自主神经对脑膜的支配功能研究进展

Meningeal lymphatic vessels (MLVs) are responsible for the drainage of waste products from the human brain. An impairment in their functionality has been associated with aging as well as brain disorders like multiple sclerosis and Alzheimer's disease. However, MLVs have only recently been described for the first time in magnetic resonance imaging (MRI), and their ramified structure renders manual segmentation particularly difficult. Further, as there is no consistent notion of their appearance, human-annotated MLV structures contain a high inter-rater variability that most automatic segmentation methods cannot take into account. In this work, we propose a new rater-aware training scheme for the popular nnU-Net model, and we explore rater-based ensembling strategies for accurate and consistent segmentation of MLVs. This enables us to boost nnU-Net's performance while obtaining explicit predictions in different annotation styles and a rater-based uncertainty estimation. Our final model, MLV$^2$-Net, achieves a Dice similarity coefficient of 0.806 with respect to the human reference standard. The model further matches the human inter-rater reliability and replicates age-related associations with MLV volume.

A comprehensive understanding of the organizational principles in the human brain requires, among other factors, well-quantifiable descriptors of nerve fiber architecture. Three-dimensional polarized light imaging (3D-PLI) is a microscopic imaging technique that enables insights into the fine-grained organization of myelinated nerve fibers with high resolution. Descriptors characterizing the fiber architecture observed in 3D-PLI would enable downstream analysis tasks such as multimodal correlation studies, clustering, and mapping. However, best practices for observer-independent characterization of fiber architecture in 3D-PLI are not yet available. To this end, we propose the application of a fully data-driven approach to characterize nerve fiber architecture in 3D-PLI images using self-supervised representation learning. We introduce a 3D-Context Contrastive Learning (CL-3D) objective that utilizes the spatial neighborhood of texture examples across histological brain sections of a 3D reconstructed volume to sample positive pairs for contrastive learning. We combine this sampling strategy with specifically designed image augmentations to gain robustness to typical variations in 3D-PLI parameter maps. The approach is demonstrated for the 3D reconstructed occipital lobe of a vervet monkey brain. We show that extracted features are highly sensitive to different configurations of nerve fibers, yet robust to variations between consecutive brain sections arising from histological processing. We demonstrate their practical applicability for retrieving clusters of homogeneous fiber architecture, performing classification with minimal annotations, and query-based retrieval of characteristic components of fiber architecture such as U-fibers.

The correct reconstruction of individual (crossing) nerve fibers is a prerequisite when constructing a detailed network model of the brain. The recently developed technique Scattered Light Imaging (SLI) allows the reconstruction of crossing nerve fiber pathways in whole brain tissue samples with micrometer resolution: The individual fiber orientations are determined by illuminating unstained histological brain sections from different directions, measuring the transmitted scattered light under normal incidence, and studying the light intensity profiles of each pixel in the resulting image series. So far, SLI measurements were performed with a fixed polar angle of illumination and a small number of illumination directions, providing only an estimate of the nerve fiber directions and limited information about the underlying tissue structure. Here, we use an LED display with individually controllable light-emitting diodes to measure the full distribution of scattered light behind the sample (scattering pattern) for each image pixel at once, enabling scatterometry measurements of whole brain tissue samples. We compare our results to coherent Fourier scatterometry (raster-scanning the sample with a non-focused laser beam) and previous SLI measurements with fixed polar angle of illumination, using sections from a vervet monkey brain and human optic tracts. Finally, we present SLI scatterometry measurements of a human brain section with 3 $μ$m in-plane resolution, demonstrating that the technique is a powerful approach to gain new insights into the nerve fiber architecture of the human brain.

Mapping the intricate network of nerve fibers is crucial for understanding brain function. Three-Dimensional Polarized Light Imaging (3D-PLI) and Computational Scattered Light Imaging (ComSLI) map dense nerve fibers in brain sections with micrometer resolution using visible light. 3D-PLI reconstructs 3D-fiber orientations, while ComSLI disentangles multiple directions per pixel. So far, these imaging techniques have been realized in separate setups. A combination within a single device would facilitate faster measurements, pixelwise mapping, cross-validation of fiber orientations, and leverage the advantages of each technique while mitigating their limitations. Here, we introduce the Scattering Polarimeter, a microscope that facilitates correlative large-area scans by integrating 3D-PLI and ComSLI measurements into a single system. Based on a Mueller polarimeter, it incorporates variable retarders and a large-area light source for direct and oblique illumination, enabling combined 3D-PLI and ComSLI measurements. Applied to human and vervet monkey brain sections, the Scattering Polarimeter generates results comparable to state-of-the-art 3D-PLI and ComSLI setups and creates a multimodal fiber direction map, integrating the robust fiber orientations obtained from 3D-PLI with fiber crossings from ComSLI. Furthermore, we discuss applications of the Scattering Polarimeter for unprecedented correlative and multimodal brain imaging.

More children and adults under the age of 40 die of brain tumor than from any other cancer. Brain surgery constitutes the first and decisive step for the treatment of such tumors. It is extremely crucial to achieve complete tumor resection during surgery, however, this is a highly challenging task, as it is very difficult to visually differentiate tumorous cells from the surrounding healthy white matter. The nerve fiber bundles constitutive of the white matter are organized in such a way that they exhibit a certain degree of structural anisotropy and birefringence. The birefringence exhibited by such aligned fibrous tissue is known to be extremely sensitive to small pathological alterations. Indeed, highly aligned anisotropic fibers exhibit higher birefringence than structures with weaker alignment and anisotropy, such as cancerous tissue. In this study, we performed experiments on thick coronal slices of a healthy human brain to explore the possibility of (i) measuring, with a polarimetric microscope (employed in the backscattering geometry to facilitate non-invasive diagnostics), the birefringence exhibited by the white matter and (ii) relating the measured birefringence to the fiber orientation and the degree of alignment. This is done by analyzing the spatial distribution of the degree of polarization of the backscattered light and its variation with the polarization state of the probing beam. We demonstrate that polarimetry can be used to reliably distinguish between white and gray matter in the brain, which might help to intraoperatively delineate unstructured tumorous tissue and well organized healthy brain tissue. In addition, we show that our technique is able to sensitively reconstruct the local mean nerve fiber orientation in the brain, which can help to guide tumor resections by identifying vital nerve fiber trajectories thereby improving the outcome of the brain surgery.

Migraine is a common disabling headache disorder characterized by recurrent episodes sometimes preceded or accompanied by focal neurological symptoms called aura. The relation between two subtypes, migraine without aura (MWoA) and migraine with aura (MWA), is explored with the aim to identify targets for neuromodulation techniques. To this end, a dynamically regulated control system is schematically reduced to a network of the trigeminal nerve, which innervates the cranial circulation, an associated descending modulatory network of brainstem nuclei, and parasympathetic vasomotor efferents. This extends the idea of a migraine generator region in the brainstem to a larger network and is still simple and explicit enough to open up possibilities for mathematical modeling in the future. In this study, it is suggested that the migraine generator network (MGN) is driven and may therefore respond differently to different spatio-temporal noxious input in the migraine subtypes MWA and MWoA. The noxious input is caused by a cortical perturbation of homeostasis, known as spreading depression (SD). The MGN might even trigger SD in the first place by a failure in vasomotor control. As a consequence, migraine is considered as an inherently dynamical disease to which a linear course from upstream to downstream events would not do justice. Minimally invasive and noninvasive neuromodulation techniques are briefly reviewed and their rational is discussed in the context of the proposed mechanism.

Understanding neuronal communication is fundamental in neuroscience but there are few methodologies offering detailed analysis for well-controlled conditions. By interfacing microElectrode arrays with microFluidics (μEF devices), it is possible to compartmentalize neuronal cultures with a specified alignment of axons and microelectrodes. This setup allows extracellular recordings of spike propagation with high signal-to-noise ratio over the course of several weeks. Addressing these μEF systems we developed an advanced, yet easy-to-use, open-source computational tool, μSpikeHunter, which provides detailed quantification of several communication-related properties such as propagation velocity, conduction failure, spike timings, and coding mechanisms. The combination of μEF devices and μSpikeHunter can be used in the context of standard neuronal cultures or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. The ability to analyze axonal signals (in a user-friendly, time-efficient, high-throughput manner) opens doors to new approaches to studies of peripheral innervation, neural coding, and neuroregeneration approaches, among many others. We demonstrate the use of μSpikeHunter in dorsal root ganglion neurons where we analyze the presence of anterograde signals in μEF devices.

There is significant paucity in the literature regarding the vertebral nerve. Moreover, descriptions of this structure are conflicting. To evaluate further the anatomy and potential clinical significance of this structure, 10 fresh adult cadavers (20 sides) underwent dissection and macroscopic observation of this structure. All specimens were found to have a vertebral nerve that originated from the stellate ganglion with the exception of two left sides (10%) in which this nerve arose from the inferior cervical ganglion. This nerve ascended posteromedial to the vertebral artery. The vertebral nerve was found to be, in essence, a long and deep gray ramus communicans that connected most commonly the stellate ganglia to C6 or C7 spinal nerves by passing through the C6 and C7 transverse foramina. Fifteen percent of sides were found to have a vertebral nerve that was plexiform in its configuration. Fifty percent were found to have very small branches that entered the fibrous capsule of adjacent zygapophyseal and intervertebral joints. Some specimens were noted to have meningeal branches of the vertebral nerve. Based on our observations, the vertebral nerve is simply a deep ramus communicans, which often provides articular and meningeal branches to the adjacent spine. As neck pain is a significant reason for physician office visits, additional knowledge of the nerves innervating the joints and adjacent meninges of the neck could be important for both surgical and medical blockade of nerve fibers.

