Fig 2: Upregulation of contractile receptors and Ang II in human cerebral arteries after organ culture (OC).The smooth muscle layers (ML, medial layer) show a dramatic increase in immunofluorescent staining of (A, B) 5-HT1B receptors, (C, D) AT1 receptors, (E, F) ETB receptors and (G, H) Ang II after 48 h of culture (48 h OC) as compared to non-incubated arteries (0 h OC). Fluorescent intensity measurements (arbitrary units, AU) were performed in the medial layer (ML), and are represented as mean ± SEM (right panel). Ang II, 5-HT1B and ETB immunofluorescence can also be seen in endothelial cells (when they are present, arrowheads). Autofluorescence in the internal lamina elastica is indicated by the asterisk.

Fig 3: The involvement of 5-HT1B and 5-HT1D receptors on the inhibitory effect of Lasmiditan. Ten minutes before the addition of 30 μM Lasmiditan: (A) 100 nM (n=4), (B) 300 nM (n=6) or (C) 1 μM (n=5) of GR127935, a 5-HT1B/5-HT1D blocker was added to the dura which subsequently stimulated with 60 mM KCl (K+). Similarly, 10 minutes before the addition of 30 μM Lasmiditan, and before the addition of 60 mM KCl (K+) (D) 100 nM (n = 5), (E) 300 nM (n = 16) or (F) 1 μM (n = 6) of GR127935, was added to the TGs which subsequently stimulated with 60 mM KCl (K+). The reduction in CGRP release caused by Lasmiditan could be inhibited by 300 nM GR127935 in the TG (p = 0.014), with a tendency observed in the dura at the same concentration. Data are shown as mean ± SEM with * p>0.05, from the paired Student’s T-test being depicted in the graph

Fig 4: Expression of 5-HT in relation to 5-HT1B/1D/1F receptors. A majority of observed TG neurons expressed 5-HT-ir in a heterogeneous granular pattern. This pattern was reminiscent of cytoplasmic vesicles and which was often visualized close to the cell membrane. 5-HT was generally weakly expressed in Aδ-fibres, Schwann cells and satellite glial cells. A 5-HT1B receptor ir was found in all observed neuronal cell bodies and more faintly in Aδ-fibres. 5-HT1B-ir was observed in an evenly distributed granular pattern across the neuron cytoplasm. 5-HT1B co-localized with 5-HT in the neuronal cytoplasm. Insert: Arrowheads mark a neuron where 5-HT is intensely expressed close to the cell membrane and 5-HT1B is more evenly distributed across the whole cell body. B A majority of observed neurons displayed 5-HT1D receptor ir in a vesicular pattern. Unlike the intense and evenly distributed ir of 5-HT1B receptor, the 5-HT1D receptor displayed both strong and weak ir in different subsets of neurons. Arrowheads mark a neuron with a fainter 5-HT1D expression than a neighbouring neuron. Furthermore, 5-HT1D displayed a distinct Aδ-fibre ir. Arrows mark a visible Aδ-fibre. Double IHC showed that 5-HT1D co-localized with 5-HT in the neuron cell bodies. 5-HT expression could be observed in 5-HT1D-positive Aδ-fibres, albeit inconsistent and faint. Insert: Arrows mark Aδ-fibre expressing both 5-HT1D and 5-HT ir. C 5-HT co-localized with the faintly positive 5-HT1F receptor in neuron cell bodies. No nerve fibre ir was observed for the 5-HT1F receptor. Insert: Arrowhead marks a 5-HT positive neuron with a faint 5-HT1F expression

Fig 5: Effect of organ culture on mRNA levels of cerebrovascular receptors in human cerebral arteries.mRNA expression for (A) 5-HT1B receptors, (B) AT1 receptors and (C) ETB receptors before (0 h OC) and after organ culture (48 h OC) are illustrated. Each bar represents mean ± SEM; females, n = 4 and males n = 8; *P<0.05.