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Abstract

Background: Symptoms associated with primary headaches are linked to cranial vascular activity and to the central nervous system (CNS).

Review: The central projections of sensory nerves from three cranial vessels are described in order to further understand pain mechanisms involved in primary headaches. Tracers that label small and large calibre primary afferent fibres revealed similar distributions for the central terminations of sensory nerves in the superficial temporal artery, superior sagittal sinus and middle meningeal artery. The sensory nerve fibres from the vessels pass through both the trigeminal and rostral cervical spinal nerves and terminate in the ventrolateral part of the C1-C3 dorsal horns and the caudal and interpolar divisions of the spinal trigeminal nucleus. The C-fibre terminations were located mainly in the superficial layers (Rexed laminae I and II), and the Aδ-fibres terminated in the deep layers (laminae III and IV). The rostral projections from the ventrolateral C1-C2 dorsal horn revealed terminations in the medial and lateral parabrachial nuclei, the cuneiform nucleus, the periaqueductal gray, the deep mesencephalic nucleus, the thalamic posterior nuclear group and its triangular part, and the thalamic ventral posteromedial nucleus. The terminations in the pons and midbrain were predominately bilateral, whereas those in the thalamus were confined to the contralateral side.

Conclusions: The observations, done in rats with the understanding that similar trigeminovascular organization exists in man, reveal vascular projections into the brainstem and some aspects of the central regions putatively involved in the central processing of noxious craniovascular signals.

Keywords

Aδ-fibres brainstem C-fibres neuronal tracing perivascular nerves trigeminal system

Introduction

No single undisputed hypothesis yet exists regarding the mechanisms behind primary headaches. The major hypotheses on the mechanisms underlying or involved in migraine are currently in question: is it a purely vascular or neurogenic disease, or does it involve both as a neurovascular disorder (1)? Although the pathogenesis of primary headaches is still unclear, it probably involves the peripheral trigeminovascular nerve and its central terminations leading into the CNS (2). The CNS itself is devoid of nociceptors, but the intracranial blood vessels are supplied with sensory nerves and receptors that may respond to thermal and mechanical stimuli (3). It is often suggested that a starting point in a migraine attack is a cortical wave of spreading depression (CSD) which is associated with local release of various molecules that can have effects on neurons, glial cells and vessels (4). These mediators may diffuse to the overlying leptomeninges and activate vascular nociceptors (5). Thus, the sensory nerve fibres around the cranial blood vessels are likely to play an important role in head pain of a migraine attack. Other initiator events involved in the start of migraine attacks are discussed but the issue is still unresolved. As an important link in the process, knowledge of the CNS connections of the sensory nerves is essential for understanding primary headache-associated intracranial and referred pain. Detailed transganglion neuronal tracing has been carried out from extra- and intracranial blood vessels to further our knowledge of the central connections of the trigeminovascular nerves, which outlined, in part, the central connections of the vascular nerves.

Innervation of the cranial vessels

Cranial blood vessels are supplied with perivascular sympathetic, parasympathetic and sensory nerve fibres (2). The sympathetic postganglionic nerve fibres originate mainly in the ipsilateral superior cervical ganglion (6), although some nerve fibres supplying the vertebral and basilar arteries originate in the inferior cervical and stellate ganglia (7). The neurotransmitters released from the sympathetic nerve fibres are noradrenaline, neuropeptide Y and adenosine triphosphate (8,9). The activation of these nerve fibres results in vasoconstriction, modulation of the cerebrovascular autoregulation, reduction of the intracranial pressure with a decrease in cerebral blood volume, and cerebrospinal fluid production (10).

The parasympathetic nerve fibres innervating cranial vessels originate in the otic and sphenopalatine ganglia and contain acetylcholine, vasoactive intestinal peptide, pituitary adenylate cyclase activating peptide (PACAP) and nitric oxide synthase (11). These parasympathetic nerve fibres mediate a major component of the vasodilator responses of cerebral arteries as demonstrated by cerebral blood flow measurements (12–14).