The objective of this article is to review the prospect of treating migraine with sphenopalatine ganglion (SPG) neurostimulation. Fuelled by preliminary studies showing a beneficial effect in cluster headache patients, the potential of treating migraine with neurostimulation has gained increasing interest within recent years, as current treatment strategies often fail to provide adequate relief from this debilitating headache. Common migraine symptoms include lacrimation, nasal congestion, and conjunctival injection, all parasympathetic manifestations. In addition, studies have suggested that parasympathetic activity may also contribute to the pain of migraineurs. The SPG is the largest extracranial parasympathetic ganglion of the head, innervating the meninges, lacrimal gland, nasal mucosa, and conjunctiva, all structures involved in migraine with cephalic autonomic symptoms. We propose two possible mechanisms of action: 1) interrupting the post-ganglionic parasympathetic outflow to inhibit the pain and cephalic autonomic symptoms, and 2) modulating the sensory processing in the trigeminal nucleus caudalis. To further explore SPG stimulation in migraineurs as regards therapeutic potential and mode of action, randomized clinical trials are warranted.

Migraine oscillates between different states in association with internal homeostatic functions and biological rhythms that become more easily dysregulated in genetically susceptible individuals. Clinical and pre-clinical data on migraine pathophysiology support a primary role of the central nervous system (CNS) through 'dysexcitability' of certain brain networks, and a critical contribution of the peripheral sensory and autonomic signalling from the intracranial meningeal innervation. This review focuses on the most relevant back and forward translational studies devoted to the assessment of CNS dysfunctions involved in primary headaches and discusses the role they play in rendering the brain susceptible to headache states. We collected a body of scientific literature from human and animal investigations that provide a compelling perspective on the anatomical and functional underpinnings of the CNS in migraine and trigeminal autonomic cephalalgias. We focus on medullary, hypothalamic and corticofugal modulation mechanisms that represent strategic neural substrates for elucidating the links between trigeminovascular maladaptive states, migraine triggering and the temporal phenotype of the disease. It is argued that a better understanding of homeostatic dysfunctional states appears fundamental and may benefit the development of personalized therapeutic approaches for improving clinical outcomes in primary headache disorders. This review focuses on the most relevant back and forward translational studies showing the crucial role of top-down brain modulation in triggering and maintaining primary headache states and how these central dysfunctions may interact with personalized pain management strategies.

No abstract

Migraine is the most common neurological disorder, and much has been learned about its mechanisms in recent years. However, the origin of painful impulses in the trigeminal nerve is still uncertain. Despite the attention paid recently to the role of central sensitisation in migraine pathophysiology, in our view, neuronal hyperexcitability depends on activation of peripheral nociceptors. Although the onset of a migraine attack might take place in deep-brain structures, some evidence indicates that the headache phase depends on nociceptive input from perivascular sensory nerve terminals. The input from arteries is probably more important than the input from veins. Several studies provide evidence for input from extracranial, dural, and pial arteries but, likewise, there is also evidence against all three of these locations. On balance, afferents are most probably excited in all three territories or the importance of individual territories varies from patient to patient. We suggest that migraine can be explained to patients as a disorder of the brain, and that the headache originates in the sensory fibres that convey pain signals from intracranial and extracranial blood vessels.

The lumbar sinuvertebral nerve (SVN) innervates the outer posterior intervertebral disc (IVD); it is thought to mediate discogenic low-back pain (LBP). Controversy, however, exists on its origins at higher (L1-L2) versus lower (L3-L5) lumbar levels. Additionally, lack of knowledge regarding its foraminal and intraspinal branching patterns and extensions may lead to iatrogenic damage. To systematically describe the origins of the L2 and L5 SVNs, their morphological variation in the intervertebral foramen (IVF) and intraspinal distribution. Dissection-based study of 20 SVNs with histological confirmation in five embalmed human cadavers. The origin, branching pattern and distribution of the L2 and L5 SVNs was investigated bilaterally in five human cadavers using dorsal and anterolateral dissection approaches. Parameters studied included somatic and/or autonomic SVN root contributions, foraminal SVN morphology and course, diameter, branching point, intraspinal distribution and IVD innervation pattern. Nerve tissue was confirmed by immunostaining for neurofilament and S100 proteins. The SVN and its origins was identified in all except one IVF at L2 and in all foramina at L5. At L2, the SVN arose in nearly 90% of sides from both somatic and autonomic roots and at L5 in 40% of sides. The remaining SVNs were formed by purely autonomic roots. The SVN arose from significantly more roots at L2 than L5 (3.1 ± 0.3 vs. 1.9 ± 0.3, respectively; p=.022). Four different SVN morphologies could be discerned in the L2 IVF: single filament (22%), multiple (parallel or diverging) filament (33%), immediate splitting (22%) and plexiform (22%) types, whereas the L5 SVN consisted of single (90%) and multiple (10%) filament types. SVN filaments were significantly thicker at L2 than L5 (0.48 ± 0.06 mm vs. 0.33 ± 0.02 mm, respectively; p=.043). Ascending SVN filaments coursed roughly parallel to the exiting spinal nerve root trajectory at L2 and L5. Branching of the SVN into ascending and descending branches occurred mostly intraspinal both at L2 and L5. Spinal canal distribution was also similar for L2 and L5 SVNs. Lumbar posterior IVDs were innervated by the descending branch of the parent SVN and ascending branch of the subjacent SVN. The SVN at L2 originates from both somatic and autonomic roots in 90% of cases and at L5 in 40% of cases. The remaining SVNs are purely autonomic. In the IVF, the L2 SVN is morphologically heterogeneous, but generally consists of numerous filaments, whereas at L5 90% contains a single SVN filament. The L2 SVN is formed by more roots and is thicker than the L5 SVN. Intraspinal SVN distribution is confined to its level of origin; lumbar posterior IVDs are innervated by corresponding and subjacent SVNs (ie, two spinal levels). Our findings indicate that L5 discogenic LBP may be mediated both segmentally and nonsegmentally in 40% of cases and nonsegmentally in 60% of cases. Failure of lower lumbar discogenic pain treatment may be the result of only interrupting the nonsegmental pathway, but not the segmental one as well. Relating SVN anatomy to microsurgical spinal approaches may prevent iatrogenic damage to the SVN and the formation of postsurgical back pain.

No abstract

Eight lesser known reflexes were grouped together because of their anatomical and physiological relationship. In all of them a branch of the vagus nerve forms a bridge between a circumscribed area of the skin, mostly the external auditory meatus, and an internal organ, namely the stomach, esophagus, lungs, heart, uterus, and some male and female sex organs. The eight reflexes are: (1) Gastroauricular phenomenon (Gaph) (Engel, 1922) in man; (2) Auricular phenomenon (Malherbe, 1958) in man; (3) Pulmonoauricular phenomenon (Deutsch, 1919) in man; (4) Auriculogenital reflex (Bradford, 1937) in cat; (5) Auriculouterine reflex (Vasiliu, 1932) in women; (6) Oculocardiac reflex (Aschner, 1967) in man; (7) Kalchschmidt's reflex in cattle (1956); and (8) Coughing attack with heartburn (Berlin, 1959) in man. The organs involved are either effector or receptor organs. The six reflexes observed in man are of diagnostic significance. Attention is also drawn to analogous reflexes in which the meningeal branch of the vagus is involved.

The microsurgical anatomy of the jugular foramen was studied in 10 fixed cadavers, each cadaver consisting of the whole head and neck. Five of the cadavers were injected with latex. The jugular foraminal region was exposed using the infratemporal fossa type A approach of Fisch and Pillsbury in five cadavers (10 sides) and the combined cervical dissection-mastoidectomy-suboccipital craniectomy approach in five cadavers (10 sides). The right foramen was larger than the left in seven cases (70%), equal in two cases (20%), and smaller in one case (10%). The dura covering the intracranial portal of the foramen had two perforations, a smaller anteromedial perforation through which passed the ninth cranial nerve (CN IX), and a larger posterolateral perforation, through which passed the 10th and 11th cranial nerves (CNs X and XI) and the distal sigmoid sinus. The perforations were separated by a fibrous septum in 16 specimens (80%). After exiting the posterior fossa, CNs IX, X, and XI all lay anteromedial to the superior jugular bulb (SJB) within the jugular foramen. The inferior petrosal sinus (IPS) entered the foramen between CNs IX and X in most cases; however, in 10% of our cases it entered the foramen between CNs X and XI, and in 10% it entered the foramen caudal to CN XI. The IPS terminated in the SJB in 90% of our cases; in 40%, the IPS termination consisted of multiple channels draining into both the SJB and internal jugular vein. This study shows that the arrangement of the neurovascular structures within the jugular foramen does not conform to the hitherto widely accepted notion of discrete compartmentalization into an anteromedial pars nervosa containing CN IX and the IPS and a posterolateral pars venosa containing the SJB, CNs X and XI, and the posterior meningeal artery.

