Neurochemical pathways that converge on thalamic trigeminovascular neurons: potential substrate for modulation of migraine by sleep, food intake, stress and anxiety

Abstract

Dynamic thalamic regulation of sensory signals allows the cortex to adjust better to rapidly changing behavioral, physiological and environmental demands. To fulfill this role, thalamic neurons must themselves be subjected to constantly changing modulatory inputs that originate in multiple neurochemical pathways involved in autonomic, affective and cognitive functions. Our overall goal is to define an anatomical framework for conceptualizing how a 'decision' is made on whether a trigeminovascular thalamic neuron fires, for how long, and at what frequency. To begin answering this question, we determine which neuropeptides/neurotransmitters are in a position to modulate thalamic trigeminovascular neurons. Using a combination of in-vivo single-unit recording, juxtacellular labeling with tetramethylrhodamine dextran (TMR) and in-vitro immunohistochemistry, we found that thalamic trigeminovascular neurons were surrounded by high density of axons containing biomarkers of glutamate, GABA, dopamine and serotonin; moderate density of axons containing noradrenaline and histamine; low density of axons containing orexin and melanin concentrating hormone (MCH); but not axons containing CGRP, serotonin 1D receptor, oxytocin or vasopressin. In the context of migraine, the findings suggest that the transmission of headache-related nociceptive signals from the thalamus to the cortex may be modulated by opposing forces (i.e., facilitatory, inhibitory) that are governed by continuous adjustments needed to keep physiological, behavioral, cognitive and emotional homeostasis.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Identification and labeling of individual thalamic trigeminovascular neurons.

(A) Neuronal responses to electrical (1 mA, 0.8 ms), mechanical (von Frey filament: 4, 63 g) and chemical (1 M KCl solution) stimulation of the dura overlying the left transverse sinus. (B) Synchronization of neuronal activity during iontophoretic injection of TMR by delivering pulses of current (1–10 nA) at 250 ms on/off intervals through the recording glass micropipette.

Figure 2. Glutamatergic innervation of thalamic trigeminovascular neurons.

Left: Immunopositive VGluT2 synaptic vesicles (green) surrounding a thalamic dura-sensitive neuron (red) labeled with TMR–dextran. Arrowheads indicate close apposition of VGluT2 positive axons and the cell body and dendrites of the labeled neuron. Upper right: Location of the dura-sensitive neuron (red star) shown at left. Number in red indicates distance from bregma (mm). Lower right: Fluorescent images showing VGluT2 axonal labeling in thalamic Po and VPM nuclei. Scale bars = 100 µm. Abbreviations: DLG, dorsal lateral geniculate; LPMR, lateral posterior thalamic, mediorostral; LPLR, lateral posterior thalamic, laterorostral part; VPL, ventral posterolateral thalamic.

Figure 3. GABAergic innervation of thalamic trigeminovascular neurons.

Left: 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. Upper right: Location of the dura-sensitive neuron (red star) shown at left. Number in red indicates distance from bregma (mm). Lower right: 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.

Figure 4. Serotoninergic innervation of thalamic trigeminovascular neurons.

Left: 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. Upper right: Location of the dura-sensitive neuron (red star) shown at left. Number in red indicates distance from bregma (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.

Figure 5. Close apposition between chemically-identified axons and thalamic trigeminovascular neurons.

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 Fig. 4). 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 Figures 1–7. Caveat: 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.

Figure 6. Noradrenergic innervation of thalamic trigeminovascular neurons.

Left: 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. Upper right: Location of the dura-sensitive neuron (red star) shown at left. Number in red indicates distance from bregma (mm). Lower right: Fluorescent image showing DBH labeling of cell bodies in the locus coeruleus (LC) of the brainstem. Scale bars = 100 µm.

Figure 7. Dopaminergic innervation of thalamic trigeminovascular neurons.

Left: Immunopositive Tyrosine 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 TH positive axons and the cell body and dendrites of the labeled neuron. Upper right: Location of the dura-sensitive neuron (red star) shown at left. Number in red indicates distance from bregma (mm). Lower right: Fluorescent image showing TH labeling of cell bodies in the hypothalamic A11 nucleus. Scale bars = 100 mm. Caveat: TH is present in noradrenergic and dopaminergic cells, thus TH-positive labeling must take into consideration these two neurotransmitters.

Figure 8. Histaminergic innervation of thalamic trigeminovascular neurons.

Left: 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. Upper right: Location of the dura-sensitive neuron (red star) shown at left. Number in red indicates distance from bregma (mm). Lower right: 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.

Figure 9. MCH innervation of thalamic trigeminovascular neurons.

Left: Immunopositive Melanin Concentrating Hormone 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 MCH positive axons and the dendrites of the labeled neuron. Upper right: Location of the dura-sensitive neuron (red star) shown at left. Number in red indicates distance from bregma (mm). Lower right: Fluorescent image showing MCH labeling of cell bodies in the lateral hypothalamus (LH). Scale bars = 100 µm. Abbreviations: ZID, zona incerta, dorsal; ZIV, zona incerta, ventral.

Figure 10. Orexinergic innervation of thalamic trigeminovascular neurons.

Left: Immunopositive Orexin A 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 OrA positive axons and the dendrites of the labeled neuron. Upper right: Location of the dura-sensitive neuron (red star) shown at left. Number in red indicates distance from bregma (mm). Lower right: Fluorescent image showing OrA labeling of cell bodies in the hypothalamic perifornical area (PeF). Scale bars = 100 µm. Abbreviation: LDVL, laterodorsal thalamic, ventrolateral.

Figure 11. Lack of innervation of thalamic trigeminovascular neurons by axons containing CGRP, Serotonin 1D receptor, Oxytocin and Vasopressin.

Left A–D: 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). Upper right A–D: Locations of the dura-sensitive neurons (red stars) shown at left. Numbers in red indicate distance from bregma (mm). Lower right A–B: 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. Lower right C: Fluorescent images showing Oxytocin labeling of cell bodies and axons in the hypothalamic paraventricular nucleus (PVN) and lateral hypothalamus (LH), respectively. Lower right D: 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.

Figure 12. (A) Schematic illustration of the neurotransmitter and neuropeptidergic systems innervating thalamic trigeminovascular neurons in VPM, Po and LP/LD.

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.