The authors have declared that no competing interests exist.
Conceived and designed the experiments: RN RB. Performed the experiments: RN VK. Analyzed the data: RN VK DB RB. Wrote the paper: RN DB RB.
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.
Historically, the thalamus was viewed as a simple relay station for sensory information from the periphery to the cortex. This view has been replaced by the concept that instead of ‘just’ transferring sensory signals from subcortical nuclei to the cortex, thalamic neurons play central role in the selection, amplification, and prioritization process that determines which type of information should be made available to the cortex at any given time
To regulate the amount of sensory signals that reach the cortex, thalamic neurons must themselves be subjected to a variety of modulatory inputs that originate in cortical, hypothalamic, brainstem, spinal and intrathalamic nuclei
Far less is known about the regulation of relay thalamic neurons by other neurotransmitters and neuropeptides
The potential release of these neurotransmitters/neuropeptides on relay thalamic nuclei suggests that the modulation of individual neurons is rather complex, likely subjected to opposing forces driven by a variety of changing external and internal conditions that require constant behavioral, physiological, and affective adjustments. Our overall goal is to understand how ‘a decision’ is made on whether or not a relay thalamic neuron fires, for how long, and at what frequency. To start answering this question, we must first determine which neuropeptides/neurotransmitters are in a position to govern the activity of individual thalamic neurons that share a common function; a task never taken before. In the current study we describe an array of neuropeptides/neurotransmitters that may modulate individual, physiologically-identified thalamic trigeminovascular neurons believed to play a role in the generation of headache perception during migraine. The understanding of this neurobiology will allow for a basis to determine functional neurotransmission between the thalamus and cortex related to multiple clinical components of migraine including pain (somatosensory cortex), cognition (frontal cortex), memory (hippocampus), altered perception (parietal cortex), interoception and awareness (insular cortex).
Experiments were approved by the Institutional Animal Care and Use Committee at Harvard Medical School and Beth Israel Deaconess Medical Center, and conducted in accordance of NIH guide for the care and use of laboratory animals. Thirty-two male Sprague-Dawley rats weighing 250–350 g were initially anesthetized with a single dose of Brevital sodium (45 mg/kg i.p.) to allow endotracheal intubation and cannulation of the right femoral vein. Each rat was then mounted on a stereotaxic frame and connected to an inhalation anesthesia system (O2/Isoflurane 2.5% for craniotomies; 1–1.2% for maintenance during the rest of the experiment, delivered at 100 ml/min). End-tidal CO2, respiratory and heart rate, blood oxygen saturation and body temperature were continuously monitored and kept within a physiological range. One craniotomy was performed at the left lambdoid suture to expose and stimulate the meninges overlying the left transverse sinus. A second craniotomy was performed at the right parietal bone to allow the introduction of a glass micropipette into the posterior thalamus for recording and juxtacellular iontophoresis of an anterograde tracer, as described previously
A glass micropipette (20–30 MΩ impedance) filled with a 3% solution of the tracer tetramethylrhodamine dextran (TMR; 3,000 MW, anionic, lysine fixable; D-3308, Invitrogen) in 0.9% NaCl, was lowered into the right posterior thalamus while searching for single-unit responses to electrical stimulation of the contralateral dura (0.8 ms, 0.5–3.0 mA, 1 Hz). Thalamic neurons responding to the electrical stimulation were additionally tested for responses induced by mechanical (calibrated von-Frey monofilament) and chemical (1 M KCl) stimulation of the dura (
(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.
Rats were injected with an overdose of pentobarbital sodium (100 mg/kg) and perfused intracardially with 200 ml heparinized saline, followed by a fixative solution consisting in 400 ml of 0.1 M phosphate buffered saline (PBS), 4% paraformaldehyde and 0.05% picric acid. Only when required for the staining protocol, rats were perfused with 200 ml of PBS followed by a fixative solution containing 75 ml of 4% ethylcarbodiimide in 0.1 M PBS. Brains were removed, soaked in the fixative solution for 2 hrs, and cryoprotected in 30% sucrose phosphate buffer for 48 hrs. Brains were then frozen and cut into serial coronal sections (60–80 µm-thick) using a cryostat (Leica). Free-floating sections were collected and mounted on slides for a rapid visualization and localization of each cell body and its dendrites using epifluorescence microscopy.
Brain sections containing successfully injected neurons were pre-incubated at room temperature in PBS containing 2% fetal bovine serum albumin (FSA) and 1% Triton X-100 for 1 hr. Sections were then incubated at 4°C for 48 hrs in the same blocking solution with one of the following primary antibodies: (i) mouse anti-Serotonin Transporter, SERT (1∶5,000 dilution; Millipore); (ii) mouse anti-Tyrosine Hydroxylase, TH (1∶5,000; Immunostar); (iii) rabbit anti-Dopamine β-Hydroxylase, DBH (1∶5,000; Immunostar); (iv) goat anti-Orexin A (1∶2,500; Santa Cruz); (v) rabbit anti-Calcitonin Gene Related Peptide, CGRP (1∶5,000; Chemicon); (vi) rabbit anti-5HT1D receptor (1∶50,000; Courtesy of Andrew Ahn, University of Florida); (vii) rabbit anti-Oxytocin (1∶10,000; Immunostar); (viii) goat anti-Vasopressin (1∶1,000; Immunostar); (ix) rabbit anti-Histamine (1∶3,000; Immunostar; ethylcarbodiimide perfusion); (x) guinea pig anti-Vesicular Glutamate Transporter 2, VGluT2 (1∶2,500; Millipore); (xi) rabbit anti-Vesicular GABA Transporter, VGaT (1∶1,000; Phosphosolutions); (xii) Melanin Concentrating Hormone, MCH (1∶1,000; Courtesy of Terry Maratos-Flier, Harvard Medical School). The sections were washed multiple times and then incubated in PBS containing 2% FSA and 0.5% Triton X-100 for 2 hrs at room temperature with the corresponding fluorescent secondary antibody (Alexa Fluor 488; Invitrogen) against the Ig’s of the animal in which the primary antibody was raised (dilution range 1∶200–1∶1,000). Immunostained sections were serially mounted on glass slides and coverslipped with fluorescent mounting media with or without DAPI counterstaining.
Digital imaging of each of the neuronal cell bodies and dendrites injected with TMR, as well as the axonal network of the different neurotransmitters/neuropeptides was performed using epifluorescence scanning microscopy that compiled 1–1.5 µm-thick scans using
Forty-seven thalamic neurons that responded to electrical, mechanical and chemical stimulation of the contralateral dura were classified as trigeminovascular neurons
Glutamatergic innervation was determined using Vesicular Glutamate Transporter 2 (
GABAergic innervation was determined using Vesicular GABA Transporter (
Serotoninergic innervation was determined using Serotonin Transporter (
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
Noradrenergic innervation was determined using the enzyme Dopamine β-Hydroxylase (
Dopaminergic innervation was determined using the enzyme Tyrosine Hydroxylase (
(
Orexinergic innervation was determined by targeting the neuropeptide orexin A (
Surprisingly, we found no evidence for presence of CGRP-positive axons in any thalamic nucleus containing trigeminovascular neurons (positive identification of CGRP fibers in the parvicellular division of the ventral posterior thalamic nucleus confirms the validity of the negative staining in the thalamic nuclei analyzed in this study) (
We processed all images containing immunohistochemical evidence for thalamic innervation of the neurotransmitter/neuropeptides, and calculated their relative density by using binary maps (ImageJ). The binary map identifies all pixels containing positive immunostaining and converts them to white pixels; the remaining black pixels are considered lack of staining. This data provide quantitative measures of density of innervation of thalamic areas where juxtacellularly labeled trigeminovascular neurons were found (
Positive Pixels | Negative Pixels | Positive Pixels (%) | Density | |
139,571 | 1,308,109 | High | ||
124,675 | 1,323,005 | High | ||
106,398 | 1,341,282 | High | ||
82,190 | 1,365,490 | High | ||
46,331 | 1,401,349 | Moderate | ||
17,578 | 1,430,102 | Moderate | ||
8,555 | 1,439,125 | Low | ||
7,153 | 1,440,527 | Low | ||
0 | 1,447,680 | Absent | ||
0 | 1,447,680 | Absent | ||
0 | 1,447,680 | Absent | ||
0 | 1,447,680 | Absent |
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
This proof-of-concept study defines a new molecular framework for a more sophisticated thinking of the complexity of factors that modulate the response properties of relay trigeminovascular thalamic neurons. Most significant was the finding that such neurons receive direct input from axons containing glutamate, GABA, dopamine, noradrenaline, serotonin, histamine, orexin and MCH but not from axons that contain oxytocin, vasopressin, CGRP or the 5HT1D receptor (
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.
The discharge mode of relay thalamocortical neurons is either burst or tonic
Vesicular glutamate transporters (VGluTs) are responsible for glutamate trafficking and for the subsequent regulated release of this excitatory neurotransmitter at the synapse. Glutamate excites relay thalamocortical neurons through NMDA receptors, if the sensory stimulus is prolong and through non-NMDA receptors if the sensory stimulus is brief
In the context of migraine, dopamine has been considered for its role in promoting hypothalamic-mediated symptoms/prodromes such as yawning and nausea
Relevant to this study is that serotonin has long been implicated in migraine pathophysiology
Because of the wide distribution of noradrenergic fibers in the brain it is difficult to assign to this neurotransmitter a specific role in certain function. Rather, it is thought to improve signal-to-noise ratio in the firing of neurons that respond to sensory stimuli
In the context of migraine, histamine has been considered for its role in causing H1 receptor mediated arterial dilatation and the consequential induction of delayed headache
The MCH system, which originates in the hypothalamus and contains GABA
The orexin system originates in the lateral hypothalamus (LH) and projects to the cortex, thalamus, brainstem, spinal cord and other hypothalamic nuclei
A large number of studies suggest that CGRP plays an important role in multiple aspects of migraine pathophysiolopgy
The thalamus is intricately connected with multiple cortical, subcortical and brainstem regions. It is viewed as an important subcortical hub with respect to functional brain networks
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