Trigeminocardiac reflex (TCR), the reproducible hypotension and bradycardia upon stimulation of the trigeminal nerve, has been reported during craniofacial surgery and during surgery within the cerebellopontine angle, petrosal sinus, orbit, and trigeminal ganglion. Whereas the falx cerebri is known to be innervated by the nervus tentorii, a recurrent branch of V1, there have been no reports to date of this response upon mechanical stimulation of the falx. We report a case of immediate, reproducible, and reflexive response of asystole upon stimulation of the falx cerebri during operative resection of a parafalcine meningioma in a 53-year-old woman. Upon recognition of the reproducible relationship between falcine stimulation and increased vagal tone, the patient was given glycopyrrolate in an effort to block cholinergic hyperactivity. After glycopyrrolate was given, no further dysrhythmias occurred. In this patient, mechanical stimulation of the falx likely resulted in the hyperactivity of the trigeminal ganglion, thereby triggering TCR. The dorsal region of the spinal trigeminal tract includes neurons from hypoglossal and vagus nerves, and projections have been seen between the vagus and trigeminal nuclei. The vagus provides parasympathetic innervation to the heart, vascular smooth muscle, and abdominal viscera. Vagal stimulation via these connections after trigeminal nerve activation likely accounts for the reflexive response of asystole seen in this patient. This is confirmed by the observation that the reflex was inhibited by the anticholinergic effects of glycopyrrolate. Awareness of TCR allows for early detection and appropriate treatment.

No abstract

The presence of a cholinergic innervation of the main cerebral blood vessels has been studied in the dog. Cholinergic nerve fibers are found in all examined arteries and veins, organized in two or in a single nerve plexus. The cerebral veins appear to be less innervated than arteries. The meaning of a cholinergic innervation in the cerebral circulation is discussed.

No abstract

The extracerebral vasculature receives a postnatal innervation of noradrenergic sympathetic axons and nociceptive sensory axons. These axons are responsive to the neurotrophin nerve growth factor (NGF), in that they possess the transmembrane receptors p140proto-trkA and p75neurotrophin receptor (NTR) which bind NGF. p75NTR-deficient mice display reduced patterns of sympathetic innervation of the pineal gland and sensory innervation of the skin (Lee et al., 1992, 1994a). The goal of this investigation was to determine whether an absence of p75 expression likewise perturbs the sympathetic and sensory innervation of the extracerebral vessels of adult mice, and if so, whether increasing levels of NGF within the target field is capable of enhancing this perturbed axon growth. Four lines of mice were used: wild-type C57Bl/6 mice, transgenic mice overexpressing NGF in the brain, p75NTR-deficient mice, and hybrid mice which overexpress NGF in the brain but lack p75NTR expression. Sympathetic and sensory innervation of the meningeal arteries were severely perturbed in p75NTR-deficient mice. Wild-type and hybrid mice displayed comparable patterns of sympathetic and sensory axons along the dural arteries. Transgenic mice, however, possessed the greatest degree of arterial innervation. These data reveal that while p75NTR expression may be a critical factor for initiating axon growth along the extracerebral vasculature during postnatal development, the sympathetic and sensory nervous systems display a remarkable degree of NGF-induced axonal plasticity, such that increased levels of NGF can ameliorate perturbed patterns of arterial innervation in p75-deficient mice.

Although not without controversy, an influence of the autonomic nervous system in headache is a matter for current debate. A possible contact site of autonomic and sensory nerves is the dura mater, where they form a dense network accompanying blood vessels. We investigated interactions between autonomic and nociceptive fibres by measuring release of calcitonin gene-related peptide (CGRP) and prostaglandin E2 (PGE2) from the dura mater, in vitro. The parasympathomimetic agent carbachol did not change basal release of CGRP or PGE2, whereas it diminished release induced by a mixture of inflammatory mediators. Norepinephrine did not change induced release of CGRP or PGE2, nor basal release of CGRP. However, basal release of PGE2 was enhanced by norepinephrine, and this enhancement was reduced by serotonin through 5-HT(1D) receptors. We conclude that sympathetic transmitters may control nociceptor sensitivity via increased basal PGE2 levels, a possible mechanism to facilitate headache generation. Parasympathetic transmitters may reduce enhanced nociceptor activity.

1. The dura mater encephali of the rat was exposed and the blood flow around branches of the medial meningeal artery was monitored with a laser Doppler flowmeter. Changes in the meningeal blood flow (MBF) following electrical stimulation of the dura mater at a parasagittal site were registered. The effects of human calcitonin gene-related peptide (h-alpha CGRP) and the CGRP antagonist (h-alpha CGRP8-37) on the MBF were tested. 2. Electrical stimulation with rectangular pulses of 0.5 ms, 10-20 V, 5-10 Hz and a duration of 30 s caused an increase of the MBF in 14 out of 16 rats tested. The increases were dependent on stimulus strength and frequency. 3. The increase in MBF was inhibited in a dose-dependent manner by topical application of 0.1 ml of h-alpha CGRP8-37 at concentrations of 10(-7) - 10(-5) M. The highest dose abolished the increase in MBF. 4. Topical administration of 0.1 ml of h-alpha CGRP at a concentration of 10(-4) M increased the basal MBF by 15% on average. 5. It is suggested that the increase in MBF following electrical stimulation of the dura mater is mediated by the release of CGRP. The contribution of the dural afferent and sympathetic and parasympathetic efferent nerve fibres to this response are discussed.

1. In cats anaesthetized with alpha-chloralose, electrical stimulation (ES) of the trigeminal ganglion produced a fall in blood pressure, a predominantly ipsilateral dilatation in the common carotid vascular bed and bilateral dilatation of the middle meningeal vascular bed. Section of the trigeminal root abolished these responses. 2. Dilatation in the middle meningeal artery was not affected by section of the cervical sympathetic trunk nor by the section of the seventh cranial nerve trunk. The dilator response was abolished by section of the spinal cord at the C3 level and by intravenous administration of bretylium (10 mg/kg) or phentolamine (5 mg/kg). The response was significantly reduced by the prior administration of papaverine (10 mg/kg). 3. Functional adrenalectomy by means of a snare placed around the nerves and blood vessels supplying the adrenal glands significantly reduced the response. Electrical stimulation of the trigeminal ganglion was accompanied by a fall in circulating levels of noradrenaline and serotonin. 4. We conclude that ES of the trigeminal ganglion produces dilatation in the middle meningeal artery partly by autoregulation during the trigeminal depressor response and partly by a reduction in the circulating levels of noradrenaline. It differs from the dilatation seen in the general carotid circulation and the cortical microcirculation, which is mediated by parasympathetic nerves. There is no evidence that antidromic release of neuropeptides from sensory nerve endings in the dura plays a part in the dilatation.

The distribution of neuropeptide- (neuropeptide Y, substance P, vasoactive intestinal peptide) and catecholamine-synthesizing enzyme-immunoreactive axons in guinea-pig trigeminal, nodose, and cervical dorsal root ganglia was studied by double-labelling immunofluorescence in controls and after extirpation of either the cervical sympathetic trunk or the stellate ganglion; tyrosine hydroxylase- and dopamine-beta-hydroxylase-immunoreactive terminals in dorsal root ganglia were ultrastructurally investigated. Six neurochemically identifiable axons innervated the trigeminal ganglion, five kinds were found in the nodose and dorsal root ganglia. Two of them (catecholaminergic with and without neuropeptide Y) were of sympathetic origin and, besides their termination at arteries, provided a direct innervation of capsule cells of the trigeminal and cervical dorsal root ganglia facing the subarachnoid space. Varicosities which were interpreted as being of sensory origin were equally numerous in all ganglia, whereas those being likely of parasympathetic origin decreased in numbers from the trigeminal to the dorsal root and nodose ganglia. It is concluded that the sensory ganglia are the target of postganglionic sympathetic, parasympathetic and primary afferent neurons, each of which are specifically organized with respect to the neurochemical phenotype and inter- and intraganglionic distribution. Among other targets, these "nervi gangliorum" appear to be intimately linked to the ganglionic capsular cells and meningeal sheaths facing the liquor spaces.

The acute-phase reaction is the multisystem response to acute inflammation. The central nervous system (CNS) mediates a coordinated set of autonomic, endocrine and behavioral responses that constitute the cerebral component of the acute-phase reaction. However, the mechanisms of immune signaling of the CNS remain controversial. Emerging evidence indicates that different parts of the acute-phase reaction are initiated by distinct mechanisms and in different brain regions. Cytokines produced as a result of local infections (for example, in the abdominal or thoracic cavities) might activate vagal sensory fibers, resulting in sickness behavior and fevers. Additionally, circulating immune stimuli might activate meningeal macrophages and perivascular microglia along the borders of the brain, eliciting the local production of prostaglandins and responses such as fever, anorexia, sleepiness, and activation of the hypothalamo-pituitary-adrenal (HPA) axis. The biological importance of these responses might favor the existence of multiple parallel CNS pathways that are engaged by cytokines.

In addition to motor axons and preganglionic axons, ventral roots contain unmyelinated or thin myelinated sensory axons and postganglionic sympathetic axons. It has been said that ventral roots channel sensory axons to the CNS. However, it now seems that these axons end blindly, shift to the pia or loop and return towards the periphery and that these units reach the CNS via dorsal roots. Sensory ventral root axons project from a variety of somatic or visceral receptors; some of them are third branches of dorsal root afferents and some seem to lack a CNS projection. Many ventral root afferents contain substance P (SP) and/or calcitonin gene-related peptide (CGRP). These fibres are not affected by neonatal capsaicin treatment and they cannot induce radicular or pial extravasation. Some thin ventral root axons are sympathetic and relate to blood vessels. Afferents containing SP and/or CGRP and sympathetic axons also occur in the spinal pia mater. The sensory axons mediate pain. They might also have vasomotor, tissue-regulatory and/or mechanoreceptive functions. The motor roots of cranial nerves IV, VI and XI contain unmyelinated axons arranged like in ventral roots outside the autonomic outflow. However, the motor root of cranial nerve V channels some unmyelinated axons into the CNS. The occurrence of thin axons in ventral roots and pia mater changes during development and ageing. After peripheral nerve injury, ipsilateral ventral roots and pia are invaded by new sensory and postganglionic sympathetic axons.

An anatomical survey of the cavernous sinus in 16 adult cadavera has been made, based on serial sections cut at 15 micron in the coronal and sagittal planes. Certain aspects of the survey are of particular interest: (1) the oculomotor, trochlear, and ophthalmic nerves do not run in the lateral dural wall of the cavernous sinus; (2) venous sinuses of the cavernous sinus flow freely in a medial direction both anterior and posterior to the dorsum sellae; (3) trabeculae in the form of collections of fine areolar tissue extend between the vascular and neural elements. The amount present is variable; (4) the horizontal section of the internal carotid artery within the cavernous sinus runs a variable course in relation to the hypophysis and the lateral dural wall; (5) the oculomotor nerve lies within a meningeal envelope as far anteriorly as the tip of the anterior clinoid process; (6) the ophthalmic nerve communicates with the oculomotor, trochlear, and abducent nerves in the anterior part of the cavernous sinus; (7) the abducent nerve may lie within a meningeal envelope in the posterior part of the cavernous sinus; (8) the greater part of the sympathetic nerve plexus around the vertical part of the internal carotid artery passes into the abducent and ophthalmic nerves. Sympathetic fibres pass into the sheaths surrounding the oculomotor and trochlear nerves. Sympathetic ganglia are suspended from the ophthalmic nerve.

Nonvisual pineal and retinal photoreceptors are synchronizing circadian and circannual periodicity to the environmental light periods in the function of various organs. Melatonin of the pineal organ is secreted at night and represents an important factor of this periodic regulation. Night illumination suppressing melatonin secretion may result in pathological events like breast and colorectal cancer. Experimental works demonstrated the role of autonomic nerves in the pineal melatonin secretion. It was supposed that mammalian pineals have lost their photoreceptor capacity that is present in submammalians, and sympathetic fibers would mediate light information from the retina to regulate melatonin secretion. Retinal afferentation may reach the organ by central nerve fibers via the pineal habenulae as well. In our earlier works we have found that the pineal organ developing from lobular evaginations of the epithalamus differs from peripheral endocrine glands and is composed of a retina-like central nervous tissue that is comprised of cone-like pinealocytes, secondary pineal neurons and glial cells. Their autonomic nerves in submammalians as well as in mammalian animals do not terminate on pineal cells, rather, they run in the meningeal septa among pineal lobules and form vasomotor nerve endings. Concerning the adult human pineal there are no detailed fine structural data about the termination of autonomic fibers, therefore, in the present work we investigated the ultrastructure of the human pineal peripheral autonomic nerve fibers. It was found, that similarly to other parts of the brain, autonomic nerves do not enter the human pineal nervous tissue itself but separated by glial limiting membranes take their course in the meningeal septa of the organ and terminate on vessels by vasomotor endings. We suppose that these autonomic vasomotor nerves serve the regulation of the pineal blood supply according to the circadian and circannual changes of the metabolic activity of the organ and support by this effect the secretion of pineal neurohormones including melatonin.

Vagus nerve stimulation (VNS) has been reported to be effective in the abortive treatment of both migraine and cluster headache. Using validated animal models of acute dural-intracranial (migraine-like) and trigeminal-autonomic (cluster-like) head pain we tested whether VNS suppresses ongoing and nociceptive-evoked firing of trigeminocervical neurons to explain its abortive effects in migraine and cluster headache. Unilateral VNS was applied invasively via hook electrodes placed on the vagus nerve. A single dose of ipsilateral or contralateral VNS, to trigeminal recording and dural-stimulating side, suppressed ongoing spontaneous and noxious dural-evoked trigeminocervical neuronal firing. This effect was dose-dependent, with two doses of ipsilateral VNS prolonging suppression of ongoing spontaneous firing (maximally by ~60%) for up to three hours, and dural-evoked (Aδ-fiber; by ~22%, C-fiber: by ~55%) responses for at least two hours. Statistically, there was no difference between ipsilateral and contralateral groups. Two doses of VNS also suppressed superior salivatory nucleus-evoked trigeminocervical neuronal responses (maximally by ~22%) for 2.5h, to model nociceptive activation of the trigeminal-autonomic pathway. VNS had no effect on normal somatosensory cutaneous facial responses throughout. These studies provide a mechanistic rationale for the observed benefits of VNS in the abortive treatment of migraine and cluster headache. In addition, they further validate these preclinical models as suitable approaches to optimize therapeutic efficacy, and provide an opportunity to hypothesize and dissect the neurobiological mechanisms of VNS in the treatment of primary headaches.

The significance of autonomic nerves reaching the pincal organ was already investigated in connection to the innervation of pinealocytes and mediating light information from the retina for periodic melatonin secretion. In earlier works we found that some autonomic nerve fibers are not secretomotor but terminate on arteriolar smooth muscle cells in the pineal organ of the mink (Mustela vison). Studying in serial sections the pineal organ of the mink and 15 other mammalian species in the present work, we investigated whether similar axons of vasomotor-type are generally present in the wall of pineal vessels, further, whether they reach the organ via the conarian nerves or via periarterial plexuses. In all species investigated, axons of perivasal nerve bundles were found to form terminal enlargements on the smooth muscle layer of pineal arterioles. The neuromuscular endings contain several synaptic and some granular vesicles. Axon terminals are also present around pineal veins. In serial sections, we found that the so-called conarian autonomic nerves reach the pineal organ alongside pineal veins draining into the great internal cerebral vein. Similar nerves present near arteries of the arachnoid enter the pineal meningeal capsule and septa by arterioles, both perivenous and periarterial nerves form terminals of vasomotor-type. The arteriomotor and venomotor regulation of the tone of the vessels of the pineal organ may serve the vascular support for circadian and circannual periodic changes in metabolic activity of the pineal tissue.

Ultrastructural, immunocytochemical, and immunoelectron microscopical examinations are reported that describe the morphology of putative sensory nerve endings in the dura mater encephali of the rat and the cat. Morphometrical measurements and reconstructions showed that in the cat the mean diameter of axons, the bare area of axolemma, and the content of mitochondria and vesicles are highly variable in dural nerve endings. Nerve fibers with a high volume density of mitochondria are thought to be sensory, while nerve fibers containing many small vesicles are considered autonomic. There is, however, a broad overlap of mitochondria-rich and vesicle-rich nerve fibers in the dura, so that discrimination between sensory and autonomic endings by these characteristics frequently fails. Whole-mount preparations treated cytochemically for detection of substance P- and calcitonin gene-related peptide-like immunoreactivity in the rat and the cat showed a network of immunopositive nerve fibers in the vicinity of dural blood vessels. Most of these peptidergic and probably sensory nerve fibers were found terminating in the dural connective tissue far from vessels. Calcitonin gene-related peptide-positive nerve fibers were much more abundant than substance P-positive fibers. Immunoelectron microscopic preparations revealed that calcitonin gene-related peptide- and substance P-like immunoreactivity is found in a small proportion of generally thin unmyelinated nerve fibers. These proportions were very similar in the rat and the cat. Summarizing the recent literature, the morphological characteristics of putative sensory nerve fibers in the dura mater are discussed in relation to their possible functional significance for neurogenic inflammation and nociception.

The physiology and pharmacology of the middle meningeal artery was investigated in cats in order to determine whether this artery was subject to normal neural and humoral control mechanisms. Carotid and middle meningeal arterial blood flows and resistances were measured in 16 cats anaesthetized with chloralose. The cervical sympathetic nerves were stimulated electrically. Stimulation of the cervical sympathetic nerves pre-ganglionically reduced blood flow in the middle meningeal artery by producing vasoconstriction in its resistance bed. The vasoconstriction was mediated via catecholamine-containing nerves, as it was abolished by prior intravenous administration of bretylium. Intravenous injections of noradrenaline or adrenaline also produced vasoconstriction in the middle meningeal arterial bed. 5-Hydroxytryptamine (5HT), on the other hand, produced a dilatation in the middle meningeal artery. We conclude that neurally or humorally released catecholamines can provide a plausible mechanism for vasoconstriction in the middle meningeal artery. The dilator effect of 5HT contrasts with the constrictor effect of the 5HT1-like receptor agonist sumatriptan and suggests a complex 5HT receptor pharmacology for the artery.

The trajectories of sympathetic nerves projecting to orbital targets were determined in adult rats with intact innervation and following acute sympathetic denervation, neonatal unilateral superior cervical ganglionectomy, or unilateral ganglionectomy on postnatal day 30. Sympathetic nerves were identified by using immunofluorescence for the noradrenergic transmitter enzyme dopamine beta-hydroxylase and by using catecholamine histofluorescence. In rats with intact innervation, sympathetic fibers travel to the orbit in association with the abducens, trochlear, and Vidian nerves. Within the retroorbital and retroocular connective tissue, the fibers redistribute to become associated with sensory-nerve branches of the trigeminal nerve, the orbital vasculature, and the periorbital sheath. Fibers reach their targets by traversing variable amounts of connective tissue of the periorbitum, the orbital septa, and the striated muscle epimysia. Following neonatal ganglionectomy, intracranial fibers of contralateral origin enter the orbit by traveling through connective tissue of the optic nerve meninges and lining the anterior lacerated foramen. These fibers travel independent of the trochlear, abducens, and Vidian nerves, but, otherwise, they use the same orbital pathways as those employed in the intact animal. In animals ganglionectomized on postnatal day 30, fibers enter the posterior portion of the orbit primarily via the optic foramen; they travel only short distances and end blindly in the periorbital sheath. These findings indicate that fascial structures are a major component of the pathways that guide sympathetic fibers to their appropriate targets both in normal development and during reinnervation following neonatal ganglionectomy. Because orbital connective tissues are termination sites of abortive fiber sprouting in older rats, developmental changes in the properties of these tissues may contribute to the absence of pathway formation in the mature animal.

The central projections of the cat superior vagal (jugular) ganglion (SVG) cells were determined using anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). WGA-HRP was injected into the SVG of 5 cats, 3 of which had the vagal nerve sectioned distal to the jugular ganglion several days previously. Following 72 h, the animals were sacrificed and the brainstem and rostral cervical spinal cord sectioned and processed with tetramethylbenzidine (TMB) and examined for anterograde transport. The vagal root afferent fibers entered the brainstem at the level of the caudal facial motor nucleus and bifurcated into descending tracts. Terminal label was identified in: the dorsal lateral subnucleus of the nucleus tractus solitarius (NTS); nucleus interpolaris and nucleus caudalis of spinal V; the ventrolateral aspect of the cuneate nucleus and; the superficial laminae (I, II) of the rostral C1-dorsal horn. To our knowledge this is the first report describing central vagal afferent terminations following injection of current axonal tract tracing substances into the isolated superior vagal ganglion. The projection of jugular ganglion cells to a region of the NTS associated with cardiovascular function is of particular interest, and may be related to ganglion cells known to innervate the cerebral vasculature and meninges.

The current view that the migraine aura arises from spasm of the major cerebral arteries and the ensuing headache from extracranial arterial vasodilatation is examined and refuted. It is proposed that the headache is due to stimulation of nociceptive nerve-endings in the walls of meningeal vessels (arterioles, venules, and particularly the dural venous sinuses); and that the aura arises from calibre changes in meningeal vessels that penetrate the outer cortex, resulting in localised inhibition or excitation. It is suggested that there are two types of migraine patients--vasodilators and vasoconstrictors.

The distribution of peptidergic nerve fibers containing substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), and neuropeptide Y (NPY) in the cerebral arteries and veins of the guinea pig was studied using immunohistochemical techniques. The ultrastructure of these immunoreactive nerve terminals was also compared. The cerebral arteries were innervated by abundant peptidergic nerve fibers with characteristic running patterns, i.e., SP fibers in a meshwork, VIP and NPY fibers in a spiral fashion. Only CGRP fibers showed both meshwork and spiral patterns. In the cerebral veins, the abundant SP fibers innervated the cortical veins, deep cerebral veins, and dural sinuses. However, CGRP, VIP, and NPY fibers in extremely low density were noted merely in the cortical veins. Electron microscopic observations demonstrated that SP-immunoreactive nerve terminals existed apart from the arterial smooth muscle cells, while VIP- and NPY-immunoreactive nerve terminals adjoined them. As for CGRP nerve terminals, some existed close to the arterial smooth muscle cells, and others were found some distance from them. These morphological characteristics observed by light and electron microscopy suggest that SP fibers are not related directly to the vasomotor function, but VIP and NPY fibers are, and that CGRP fibers have a more complicated function. The distribution patterns of the peptidergic nerve fibers are consistent with the suggestion that vasomotor peptidergic fibers may function actively on cerebral arteries and passively on cerebral veins and that SP fibers regarded as sensory fibers may provide information regarding cerebral vascular conditions, innervating every part of both cerebral arteries and veins.

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This study was designed to identify the location of neurons giving rise to fibers innervating the posterior fossa dura in the cat using horseradish peroxidase (HRP, Sigma, Type VI). Investigations since the 19th century have implicated innervation by cranial nerves V, VII, IX, X and XII and the upper cervical nerves, C1-3. The meninges of the posterior fossa of 14 cats was exposed using one of three surgical approaches: (1) a suboccipital craniectomy and C1 laminectomy, (2) a parieto-occipital craniectomy with removal of the occipital lobe and bony tentorium exposing the meninges over the cerebellum, or (3) an anterior approach through the upper neck, exposing the dura of the ventral surface of the caudal brainstem. A unilateral, curvilinear incision was made in the dura and HRP was applied to the exposed dural edges. Following 48 hours the animals were sacrificed and fixed by perfusion. Cranial nerve ganglia of V, VII, IX, X, dorsal root ganglia (DRG) of C1-3, and superior cervical ganglia (SCG) were removed bilaterally, sectioned and processed with tetramethylbenzidine (TMB). HRP labeled cells were located bilaterally, always more ipsilaterally, in DRG of C1, C2, C3, and SCG with application of HRP to all three regions of the dura. Labeled cells were also located in trigeminal ganglia and superior ganglia of CN X, occasionally bilaterally, depending on the site of application. No HRP was ever identified in neurons of the geniculate ganglion, inferior ganglion of CN X or superior or inferior ganglia of CN IX. This information is valuable to an understanding of the innervation of intracranial structures and the problems of head pain.

We tested the hypothesis that the 5HT(1D)R, the primary antinociceptive target of triptans, is differentially distributed in tissues responsible for migraine pain. The density of 5HT(1D)R was quantified in tissues obtained from adult female rats with Western blot analysis. Receptor location was assessed with immunohistochemistry. The density of 5HT(1D)R was significantly greater in tissues known to produce migraine-like pain (i.e. circle of Willis and dura) than in structures in which triptans have no antinociceptive efficacy (i.e. temporalis muscle). 5HT(1D)R-like immunoreactivity was restricted to neuronal fibres, where it colocalized with calcitonin gene-related peptide and tyrosine hydroxylase immunoreactive fibres. These results are consistent with our hypothesis that the limited therapeutic profile of triptans could reflect its differential peripheral distribution and that the antinociceptive efficacy reflects inhibition of neuropeptide release from sensory afferents. An additional site of action at sympathetic efferents is also suggested.

In order to determine the anatomical distribution of cells concerned with relaying craniovascular nociception, local cerebral glucose utilization was determined by the 2-deoxyglucose method in tissue autoradiographs of the alpha-chloralose anesthetized cat. The superior sagittal sinus was carefully lifted from the brain by sectioning the dura laterally and the falx inferiorly and suspending the sinus across two platinum hook electrodes for stimulation. The sinus was stimulated electrically and its effect on caudal brainstem, upper cervical spinal cord and diencephalic metabolic activity determined. Stimulation of the sinus caused increased metabolic activity in the trigeminal nucleus caudalis, in the cervical dorsal horn and in a discrete area in the dorsolateral spinal cord at the second cervical segment. Metabolic activity was also increased in the ventrobasal thalamus, specifically in the ventroposteromedial (188%) nuclear group, in the medial nucleus of the posterior complex (70%) and the intralaminar complex (49%). There was no change in the surrounding thalamus, lateral geniculate nucleus or overlying cerebral cortex. These increases in 2-deoxyglucose utilisation were blocked by bilateral trigeminal ganglion ablation. The dorsolateral area activated in the spinal cord corresponds to a hitherto unrecognised group of cells in or near the lateral cervical nucleus that may form an important relay for craniovascular nociception. Further electrophysiological studies with glass coated tungsten microelectrodes have characterised the cells in these regions of the thalamus to be responsible for relaying nociceptive information. An understanding of the connections and properties of the neurons that subserve craniovascular pain is an essential prerequisite to understanding the complex pathophysiology of migraine.

Agonists at serotonin 1D (5-HT1D) receptors relieve migraine headache but are not clinically used as general analgesics. One possible explanation for this difference is that 5-HT1D receptors are preferentially expressed by cranial afferents of the trigeminal system. We compared the distribution of 5-HT1D receptor-immunoreactive (5-HT1D-IR) peripheral afferents within the trigeminal ganglion (TRG) and lumbar dorsal root ganglion (DRG) of the rat. We also examined the neurochemical identity of 5-HT1D-IR neurons with markers of primary afferent nociceptors, peripherin, isolectin B4, and substance P, and markers of myelinated afferents, N52 and SSEA3. We observed a striking similarity in the size, distribution, and neurochemical identity of 5-HT1D-IR neurons in TRG and lumbar DRG afferents. Furthermore, the vast majority of 5-HT1D-IR neurons are unmyelinated peptidergic afferents that distribute peripherally, including the dura, cornea, and the sciatic nerve. In the central projections of these afferents within the trigeminal nucleus caudalis and the spinal cord dorsal horn, 5-HT1D-IR fibers are concentrated in laminas I and outer II; a few axons penetrate to lamina V. At the ultrastructural level, 5-HT1D receptors in the spinal cord dorsal horn are localized exclusively within dense core vesicles of synaptic terminals. We observed scattered 5-HT1D-IR neurons in the nodose ganglia, and there was sparse terminal immunoreactivity in the solitary nucleus. The visceral efferents of the superior cervical ganglia did not contain 5-HT1D immunoreactivity. Our finding, that 5-HT1D receptors are distributed in nociceptors throughout the body, raises the possibility that triptans can regulate not only headache-associated pain but also nociceptive responses in extracranial tissues.

Using immunohistochemistry, we studied the origins and pathways of parasympathetic and sensory nerve fibers to the pial arteries in four squirrel monkeys. Following its application to the surface of the middle cerebral artery, the retrograde axonal tracer True Blue accumulated in parasympathetic neurons of the sphenopalatine ganglion and the internal carotid ganglion. The latter is strategically located where the internal carotid artery enters the cranium. Fibers from the sphenopalatine ganglion reach the internal carotid artery in the cavernous sinus region after running as rami orbitales. Before reaching the internal carotid artery, the fibers bypass aberrant sphenopalatine ganglia, with the most distant, the cavernous ganglion, being located in the cavernous sinus region. True Blue also accumulated in sensory neurons of the ophthalmic and maxillary divisions of the trigeminal ganglion and in sensory neurons of the internal carotid ganglion. Fibers from the ophthalmic division of the trigeminal ganglion reach the internal carotid artery as a branch through the cavernous sinus, bypassing the cavernous ganglion. Fibers from the maxillary division also bypass the cavernous ganglion after reaching it via a recurrent branch of the orbitociliary nerve. Thus, the cavernous ganglion forms a confluence zone for parasympathetic and sensory fibers in the region. In addition, parasympathetic and sensory fibers leave the confluence zone to follow the abducent and trochlear nerves backward to the basilar artery and tentorium cerebelli, respectively. Clinical implications are discussed.

Neurologic signs of increased parasympathetic outflow to the head often accompany migraine attacks. Because increased parasympathetic outflow to the cranial cavity induces vasodilation of cerebral and meningeal blood vessels, it can enhance plasma protein extravasation and the release of proinflammatory mediators that activate perivascular nociceptors. We recently showed that activation of intracranial perivascular nociceptors induces peripheral and central sensitization along the trigeminovascular pathway and proposed that these sensitizations mediate the intracranial hypersensitivity and the cutaneous allodynia of migraine. The present study investigates possible parasympathetic contributions to the generation of peripheral and central sensitization during migraine by applying intranasal lidocaine to reduce cranial parasympathetic outflow through the sphenopalatine ganglion. In the absence of migraine, patients were pain-free, and their skin sensitivity was normal. Their mean baseline pain thresholds were less than 15 degrees C for cold, more than 45 degrees C for heat, and more than 100 g for mechanical pressure. Their mean pain score was 7.5 of 10 (standard deviation, 1.4) during untreated migraine and 3.5 of 10 (standard deviation, 2.4) after the nasal lidocaine-induced sphenopalatine ganglion block (P <.0001). Most patients developed cutaneous allodynia during migraine, and their mean pain thresholds changed to more than 25 degrees C for cold, less than 40 degrees C for heat, and less than 10 g for mechanical pressure. Following the nasal lidocaine administration (sphenopalatine ganglion block), this allodynia remained unchanged in spite of the pain relief. These findings suggest that cranial parasympathetic outflow contributes to migraine pain by activating or sensitizing (or both) intracranial nociceptors, and that these events induce parasympathetically independent allodynia by sensitizing the central nociceptive neurons in the spinal trigeminal nucleus.

The distribution of calcitonin-gene-related peptide-like immunoreactivity (CGRP-IR) was studied in sections of decalcified rat head and selected whole-mount preparations in order to address the complex peptidergic innervation patterns in peripheral cephalic specialized zones and to examine neuronal ganglia in situ. Labeled neuron somata in trigeminal, glossopharyngeal, and vagal ganglia comprised a large proportion of small to medium size type B ganglion cells. Parasympathetic ganglia (ciliary, otic, sphenopalatine, submandibular) revealed a small population of labeled somata and numerous perisomatic IR axons, whereas sympathetic ganglion cells (superior cervical) were devoid of label though richly innervated by perisomatic IR axons. The gustatory geniculate ganglion contained only a few labeled neurons and axons. Coarse peripheral CGRP-IR axons were traced to skeletal muscle motor end plates (e.g., lingual, tensor tympani, etc.), and thin sensory axons most densely innervated the cornea, iris, general integument, all mucosal epithelia lining the tympanic, nasal, sinus and oropharyngeal cavities, and the cerebral meninges. Blood vessels, glands, ducts, and their orifices were often heavily innervated, and specific specializations and exceptions are discussed. Distinctive patterns of IR innervation characterized the various specialized sensory systems, including 1) cochlear and vestibular hair cells; 2) lingual, palatal, oropharyngeal, and laryngoepiglottal taste buds; 3) main olfactory epithelium and axons projecting to glomeruli in specific sectors of main olfactory bulb; 4) septal-olfactory organ; 5) vomeronasal organ; and 6) the nervus terminalis system. Secretory epithelia (ciliary body, choroid plexus, and stria vascularis) were notably lacking in CGRP-IR. Despite the multiplicity of functionally distinct CGRP neuronal and axonal populations, certain generalizations merit consideration. The extensive innervation of chemosensory nasal and oral epithelia may contribute to specific chemical sensitivities (e.g., relating to olfactory and gustatory senses) as well as evoking "nociceptive" responses to chemical irritants as part of a "common chemical sense." An efferent role for some of these peptidergic afferent axons may also be inferred from their specific distributions. Sites involved in regulating access to and sensitivity of sense organs to external stimuli (e.g., cochlear and vestibular hair cells, taste bud orifices, and main olfactory epithelium) are heavily innervated. Other IR axons are in position to exert control over airflow through nasal turbinates, glandular secretion, blood circulation, and duct transport systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Parasympathetic innervation of meninges and ability of carbachol, acetylcholine (ACh) receptor (AChR) agonist, to induce headaches suggests contribution of cholinergic mechanisms to primary headaches. However, neurochemical mechanisms of cholinergic regulation of peripheral nociception in meninges, origin place for headache, are almost unknown. Using electrophysiology, calcium imaging, immunohistochemistry, and staining of meningeal mast cells, we studied effects of cholinergic agents on peripheral nociception in rat hemiskulls and isolated trigeminal neurons. Both ACh and carbachol significantly increased nociceptive firing in peripheral terminals of meningeal trigeminal nerves recorded by local suction electrode. Strong nociceptive firing was also induced by nicotine, implying essential role of nicotinic AChRs in control of excitability of trigeminal nerve endings. Nociceptive firing induced by carbachol was reduced by muscarinic antagonist atropine, whereas the action of nicotine was prevented by the nicotinic blocker d-tubocurarine but was insensitive to the TRPA1 antagonist HC-300033. Carbachol but not nicotine induced massive degranulation of meningeal mast cells known to release multiple pro-nociceptive mediators. Enzymes terminating ACh action, acetylcholinesterase (AChE) and butyrylcholinesterase, were revealed in perivascular meningeal nerves. The inhibitor of AChE neostigmine did not change the firing Trigeminal nerve terminals in meninges, as well as dural mast cells and trigeminal ganglion neurons express a repertoire of pro-nociceptive nicotinic and muscarinic AChRs, which could be activated by the ACh released from parasympathetic nerves. These receptors represent a potential target for novel therapeutic interventions in trigeminal pain and probably in migraine.

To understand the mechanism of action of oxygen treatment in cluster headache. Trigeminal autonomic cephalalgias, including cluster headache, are characterized by unilateral head pain in association with ipsilateral cranial autonomic features. They are believed to involve activation of the trigeminovascular system and the parasympathetic outflow to the cranial vasculature from the superior salivatory nucleus (SuS) projections through the sphenopalatine ganglion, via the greater petrosal nerve of the VIIth (facial) cranial nerve. Cluster headache is remarkably responsive to treatment with oxygen, and yet our understanding of its mode of action is unknown. Combining models of trigeminovascular nociception and a novel approach that activates the trigeminal-autonomic reflex, using SuS/facial nerve stimulation, we explored the effect of oxygen on trigeminal nerve activation as well as on autonomic responses through blood flow observations of the lacrimal duct/sac. Meningeal vasodilation and neuronal firing in the trigeminocervical complex (TCC), in response to dural electrical stimulation, was unaffected by treatment with 100% oxygen. Stimulation of the SuS via the facial nerve caused only marginal changes in dural blood vessel diameter, but did result in evoked firing in the TCC. Two populations of neurons were characterized, those responsive to 100% oxygen treatment, with a maximal inhibition of 33%, 20 minutes after the start of oxygen treatment (t(15) = 4.4, P < .0001). A second population of neurons were not inhibited by oxygen and tended to have shorter latency. Oxygen also inhibited evoked blood flow changes in the lacrimal sac/duct caused by SuS stimulation. The data provide the first systematic, experimental evidence for a mechanism of action of oxygen in cluster headache. The data show oxygen has no direct effect on trigeminal afferents, acting specifically on the parasympathetic/facial nerve projections to the cranial vasculature to inhibit both evoked trigeminovascular activation and activation of the autonomic pathway during cluster headache attacks. Moreover, the studies begin to characterize a novel laboratory model for the most painful primary headache syndrome known--cluster headache.

To trace the path taken by the putative postganglionic secretomotor fibres to the lacrimal gland the contents of the orbital and pterygopalatine fossa were removed whole, cut coronally into slabs and embedded in resin. Thin sections were cut at varying intervals to reconstruct the pathway taken. One group of rami orbitales issuing from the pterygopalatine ganglion passed dorsally adjacent to the lateral wall of the orbit, joined the retro-orbital plexus at the apex, and 5-10 rami lacrimales advanced from the plexus to enter the gland. An accessory ophthalmic artery, a branch of the middle meningeal artery, entered the orbit through the superior fissure orbital joining the ophthalmic or lacrimal artery. Perivascular nerves of the artery continued to the gland as supplementary rami lacrimales and in some orbits others served the vasculature of the eye and orbit. The nerves are presumably derived from the middle meningeal supply and may include otic parasympathetic fibres. The route taken by parasympathetic nerves serving the human lacrimal gland is demonstrated here for the first time and apart from the perivascular meningeal artery source, it is similar to that described in monkeys. The traditional assumption that secretomotor nerves pass to the gland via the zygomatic and lacrimal nerves is therefore unlikely and clinical measures to reduce lacrimation based on that assumption and involving severance of ophthalmic branches is not indicated.

The aims of the present study were to determine if there is neuronal Cocaine and amphetamine regulated transcripts (CART) peptide expression (CART+) in parasympathetic (sphenopalatine (SPG); otic (OG)) and sensory (trigeminal (TG)) ganglia of the head and to examine the neurochemical phenotype (calcitonin gene-related peptide (CGRP), neurofilament 200 (NF200), isolectin B4 (IB4) binding, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY) and enkephalin (ENK) immunoreactivity) and projection targets (lacrimal gland (LG), parotid gland (PG), nasal mucosa (NM), temporomandibular joint (TMJ), middle cerebral artery (MCA) and middle meningeal artery (MMA)) of CART expressing neurons in these ganglia. We found CART+ neurons in both the SPG (5.25±0.07%) and OG (4.32±0.66). A significant proportion of these CART+ neurons contained VIP, NPY or ENK (34%, 26% and 11%, respectively). SPG neurons retrogradely labelled from the lacrimal gland (29%) were CART+, but we were unable to demonstrate CART+ labelling in any of the SPG or OG neurons labelled from other targets. This supports a role for CART peptides in lacrimation or regulation of vascular tone in the lacrimal gland, but not in salivation or nasal congestion. CART+ neurons were also present in the trigeminal ganglion (1.26±0.38%), where their size distribution was confined almost completely to neurons smaller than 800 μm2 (mean=410 μm2; 98%<800 μm2), and were almost always CGRP+, but did not bind IB4. This is consistent with a role for CART peptides in trigeminal pain. However, there were few CART+ neurons amongst any of the trigeminal neurons retrogradely labelled from the targets we investigated and thus we cannot comment on the tissue type where such pain may have originated. Our study shows that some specialization of CART peptide expression (based on neurochemical phenotype and target projection) is evident in sensory and parasympathetic ganglia of the head.

Dynorphin B (dyn B) in trigeminal ganglion cells and in perivascular nerve fibers in pial arteries was demonstrated in rat, guinea-pig, and monkey by immunohistochemistry. The pathway from the trigeminal ganglion, which runs via the nasociliary nerve and ethmoidal foramen to the pial arteries, was shown in rat by retrograde tracer technique and nerve section. In the guinea-pig the peptide was demonstrated to coexist with substance P and calcitonin gene-related peptide in neurons of the trigeminal ganglion and pial nerve fibers, i.e., it was present in cerebrovascular sensory nerves with primarily nociceptive function. Another finding in guinea-pig was a coexistence of dyn B with vasoactive intestinal polypeptide in the pial nerve fibers and neurons of the sphenopalatine ganglion, indicating a presence also in parasympathetic nerves to the cerebral vessels. No vasomotor effect of dyn B could be detected in isolated segments of rat pial arteries, which rules out a direct postsynaptic effect on vascular tone. The peptide did not display a prejunctional modulatory action on the adrenergic nerves present in the vessels. The function of dyn B in the cerebrovascular nerves is discussed.

Endogenous acetylcholine (ACh) levels and choline acetyltransferase (ChAT) activity were measured in several vascular segments (major cerebral arteries, cortical pial vessels, and peripheral arteries) and nervous tissues [including the sphenopalatine ganglion (SPG)] in the rat. The effects of uni- or bilateral surgical ablation of the SPG, a putative origin of the cholinergic cerebrovascular innervation, were investigated on these two specific cholinergic markers at various postoperative times. ChAT activity and ACh levels were enriched in the cerebral as compared to the peripheral arteries. Among the cerebrovascular tissues tested, ACh levels were particularly high in the circle of Willis and the vertebrobasilar segments and, to a lesser extent, in the middle cerebral artery. Lower levels were found in the small pial vessels and choroid plexus. Overall, ChAT activity measured in different arterial beds paralleled the distribution of ACh. Following uni- or bilateral removal of the SPG, slight reductions (18-36%, statistically not significant) were observed in ChAT activity in rostral cerebral arteries and pial vessels overlying the frontal cortex. Similarly, bilateral ganglionectomy resulted in minor decreases (11-22%, not significant) in the cerebrovascular contents of ACh in these same vascular segments. These results clearly show that the SPG does not or only partly contributes to the cholinergic fibers that supply the cerebrovascular bed.

The human otic ganglion (OG) is not readily accessible during ordinary anatomical teaching courses because of insufficient time and severe difficulties encountered in dissection. Accordingly, most anatomical descriptions of its location, relation to neighbouring structures, size and shape are supported only by drawings, but not by photographs. The aim of this study has been to present the OG with associated roots and branches in dissected anatomic specimens. Following cumbersome dissection and precise photo-documentation, a detailed analysis of location, syntopy and morphology was performed. We carried out this study in 21 infratemporal fossae of 18 cadavers and were able to identify the OG, the mandibular-, the inferior alveolar- and the lingual nerve in all of them. We found no significant variation regarding the location of the GO in the infratemporal fossa and its syntopy to the adjacent structures. An OG resembling the classic description was found only in 90.50% of the cases. All 3 roots (parasympathetic, sympathetic and sensory) could be identified only in 82.3% of the specimens. The established presence of ganglionic branches varied from 0% (communicating rami to the meningeal branch of the mandibular nerve, to the greater petrosal nerve and to the lingual nerve) to 90% (r. communicans to n. canalis pterygoideus). We conclude that precise knowledge of this enormous variety might be very helpful not only to students of medicine and dentistry during anatomical dissection courses, but also to head and neck surgeons, ear-nose-throat specialists and neurosurgeons when treating pathology of pre- and postganglionic fibres.

Peripheral sources of cerebral vascular innervation have been investigated with retrograde and anterograde neuronal tracing of wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP) in the rat. For retrograde identification of sources of innervation, WGA-HRP was applied to the exposed basilar artery through a fine slit in the overlying meninges, and sections of brain and peripheral ganglia were reacted with tetramethylbenzidine for detection of the tracer. A high density of tetramethylbenzidine reaction product was observed around the basilar artery and in the surrounding pial tissue, but the application sites were not completely selective since some tracer always had spread into the ventral brain stem. Retrogradely labelled cell bodies were identified in the superior cervical, stellate, first and second spinal, and trigeminal ganglia, i.e. these ganglia may represent origins of basilar artery innervation. In a second series of experiments, microinjections of WGA-HRP were placed into the indicated ganglia to obtain anterograde labelling of nerve fibres on whole-mounts of the cerebral vessels. Injections into trigeminal ganglia labelled nerve fibres on the ipsilateral half of the circle of Willis, as well as the contralateral anterior cerebral artery and the rostral part of the basilar artery. The first and second spinal ganglia projected to the vertebrobasilar arteries, while the ipsilateral part of the internal carotid (outside the circle of Willis) received fibres from the second spinal ganglion. Nerve fibres originating in trigeminal and spinal ganglia were organised in bundles, and between these a sparse plexus of thin single fibres appeared. Injection of WGA-HRP into superior cervical ganglion labelled a plexus of nerve fibres on the ipsilateral circle of Willis and the (rostral) basilar artery. These experiments demonstrated the origin and distribution of sympathetic and sensory innervation to major cerebral arteries in the rat.

To reinvestigate the innervation pattern of the dura mater of rat and human middle cranial fossa, the morpho-functional substrate of headache generation, and adjacent extracranial tissues with neuronal in vitro tracing. This study was initiated by recent structural and functional findings of meningeal afferent fibers which innervate the cranial dura mater and may project to extracranial tissues. Anterograde and retrograde neuronal in vitro tracing was made in formaldehyde fixed hemisected rat and human skulls. The fluorescent tracer DiI was applied to proximally cut meningeal nerves in rat and to distal branches of the spinosus nerve in human calvaria lined by dura mater. After several weeks, the dura mater and deep extracranial tissues were examined with fluorescence microscopy. In addition to a network of meningeal nerve fibers, several fiber bundles were observed, leaving the skull through emissary canals and fissures to innervate the pericranial temporal, parietal, and occipital periosteum. Traced fibers were seen spreading into deep layers of the temporal and upper neck muscles. Retrograde neuronal tracing revealed labeled cell bodies exclusively in the mandibular and maxillary division of the rat trigeminal ganglion, and centrally projecting fibers were identified in the spinal trigeminal tract. Electron microscopy of the cross-sected spinosus nerve showed myelinated and unmyelinated axons with similar numbers in human and rat. We conclude that a proportion of meningeal afferents innervates extracranial tissues like periosteum and pericranial muscles via collaterals projecting through the skull. These afferents may be nociceptive, some may subserve proprioceptive functions. The finding of extracranial projections of meningeal afferents may be important for our understanding of extracranial impacts on headache generation and therapy.

Peripheral neurostimulation within the trigeminal nerve territory has been used for pain alleviation during migraine attacks, but the mechanistic basis of this non-invasive intervention is still poorly understood. In this study, we investigated the therapeutic role of peripheral stimulation of the trigeminal nerve, which provides homosegmental innervation to intracranial structures, by assessing analgesic effects in a nitroglycerin (NTG)-induced rat model of migraine. As a result of neurogenic inflammatory responses in the trigeminal nervous system, plasma protein extravasation was induced in facial skin by applying noxious stimulation to the dura mater. Noxious chemical stimulation of the dura mater led to protein extravasation in facial cutaneous tissues and caused mechanical sensitivity. Trigeminal ganglion (TG) neurons were double-labeled via retrograde tracing to detect bifurcated axons. Extracellular recordings of wide dynamic range (WDR) neurons in the spinal trigeminal nucleus caudalis (Sp5C) demonstrated the convergence and interaction of inputs from facial tissues and the dura mater. Peripheral neurostimulation of homotopic facial tissues represented segmental pain inhibition on cephalic cutaneous allodynia in the migraine model. The results indicated that facial territories and intracranial structures were directly connected with each other through bifurcated double-labeled neurons in the TG and through second-order WDR neurons. Homotopic stimulation at the C-fiber intensity threshold resulted in much stronger inhibition of analgesia than the same intensity of heterotopic stimulation. These results provide novel evidence for the neurological bases through which peripheral neurostimulation may be effective in treating migraine in clinical practice.

The trigeminal sensory innervation of the major cerebral vessels is thought to carry the nociceptive information during a migraine headache, and this pain is usually referred to the forehead area. Using retrograde tracing techniques, we have described the distribution of sensory trigeminal cells that innervate the middle cerebral artery (MCA) and the forehead. Nearest-neighbor analysis of the ophthalmic division of the trigeminal ganglion revealed that cells innervating the forehead tend to be clumped around individual cells that innervate the MCA. An average of less than 1 cell per animal was found to project divergent collaterals to both areas. The close association of ganglion cell bodies innervating the cerebral vasculature and those innervating forehead areas may underlie the convergence of their central processes onto common brain-stem trigeminal nucleus cells, and thus the referral of headache pain. In contrast to the lack of ganglion cells with axonal collaterals to the cerebral vasculature and forehead, a significant population of cells that innervate the MCA also have collateral projections to other cerebral arterial branches (branches of the middle meningeal artery), as well as the surrounding dura. Thus, the innervation targets of individual trigeminal cells are very widespread intracranially (including arteries and dura), but separate cells in the ophthalmic division innervate extracranial targets.

Prior studies have documented a trigeminal (V) mandibular primary afferent projection to the dorsomedial portion of the contralateral medullary and cervical dorsal horns in cat, hamster, and rat. We now report the existence of a much more substantial V ophthalmic primary afferent projection to the ventrolateral portion of contralateral medullary and cervical dorsal horns in rat. Horseradish peroxidase (HRP) injections into the V ganglion or V brainstem complex anterogradely labeled a fascicle of primary afferent axons that exited the caudal ventrolateral V spinal tract to form a rostrocaudally continuous, transversely oriented, V primary afferent decussation. These fibers terminated most heavily in laminae III-V of the ventrolateral dorsal horn in contralateral caudal medulla and the first and second cervical segments. Retrograde tracing with diamidino yellow (DY) or fluorogold and anterograde tracing with Phaseolus vulgaris leucoagglutinin also demonstrated a substantial commissural projection of central origin in medullary dorsal horn laminae I-VII. The latter projection had a more diffuse trajectory and termination pattern than that of the V primary afferent decussation. Unilateral HRP injections into medullary and cervical dorsal horns also retrogradely labeled V primary afferent collaterals contralateral to the injection site in corresponding regions of dorsal horn, and also in ventromedial interpolaris, oralis, and principalis, rostral to their decussation. Axons (1.5 +/- 0.8 microns mean diameter; 0.4-3.9 microns range) therefore terminated both ipsi- and contralateral to their cells of origin. These HRP injections also labeled an average of 40.4 +/- 13.0 V ganglion cells (mean +/- SD, corrected for split somata) in dorsomedial, ophthalmic regions of the contralateral ganglion. Their mean diameter was slightly larger than that of cells labeled ipsilaterally (29.9 vs. 26.3 microns). Double-labeling studies assessed possible ophthalmic receptor surfaces innervated by centrally crossing primary afferents. DY was injected into right medullary and cervical dorsal horns, and HRP was applied to either the left cornea, the ethmoid nerve, or the dura overlying cerebral cortex. Though DY labeled from 75 to 125 left ganglion cells per animal, no cells were double-labeled. All of these findings suggest that nociceptive-specific ganglion cells are not a source of the crossed ophthalmic primary afferent projection. Unilateral transection of the infraorbital nerve on the day of birth did not alter the crossed primary afferent projection to the partially deafferented side of the brainstem. This is further evidence of an absence of central sprouting in spared V primary afferents following neonatal V deafferentation.

Alterations in cortical excitability are implicated in the pathophysiology of migraine. However, the relationship between cortical spreading depression (CSD) and headache has not been fully elucidated. We aimed to identify the corticofugal networks that directly influence meningeal nociception in the brainstem trigeminocervical complex (Sp5C) of the rat. Cortical areas projecting to the brainstem were first identified by retrograde tracing from Sp5C areas that receive direct meningeal inputs. Anterograde tracers were then injected into these cortical areas to determine the precise pattern of descending axonal terminal fields in the Sp5C. Descending cortical projections to brainstem areas innervated by the ophthalmic branch of the trigeminal nerve originate contralaterally from insular (Ins) and primary somatosensory (S1) cortices and terminate in laminae I-II and III-V of the Sp5C, respectively. In another set of experiments, electrophysiological recordings were simultaneously performed in Ins, S1 or primary visual cortex (V1), and Sp5C neurons. KCl was microinjected into such cortical areas to test the effects of CSD on meningeal nociception. CSD initiated in Ins and S1 induced facilitation and inhibition of meningeal-evoked responses, respectively. CSD triggered in V1 affects differently Ins and S1 cortices, enhancing or inhibiting meningeal-evoked responses of Sp5C, without affecting cutaneous-evoked nociceptive responses. Our data suggest that "top-down" influences from lateralized areas within Ins and S1 selectively affect interoceptive (meningeal) over exteroceptive (cutaneous) nociceptive inputs onto Sp5C. Such corticofugal influences could contribute to the development of migraine pain in terms of both topographic localization and pain tuning during an attack.

Using immunoperoxidase labeling (IPL) and immunofluorescence labeling (IFL) methods, and each followed by NADPH diaphorase (NADPHd) histochemical staining in the same specimen, colocalization of choline acetyltransferase (ChAT) and NADPHd, indicative of nitric oxide synthase (NOS), in cerebral pial arteries and the sphenopalatine ganglia (SPG) of the cat was examined. In addition, retrograde axonal tracing using true blue was performed to determine if cerebral perivascular nerves containing ChAT and NADPHd originate in the SPG. Consistent results were obtained from IPL and IFL methods, indicating that the middle cerebral artery (MCA) and the circle of Willis received dense ChAT-immunoreactive (I) and NADPHd bundles and fine fibers. Almost all ChAT-I fibers and NADPHd fibers were found to be coincident in the arteries examined. A few fine fibers exhibited only NADPHd staining. In the SPG, approximately half of the ganglionic cells were both ChAT-I and NADPHd positive, while the remaining cells were positively only for NADPHd staining. One week after application of true blue on the middle cerebral arteries (MCA), the fluorescent true blue was found in the ganglionic cells of the SPG. Some of the true blue-positive cells contained both ChAT-immunoreactivity and NADPHd staining. These results provide morphological evidence indicating that all ChAT-I fibers in the MCA and the circle of Willis contain NOS, and that these fibers originate in the SPG, although not all NOS-I ganglionic cells in the SPG send fibers to pial vessels. These results also support the hypothesis that acetylcholine (ACh) and nitric oxide (NO) are synthesized and co-released in the same neurons in cerebral perivascular nerves. Based on the reported findings that NO mediates a major component of neurogenic vasodilation, and that ACh acts as a modulator, the present results demonstrate the presence of a cholinergic, nitric oxidergic innervation in cerebral arteries of the cat.

Essential hypertension is associated with hyperactivity of the sympathetic nervous system and hypoperfusion of the brainstem area controlling arterial pressure. Sympathetic and parasympathetic innervation of vertebrobasilar arteries may regulate blood perfusion to the brainstem. We examined the autonomic innervation of these arteries in pre‐hypertensive (PHSH) and hypertensive spontaneously hypertensive (SH) rats relative to age‐matched Wistar rats. Our main findings were: (1) an unexpected decrease in noradrenergic sympathetic innervation in PHSH and SH compared to Wistar rats despite elevated sympathetic drive in PHSH rats; (2) a dramatic deficit in cholinergic and peptidergic parasympathetic innervation in PHSH and SH compared to Wistar rats; and (3) denervation of sympathetic fibres did not alter vertebrobasilar artery morphology or arterial pressure. Our results support a compromised vasodilatory capacity in PHSH and SH rats compared to Wistar rats, which may explain their hypoperfused brainstem.