Sensory fibres innervating the cranial vessels originate mainly in the trigeminal ganglion (TG), but also partly from rostral cervical dorsal root ganglia (DRG). The perivascular sensory nerve fibres contain to a varying degree calcitonin gene-related peptide (CGRP), substance P, neurokinin A, PACAP and nitric oxide synthase (11). In humans, the levels of CGRP in the jugular vein are increased in the headache phase of migraine and cluster headache; treatment with a triptan aborts both the pain and the elevated CGRP (15–17). There are differences in opinion (18), but CGRP is now established to play an important role in the pathogenesis of headache (19,20).

Primary afferent fibres in the cranial vasculature

Most of the sensory information from the face and head is conveyed to the CNS via the fifth cranial nerve, the trigeminal nerve. The peripheral sensory fibres of all three branches originate in the pseudounipolar neurons of the TG. The central branches of these neurons enter the pontine region of the brainstem. The central branches may divide into an ascending branch that terminates in the main sensory nucleus and a descending branch that terminates in the spinal trigeminal nucleus (21). Almost equal numbers of unmyelinated and myelinated fibres are found in the trigeminal nerve (22).

The first branch of the trigeminal nerve innervates the temporal area, which is the most frequent location of pain in primary headaches (3,23). The large cerebral arteries, the cortical pial vessels and the meningeal arteries in the dura mater are innervated by nerve fibres that arise from the ophthalmic branch of the TG. In migraine patients, the pain can sometimes be relieved by percutaneous radiofrequency trigeminal ganglio-rhizolysis, supporting an essential role for signals transmitted through the trigeminal nerve in migraine pain (24). Cortical spreading depression can activate trigeminal neurons in rats (25,26). Animal experiments and clinical evidence suggest that CNS dysfunction (e.g. mutation in an ion channel gene) initiates migraine attacks and that part of the clinical expression of a migraine attack is dependent on the trigeminovascular system (1).

To reveal the neural mechanisms involved in migraine attacks, many studies have focused on sensory innervation of the cerebral vessels and dura mater by the middle meningeal artery (27). Stimulation of the TG results in C-fibre-dependent neurogenic plasma extravasation in the dura mater but not the brain (28). Two types of tracers have shown complementary information: (i) wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) on cerebral arteries retrogradely labels neurons in the TG (7,29), and (ii) true blue application on the superficial temporal artery (STA), middle meningeal artery (MMA) and middle cerebral artery (MCA) labels neurons in the TG and C2 DRG (30–32). The true blue tracing has also shown co-localization between the tracer and the different neuronal messenger molecules in neurons of the cranial autonomic ganglia, TG and DRG. Following injections of WGA-HRP into the TG, labelled nerve fibres were observed in the ipsilateral half of the circle of Willis, the contralateral anterior cerebral artery and the rostral part of the basilar artery (7).

The human STA, MMA and cerebral arteries are supplied by a network of nerve fibres that display immunoreactivity for neuropeptides involved in nociception, including substance P and CGRP (2). The superior sagittal sinus (SSS) is a pain-sensitive intracranial venous vessel and many experimental studies have used stimulation of the SSS as a model for investigating the anatomy and physiology of the trigeminovascular pain structures. Stimulation of the SSS causes referred pain to the first division of the trigeminal nerve (23). Functional vasodilator receptors for CGRP have been found in the human MMA (33,34). Trigeminal activation causes selective long-lasting blood flow enhancement within the MMA (25), which is in line with the presence of the receptor proteins in the smooth muscle cells of the vasculature (35). Using retrograde tract tracing methods, the sensory nerves to the MMA have been shown to originate from both the TG and rostral two to three cervical spinal DRG (32,36).

So far, sensory nerves innervating cerebral vessels clearly terminate in a region extending from the caudal portion of the spinal trigeminal nucleus (Sp5C) in the medulla to the lower end of the C2 spinal dorsal horn, a region referred to as the ‘trigeminocervical complex’ (37). Electrical stimulation of the SSS causes increased metabolic activity and blood flow in the Sp5C and spinal dorsal horn in segments C1 and C2 (38–40). Nociceptive electrical and mechanical stimulation of the SSS results in c-Fos expression in the Sp5C and C1 and C2 spinal dorsal horns (41,42). Stimulation of the SSS also activates neurons in the Sp5C and C1 and C2 dorsal horns in cats (42) and monkeys (43). In WGA-HRP transganglionic tract tracing studies of rats and squirrel monkeys, the sensory nerves of the MCA mainly project to the trigeminal brainstem nuclear complex. The sensory fibres of the basilar artery in the rat mainly project to the C2 spinal dorsal horn, the cuneate nucleus (Cu), the dorsal motor nucleus of the vagus and the nucleus of the solitary tract (29,44).

Although accumulating data indicate that the central terminations of some cranial vessel nerves are located in the rostral spinal cord and caudal brainstem, the exact location of the central projections of the sensory innervation of many cranial vessels is still unknown. To understand the pathogenesis of headache disorders, it was necessary to investigate the central projections of sensory nerves from three cranial vessels in a series of studies using tract tracing techniques. The projecting locations of sensory nerves were studied using WGA-HRP and cholera toxin subunit b (CTb) as transganglionic tracers to label both thin nociceptive (unmyelinated C and small myelinated Aδ) and thick low threshold mechanoreceptive (large and small myelinated A) fibres (45–47).

Central projections of sensory nerves in the superficial temporal artery

For both CTb and WGA-HRP, numerous labelled cell bodies were observed in the ipsilateral TG and C2 DRG after deposition of the tracers on the STA (47). The size of the labelled cell bodies varied. In the WGA-HRP series, labelled ganglion cells were mainly small or medium-sized (8–25 µm in diameter), whereas CTb-labelled cells were large (8–45 µm in diameter). In the TG, labelled cells were concentrated in the dorsal half of the medial area, a region known to give rise to the first (ophthalmic) trigeminal nerve branch.

In the CTb series, transganglionic labelling was observed in the ipsilateral C1-C3 spinal dorsal horn, Sp5C and interpolar spinal trigeminal nucleus (Sp5I), with a density peak in the middle of C2 (47). In the cervical spinal dorsal horn, CTb-labelled terminations were located in Rexed laminae III and IV. In the Sp5C, CTb-labelled terminations were located superficially in the rostrodorsal portion. In the Sp5I, the CTb labelling was localized in the lateral and ventral peripheral region. Sparse labelling was found in the dorsolateral Cu.

Following WGA-HRP application on the adventitia of the STA, transganglionic labelling was found in the ipsilateral C1-C3 spinal dorsal horn, the Sp5C and the Sp5I (47). In the dorsal horn, labelled terminations started to appear in the middle of C3 and extended to the rostral end of C1, with the densest labelling in the rostral portion of C2. Labelling was localized to the ventrolateral region of the dorsal horn. WGA-HRP labelling was observed predominantly in laminae I and II. The labelled areas formed a continuous wedge-shaped column through layers I to IV in the ventrolateral portion of the C1-C3 dorsal horn. In the brainstem, transganglionic WGA-HRP labelling was prominent in the Sp5C and located in the superficial layers at different rostrocaudal levels. In the Sp5I, labelling was found in the caudolateral portion.

In control experiments, CTb or WGA-HRP was applied to a branch of the facial nerve; many labelled cell bodies were observed in the ipsilateral facial nucleus, but no terminal labelling was found in any part of the brainstem or spinal cord (47).

Central projections of sensory nerves in the superior sagittal sinus

At the sites of tracer application on the SSS (a midline structure), intense CTb or WGA-HRP labelling was, in most cases, limited to the adventitia of the SSS and the immediately adjacent dura mater (45). The tracer spread beyond the immediately adjacent dura in only one of the WGA-HRP experiments. The transganglionic labelling pattern in this context was somewhat different from that of the other experiments (see below).

Following CTb or WGA-HRP application, labelled cell bodies (8–40 µm in diameter) were observed in the bilateral TG and C2 DRG. Most cells labelled with WGA-HRP were small to medium-sized, whereas most of those labelled with CTb were large. In the TG, the labelled cell bodies were mainly located in the dorsomedial portion that gives rise to the first branch of the trigeminal nerve.

For both CTb and WGA-HRP, labelled terminations were observed in the ventrolateral portion of the C1-C3 dorsal horn, Sp5C and Sp5I, with density peaks in the C2 dorsal horn and the caudal portion of the Sp5C (45). With CTb, labelled terminations were found in the lateral-most portion of the Cu. The CTb labelling in the spinal cord and brainstem was consistently denser and covered a broader area than WGA-HRP labelling. In the dorsal horn of segments C1-C3, CTb labelling was mostly found in laminae III-IV, whereas WGA-HRP labelling was located predominantly in the superficial layers (laminae I and II).

In the experiment in which WGA-HRP spread beyond the dura mater immediately adjacent to the SSS, labelled terminations were detected in more medial areas of the superficial C1-C3 dorsal horn in addition to the labelling pattern described above (45).

Central projections of sensory nerves in the middle meningeal artery

For both CTb and WGA-HRP, retrogradely labelled cell bodies were observed ipsilaterally in the TG and C2 DRG after application of the tracers on the MMA (46). In the TG, labelled cell bodies were predominantly located in the dorsomedial area but were also evident in the dorsal middle area, the two areas giving rise to ophthalmic and maxillary nerves, respectively. With CTb, both small and large cell bodies were labelled in the ganglia, whereas WGA-HRP labelled cell bodies small to medium in size. Recent immunocytochemistry were revealed that the small to medium-sized neurons store CGRP and send C-fibres to layers I-II in the brainstem; the large neurons store the CGRP receptor elements and is associated with the small myelinated Aδ-type of fibres (22).

With both CTb and WGA-HRP, transganglionic labelling was confined to the side where tracer was applied. In DRG segment C3, labelled terminations were very sparse. From C2 and rostrally, the labelling gradually became denser, with a labelling density peak extending from rostral C1 through caudal Sp5C (46). Moderate amounts of terminal labelling were found in Sp5I. In segments C1-C3, the labelled terminations were located in the ventrolateral region of the spinal dorsal horn for both CTb and WGA-HRP, but in different layers. The CTb-labelled terminations were mainly located in laminae III and IV, whereas WGA-HRP labelling was located just superficially to the CTb terminations (laminae I and II). If overlapped, the CTb and WGA-HRP labelling in segments C1-C3 forms a continuous wedge-shaped column throughout layers I to IV in the lateral portion of the dorsal horn.

In summary, the distribution of tracer-labelled central terminations was very close in all three vessels. The sensory nerve fibres originated from the vessels and went through both the trigeminal and spinal nerves. The fibres terminated in the ventrolateral part of the C1-C3 dorsal horns, the caudal and interpolar divisions of the spinal trigeminal nucleus with a focus from C2 to the caudal spinal trigeminal nucleus.

Retrograde labelling in the TG and C2 DRG

Labelled cell bodies were found in both the TG and C2 DRG following CTb or WGA-HRP application on the STA, SSS and MMA. Other studies have reported craniovascular innervation from both the TG and C2 DRG (29–32). Injections of WGA-HRP into the TG resulted in labelled nerve fibres on the ipsilateral half of the circle of Willis, the contralateral anterior cerebral artery, and the rostral portion of the basilar artery, whereas the C2 DRG neurons project to the vertebrobasilar arteries (7). Thus, substantial evidence indicates that sensory information from the cranial vessels is transmitted to the CNS via branches of both the trigeminal and cervical spinal nerves.

The first of the three trigeminal nerve branches, the ophthalmic nerve, innervates the temporal area, which is a common location of pain in primary headaches (3,23). CGRP, substance P and PACAP are potent vasodilators that localize in the trigeminovascular system (48). Stimulation of the TG elicits the release of CGRP and substance P and increases both extracerebral and regional cerebral blood flow in cats and monkeys (49–51). On the other hand, PACAP originates mainly in the parasympathetic ganglia with only minor expression in the TG (11). Furthermore, the responses to electric stimulation of SSS in the Sp5C and C1-C2 dorsal horns are lost following ablation of the TG (39). Taken together, these data support an essential role of signals transmitted through the trigeminal nerve in migraine pain, possibly including trigeminal primary afferent projections to the cervical spinal dorsal horn (52). The importance of the fibres entering through the cervical spinal ganglia is unclear in this context. Comparing the tracing data for all three vessels, both similarities and differences are seen in the distribution of labelled cell bodies in the TG. In cranial artery tracing studies, CTb and WGA-HRP labelled cell bodies predominantly located in the dorsomedial area of the TG, from which the first branch of the trigeminal nerve originates, whereas tracer application on the MMA labelled cell bodies in both the dorsomedial and dorsal central areas of the TG, which give rise to the first and second trigeminal nerve branches, respectively.

The central terminations of cranial vascular sensory fibres

Following CTb or WGA-HRP application on the STA, SSS and MMA, labelled terminations were demonstrated in the rostral three cervical spinal cord segments in the SP5C and SP5I (45–47). The Sp5C and dorsal horn in the rostral cervical spinal cord, the trigeminocervical complex, exhibit similar anatomical and physiological characteristics (37). The spinal trigeminal nucleus, especially Sp5C, is an essential component in the transmission and processing of pain and thermal sensations from the head and face (53). Following WGA-HRP application on MCA and the basilar artery, primary sensory fibres were detected in the C2 dorsal horn (29). Electrical stimulation of the MMA led to c-Fos expression in the C1-C2 dorsal horn and Sp5C, and evoked cell firing in the Sp5C and C2 dorsal horn (54–56). Stimulation of the SSS increased blood flow in the area between the C2 dorsal horn and Sp5C (39), and increased neuronal activity in the Sp5C (54,57).

Comparing the findings in the STA, SSS and MMA experiments, the distribution of transganglionic labelling patterns in the trigeminal nuclei and cervical spinal cord are similar. For all three vessels, thin (WGA-HRP labelled) and large (CTb labelled) afferents form a column extending through laminae I-IV of the rostral cervical spinal dorsal horn, similar to the termination pattern of corresponding afferents from the hind paw skin (58). However, certain differences are evident in the termination of the different perivascular nerves. First, the location of the transganglionic labelling density peak is different. In the STA study, a single density peak was evident in the C2 dorsal horn, whereas two density peaks could be discerned in the SSS study, one at the level of the rostral C2 and another in the caudal portion of Sp5C. In the MMA study, the labelled terminations displayed a single density peak extending across the rostral C1 dorsal horn and caudal Sp5C. Second, the mediolateral orientation of the transganglionic labelling in the cervical spinal dorsal horns was slightly different. The terminations from the MMA and SSS are both located in the lateral-most portion of the dorsal horn, whereas those from the STA are located slightly medial to those from the MMA and SSS.

Possible significance of primary afferent fibres with different diameters

Both CTb and B-HRP efficiently label myelinated A-fibres, which originate at the spinal levels, to a large extent, from low threshold mechanoreceptors and terminate in the deep layers (laminae III-IV) of the dorsal horn. In contrast, WGA-HRP predominantly labels unmyelinated C-fibres, including nociceptive afferents, which terminate in the superficial layers (laminae I and II) of the spinal dorsal horn (58–61).

Although early studies reported that the meningeal innervation is composed of small myelinated or unmyelinated axons (62) and that dural stimulation results only in painful sensations (3), reports demonstrate that dural nerves contain a substantial number of myelinated axons, approximately one-third of which can be classified as Aβ-fibres (63,64). Moreover, the dural A-fibres with faster conduction velocities display mechanical response properties different from those of the slower conducting A-fibres (presumably Aδ nociceptors) (63). Thus, in addition to nociceptive afferents, the dural innervation also includes a group of large calibre fibres that probably serve a function other than nociception. The findings further substantiate this notion by demonstrating the presence of a population of primary afferent fibres in the three vessels that, similar to low threshold mechanoreceptors in the skin, take up and transport CTb and terminate in the deep layers of the spinal and trigeminal (Sp5C) dorsal horns. The functional role of these large calibre craniovascular afferents is not yet clear, though large calibre dural afferents have been suggested to signal transient mechanical stimuli during, for example, sudden head movements (64).

Higher central nervous centres involved in headache pain processing

The expression of pain in primary headaches is putatively associated with activity in intracranial perivascular sensory nerve fibres, which originate in the TG and project to the trigeminocervical complex. However, it should be noted that the morphological methods cannot distinguish between neurons that receive direct input from the meninges and the vessels, and those that do not but have the same anatomical localization. The sensory signals conveyed to this region are then transmitted to higher brain centres in order to process the signals and form the pain sensations. Therefore, identifying the CNS regions that process nociceptive signals from the trigeminovascular system is important (2,22).

Evidence from animal experiments indicates that many regions in the brainstem and diencephalon may be involved in migraine pathophysiology (65–68). Using positron emission tomography (PET) and functional magnetic resonance imaging (fMRI), several regions in the human brainstem, thalamus, hypothalamus and cerebral cortex have been found to have increased activity during somatic nociceptive stimuli and in migraine attacks (69–72). In one study, seven healthy volunteers were examined during an acute experimental pain state; using PET, increased regional cerebral blood flow was found bilaterally in the insula, anterior cingulate cortex and cerebellum (73).

Although accumulating evidence suggests that certain CNS areas play important roles in headache pain, to what extent such activities depend on direct input from trigeminocervical neurons relaying nociceptive signals from the perivascular nerves located in cranial blood vessels is unclear. Therefore, to further understand the neural mechanisms behind primary headaches, it is important to know where neurons in the trigeminocervical complex project in the brainstem and thalamus. Such knowledge may also be of value to the development of novel strategies for relieving headaches. Therefore, after establishing that the major termination site of the cranial perivascular sensory innervation is located in the C1-C2 dorsal horn, the aim was to investigate the projections to the brainstem and thalamus from the ventrolateral C1-C2 dorsal horn (74). A similar study injecting biotinylated dextran amine (BDA) into the Sp5C region revealed complementary information (75). It is, however, worth pointing out that the work was performed in rodents, with the view that similar neuroanatomical organization exists in man.

Projections from the ventrolateral C1-C2 dorsal horn

To visualize the ascending projections from the ventrolateral C1-C2 dorsal horn, they were labelled with the anterograde tracer BDA (74). BDA injections in the ventrolateral C1 or C2 dorsal horn were aimed at either laminae I-II or laminae III-IV (74). However, the injections in the superficial dorsal horn remained confined to laminae I-II, but the injections centred in lamina III-IV spread along the needle track into the overlying superficial dorsal horn. Thus, the injections confined to the dorsal horn formed two separate groups: (i) rats with BDA injections confined to laminae I-II; and (ii) rats with BDA injections extending through laminae I-IV. Two additional rats had BDA injections in laminae I-IV that also extended into the adjacent lateral cervical nucleus (LCN).

Following the BDA injections into laminae I-II, anterogradely labelled terminal-like varicosities were evident in several nuclei in the pons, midbrain and thalamus. In the pons, relatively dense terminal labelling was detected in the lateral parabrachial nucleus (LPB), and labelling was sparser in the medial parabrachial nucleus (MPB). Terminal labelling was also detected in the caudal portion of the pontine reticular nucleus (PnC) and in the subcoeruleus nucleus (SubC). In the midbrain, moderate labelling was detected in the cuneiform nucleus (CnF), the lateral and ventrolateral portions of the periaqueductal gray (PAG), the intermediate gray layer of the superior colliculus (InG), and the deep mesencephalic nucleus (DpMe). Sparse labelling was evident in the anterior pretectal nucleus (APT). In the thalamus, dense labelling was detected in the triangular portion of the posterior nuclear group (PoT) and the ventral posteromedial nucleus (VPM). In one of the rats, moderate labelling was also evident in the posterior nuclear group (Po) and the ventromedial nucleus (VM). Labelling in the pons and the midbrain was bilateral except for the DpMe and APT, whereas labelling in the DpMe, APT and thalamus was confined to the side contralateral to the BDA injection.

Following the BDA injections in laminae I-IV, anterograde labelling was more extensive in the brainstem and thalamus (74). In addition to labelling the nuclei that were also labelled following laminae I-II injections, sparse to moderate labelling was detected in the deep gray layer of the superior colliculus (DpG) in the midbrain, and the suprageniculate nucleus (SG), posterior intralaminar thalamic nucleus (PIL), and ventral posterolateral nucleus (VPL) in the thalamus. The density of terminal-like labelling was higher in most nuclei after laminae I-IV injections compared to laminae I-II injections.

In the two rats in which the BDA injections in laminae I-IV extended into the LCN, the distribution of anterograde labelling was generally similar to that of injections confined to laminae I-IV (74). However, anterograde labelling in the central nucleus (CIC) and external cortex (ECIC) of the inferior colliculus was only detected when the BDA injections extended into the LCN.

Projections from the medullary trigeminal nucleus caudalis (Sp5C)

Following BDA injections into the Sp5C region (‘trigeminal nucleus caudalis’) using the same method as above, Noseda and colleagues observed that the Sp5C neurons project to the commissural subnucleus of the solitary tract, the A5 cell group region/superior salivatory nucleus, lateral PAG matter, inferior colliculus and parabrachial nuclei (75). Trigeminothalamic afferents were restricted to the posterior group and ventroposteromedial thalamic nuclei. Some of these areas also showed immunoreactivity for 5-HT_1D and CGRP, two molecules tightly linked with migraine pathophysiology and treatment.

Projections from the ventrolateral C1/C2 dorsal horn

Following BDA injections into the superficial ventrolateral C1/C2 dorsal horn, extensive anterograde labelling was detected in the LPB, CnF and PAG in the brainstem (74). Injection into Sp5C also revealed terminations in the PAG (75). Terminations in these nuclei were also reported in studies of spinal and trigeminal projections in rats, cats and monkeys (76–79). Most of the labelling in the thalamus following BDA injections was located in the PoT and VPM. A similar distribution of terminations has been noted by others following superficial tracer injections into the spinal dorsal horn (80) and Sp5C (75).

However, several brainstem and thalamic nuclei that are considered to be important for the processing and modulation of nociceptive signals were devoid of anterograde labelling in our study, including the nucleus of the solitary tract, the locus coeruleus and raphe nuclei in the brainstem, and the intralaminar nuclei and nucleus submedius (Sm) in the thalamus. In addition, no labelled terminals were found in any part of the hypothalamus (74). With the exception of the Sm, all of the above-mentioned nuclei or regions have been shown to receive projections from the cervical or medullary dorsal horns in different species (77,81). The absence of anterograde labelling in these nuclei may be due to either an absence of such projections from this particular part of the dorsal horn, or our small injections failing to label neurons with such projections.

Nuclei in the brainstem and thalamus possibly involved in primary headaches

The next important issue to consider is what possible role, if any, the nuclei identified as targets of ascending projections from the dorsolateral C1/C2 and Sp5C may play in primary headaches. As lamina I neurons serve a critical role in conducting nociceptive signals to higher centres (82,83), it appears likely that at least some of the brainstem and thalamic areas critical for primary headaches are found among those anterogradely labelled following BDA injections into laminae I-II. Therefore, the discussion below is focused on the nuclei that receive significant projections from the lateral superficial dorsal horn of segments C1-C2.

The PAG receives input from nociceptive neurons in the spinal cord and sends projections to thalamic nuclei that process nociception (76,81,84). The PAG is a major component of a descending pain inhibitory system. Activation of this system inhibits nociceptive neurons in the dorsal horn of the spinal cord (85). Neurons in the PAG respond to noxious stimulation of cranial vessels (65–68), and electrical or chemical stimulation of the SSS induces a dramatic increase in c-Fos expression in the ventrolateral PAG (65,68,86). High-resolution fMRI has demonstrated that patients with episodic migraine have impaired iron homeostasis in the PAG during attacks, indicating a role of the PAG as a possible ‘generator’ of migraine attacks (87). Our findings support the assertion that the PAG is an important locus for the generation and/or modulation of pain in primary headaches (74).

Figure 1.

Schematic summarizing the available data. Sensory nerves in the three cranial blood vessels terminate in the ventrolateral C1/C2 spinal dorsal horn (DH), