine are not fixed, but rather dynamic in nature, and that function, sensitivity and even anatomy of the brainstem may fluctuate throughout the migraine cycle. However, while the brainstem has been heavily implicated in migraine pathogenesis, it has been poorly studied by neural imaging investigations. Likewise, the spontaneous and episodic nature of migraines has made it difficult to study the migraine cycle in its entirety. As such, this thesis aimed to explore brainstem anatomy, functional connectivity, and sensitivity to noxious stimuli, in migraineurs across the entire migraine cycle. The investigations performed in this thesis, consistently revealed key brainstem pain-processing regions, such as the spinal trigeminal nucleus and periaqueductal grey matter, as having altered anatomy, functional connectivity, and sensitivity across the migraine cycle. Most intriguingly, these changes were most dramatically altered in the 24 hours immediately prior to migraine onset. This suggests the observed changes in these brainstem regions may underlie the processes causing migraine initiation. Furthermore, these changes were dynamic in nature, supporting the notion of a “cycling” migraine brain. This opens new avenues for migraine research which will hopefully lead to better prophylactic treatments, capable of controlling these fluctuations that underlie the disorder.

Migraine is the leading cause of neurological disability worldwide, with many treatments shown to be ineffective. Enhancing our understanding of mechanism of migraine may lead to the development of effective, migraine-specific treatments. Elevated Gamma-amino butyric acid (GABA) levels have been observed in people with migraine but not other pain conditions, suggesting GABA levels may indicate a mechanism responsible for migraine. Advances in magnetic resonance imaging (MRS), such as MEGA-PRESS, enable direct measurement of GABA in the human brain, although there is no agreed best practice for data acquisition. The thesis aims were to: 1) advance understanding of the role of GABA in migraine (and pain conditions) 2) improve the standardisation of methods for quality assessment, and accurate measurement and reporting of GABA in studies using MEGA-PRESS. Initially, GABA+ levels appeared to be unique to migraine when compared to other pain conditions in a meta-analysis (Chapter 2). However, a direct comparison of GABA+ levels in people with migraine, whiplash-headache and back pain compared to controls found GABA+ levels were elevated across all pain groups (Chapter 3). Finally, an increase in GABA+ levels over time were shown to be associated with decreased clinical measures of migraine (Chapter 3). Together these findings suggest GABA+ levels are a general pain-related measure in migraine and pain. To improve standardisation two new tools were developed. The MRS-Q a 12-point quality assessment tool for spectroscopy studies (Chapter 2) and The Comprehensive Guide to MEGA-PRESS (Chapter 5), an evidence-based guideline for best practice in GABA measurement. Together, the findings advance what is known about GABA in migraine and pain, and serves to shape future research into GABA through providing methods to standardise quality assessment, reporting and measurement of GABA using MEGA-PRESS.

Migraine is a painful neurological disease that affects at least 12% of the Australian population. It is generally characterized by recurrent head pain usually accompanied by nausea, vomiting, neurological disturbance, photo-and phonophobia. Migraine has been classified by the international headache society (IHS) into two most common types, migraine with aura (MA) and migraine without aura (MO). The underlying pathophysiology of this debilitating disease is still partially understood and there are no known diagnostic markers for these common types of migraine. Current diagnosis of migraine is based on patient reported symptoms. Studies have shown several health conditions such as epilepsy, depression and stroke to be co-morbid with migraine. Current migraine treatments work with varying efficacy and often have adverse side effects. A greater understanding of this debilitating and painful disease is thus pertinent for developing new and improved migraine treatment. Both familial clustering and twin studies have shown evidence for significant genetic mechanisms to underlie migraine pathogenesis. Migraine is thus currently defined as a complex multifactorial disorder which involves an interaction between genetic and environmental factors. We have not yet identified all migraine genes but a number of genes, causative mutations and susceptibility variants have been identified and are already of significant clinical relevance. Currently the detection of 3 rare subtypes of migraine (Familial Hemiplegic Migraine 1, 2 and 3) and several related conditions with symptom overlap (Episodic Ataxia 2, Spinocerebellar Ataxia Type 6 and Cerebral Autosomal Dominant Arteriopathy with Sub cortical Infarcts and leucoencephalopathy (CADASIL) is undertaken by sequencing, with susceptibility variants for common types of migraine detected by sequencing or genotyping.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
Full Text

La migraine est un trouble neurologique qui touche plus d'un milliard d'individus dans le monde, principalement des femmes. Chez 3 % des patients, la fréquence des crises de migraine augmente, et d'épisodique, la migraine devient chronique. Les mécanismes de cette chronicisation sont encore incertains, et la migraine reste une affection difficile à traiter. Des études récentes ont suggéré l'implication du L-tryptophane (L-Trp), un acide aminé essentiel, et de ses métabolites dans la physiopathologie de la migraine. En effet, la privation de L-Trp aggrave l'intensité des maux de tête, alors que son apport régulier réduit le risque de crise. Notre objectif est de comprendre le mécanisme de l'effet de la dérégulation de la Trp dans la migraine. D'étudier la contribution de la L-Trp dans la chronicisation de la migraine ainsi que dans l'apparition de symptômes sensoriels associés (allodynie mécanique et photophobie) et la comorbidité de la migraine avec des troubles psychiatriques (dépression et anxiété). De plus, nous explorons l'effet du déficit en L-Trp sur l'efficacité des médicaments anti-migraineux actuellement établis
Migraine is a neurological disorder that affects over a billion individuals globally, primarily women. In 3% of patients, the frequency of migraine attacks increases, and from episodic, the migraine becomes chronic. The mechanisms of this chronicization are still uncertain, and migraine is still a tough condition to treat. Recent studies have suggested the involvement of L-tryptophan (L-Trp), an essential amino acid, and its metabolites in the pathophysiology of migraine. Indeed, deprivation of L-Trp aggravates the intensity of headaches, while its regular intake reduces the risk of attack. Our aim is to underatand the mechanism of the effect of Trp deregulation in migraine. To study the contribution of L-Trp in the chronicization of migraine as well as in the appearance of associated sensory symptoms (mechanical allodynia and photophobia) and the comorbidity of migraine with psychiatric disorders (depression and anxiety). In addition, we explore the effect of L-Trp deficit on the efficacy of currently established anti-migraine medications