Histaminergic innervation of thalamic trigeminovascular neurons.

<p><u>Left</u>: Immunopositive Histamine axons (green) surrounding a thalamic dura-sensitive neuron (red) labeled with TMR–dextran. Nuclear counterstaining was performed with DAPI (blue). Arrowheads indicate close apposition of Histamine positive axons and the cell body and dendrites of the labeled neuron. <u>Upper right</u>: Location of the dura-sensitive neuron (red star) shown at left. Number in red indicates distance from <i>bregma</i> (mm). <u>Lower right</u>: Fluorescent image showing Histamine labeling of cell bodies in the dorsal and ventral tuberomammillary nuclei of the hypothalamus (DTM and VTM). Scale bars = 100 µm.</p

Relative density of thalamic innervation by neurotransmitters and neuropeptides.

<p>Quantitative analysis using binary maps: >5% of positive (white) pixels per image indicates high density, 1–5% indicates moderate density, and <1% indicates low density of innervation. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103929#pone.0103929.s008" target="_blank">Figs. S8</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103929#pone.0103929.s009" target="_blank">S9</a> for actual binary maps.</p

Lack of innervation of thalamic trigeminovascular neurons by axons containing CGRP, Serotonin 1D receptor, Oxytocin and Vasopressin.

<p><u>Left A–D</u>: Thalamic dura-sensitive neurons (red) labeled with TMR–dextran and nuclear counterstain with DAPI (blue). Note the absence of axonal immunoreactivity to CGRP (A), Serotonin 1D receptor (B), Oxytocin (C) and Vasopressin (D). <u>Upper right A–D</u>: Locations of the dura-sensitive neurons (red stars) shown at left. Numbers in red indicate distance from <i>bregma</i> (mm). <u>Lower right A–B</u>: Fluorescent images showing CGRP (A) and Serotonin 1D receptor (B) immunopositive axons in the parvicellular division of the ventral posterior thalamic nucleus (VPpc) and the spinal trigeminal nuclei (SpVC/SpVI; caudal/interpolar), respectively. <u>Lower right C</u>: Fluorescent images showing Oxytocin labeling of cell bodies and axons in the hypothalamic paraventricular nucleus (PVN) and lateral hypothalamus (LH), respectively. <u>Lower right D</u>: Fluorescent images showing Vasopressin labeling of cell bodies in the PVN and circular (Cir) nuclei of the hypothalamus, and axons in the LH. Scale bars = 100 µm.</p

Serotoninergic innervation of thalamic trigeminovascular neurons.

<p><u>Left</u>: Immunopositive Serotonin Transporter axons (green) surrounding a thalamic dura-sensitive neuron (red) labeled with TMR–dextran. Nuclear counterstaining was performed with DAPI (blue). Arrowheads indicate close apposition of SERT positive axons and the cell body and dendrites of the labeled neuron. <u>Upper right</u>: Location of the dura-sensitive neuron (red star) shown at left. Number in red indicates distance from <i>bregma</i> (mm). Scale bars = 100 µm. Since SERT does not stain cell somas, it was not possible to use this marker to identify the serotoninergic neurons in the raphe nuclei that project to the thalamic nuclei containing trigeminovascular neurons. Abbreviations: Hb, habenula; MD, mediodorsal thalamic; CL, centrolateral thalamic.</p

Noradrenergic innervation of thalamic trigeminovascular neurons.

<p><u>Left</u>: Immunopositive Dopamine β-Hydroxylase axons (green) surrounding a thalamic dura-sensitive neuron (red) labeled with TMR–dextran. Nuclear counterstaining was performed with DAPI (blue). Arrowheads indicate close apposition of DBH positive axons and the cell body and dendrites of the labeled neuron. <u>Upper right</u>: Location of the dura-sensitive neuron (red star) shown at left. Number in red indicates distance from <i>bregma</i> (mm). <u>Lower right</u>: Fluorescent image showing DBH labeling of cell bodies in the locus coeruleus (LC) of the brainstem. Scale bars = 100 µm.</p

(A) Schematic illustration of the neurotransmitter and neuropeptidergic systems innervating thalamic trigeminovascular neurons in VPM, Po and LP/LD.

<p>The peripheral (meningeal nociceptors) and central (trigemino-thalamic) components of the trigeminovascular pathway are shown in red. The neurotransmitter and neuropeptidergic systems are color coded as follow: (a) Glutamate from SpVC/C1-2 in red; (b) GABA from Rt in yellow; (c) Noradrenalin from LC in blue; (d) Serotonin from raphe magnus (RMg) and dorsal raphe (DR) in green; (e) Histamine from DTM and VTM in orange; (f) Melanin Concentrating Hormone from LH in purple; (g) Orexin from PeF in black; (h) Dopamine from A11 in brown. (B) The diverse neurochemical pathways that converge on thalamic trigeminovascular neurons and the probability that many of them modulate neuronal activity in the same direction under certain conditions (e.g., sleep deprivation, wakefulness, food withhold, stress, anxiety) and in opposite directions under other conditions (e.g., food intake, sleep) define a sophisticated neuroanatomical network that may help us conceptualize how sensory, physiological, cognitive and affective conditions trigger, worsen or improve migraine headache.</p

GABAergic innervation of thalamic trigeminovascular neurons.

<p><u>Left</u>: Immunopositive VGaT synaptic vesicles (green) surrounding a thalamic dura-sensitive neuron (red) labeled with TMR–dextran. Nuclear counterstaining was performed with DAPI (blue). Arrowheads indicate close apposition of VGaT positive axons and the cell body and dendrites of the labeled neuron. <u>Upper right</u>: Location of the dura-sensitive neuron (red star) shown at left. Number in red indicates distance from <i>bregma</i> (mm). <u>Lower right</u>: Fluorescent images showing VGaT axonal labeling in thalamic Po and VPM nuclei. Scale bars = 100 µm. Abbreviations: eml, external medullary lamina; ic, internal capsule; ZI, zona incerta.</p

Close apposition between chemically-identified axons and thalamic trigeminovascular neurons.

<p>Images from the original z-stack (obtained every 1 µm) were used to create orthogonal views in the y–z and x–z planes. The three views provide evidence that SERT immunopositive fibers (green) may contact cell bodies, proximal and distal dendrites of trigeminovascular neurons in Po (red; as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103929#pone-0103929-g004" target="_blank">Fig. 4</a>). Note that some green-labeled axons and red-labeled soma or dendrites are in the same focal plane (yellow). To see similar images for all the neurotransmitters and neuropeptides identified in this study, see Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103929#pone-0103929-g001" target="_blank">Figures 1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103929#pone-0103929-g007" target="_blank">7</a>. <i>Caveat</i>: proximity between the chemically-identified axons and the TMR-labeled trigeminovascular thalamic neurons suggests that they are innervated by the different neuropeptides/neurotransmitters. Definitive evidence for actual synapses, however, requires tissue examination with electron microscopy. Scale bar = 50 µm.</p

Somatotopic activation triggered by combined corneal pain and eye blink.

<p>The Pain+Blink condition activated contralateral S1 (Max zstat  = 4.9 at 56, −14, 43) and bilateral M1 (Max zstat  = 9.9 at 52, 2, 31) in regions corresponding to the eye in the Penfield sensory and motor homunculi <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044643#pone.0044643-Penfield2" target="_blank">[19]</a> (p<0.0001, uncorrected for multiple comparisons). The No Pain+Blink condition activated bilateral M1 (Max zstat  = 7.4 at 44, −4, 43), but not S1. Investigations were restricted to the non-shaded areas in the activation maps, which correspond to bilateral pre- (blue) and post-central gyri (red) as highlighted in the underlying brain slices and colored squares (dashed squares denote absent activation). Note that the boundaries of these probabilistic-defined areas overlap with other regions, such as supplementary motor area, middle frontal gyrus, and supramarginal gyrus. Of note, the supplementary motor area has previously been associated with voluntary blinking <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044643#pone.0044643-Chung1" target="_blank">[13]</a>, and was active in both conditions along the midline of coronal slice y = −14 (only shown for Pain+Blink in figure). A = anterior; L = left; P = posterior; R = right.</p

Time-to-peak determined from a 2 gamma function fit of the average responses contra/ipsi for VAS of 3 and 7 (see text).

<p>Time-to-peak determined from a 2 gamma function fit of the average responses contra/ipsi for VAS of 3 and 7 (see text).</p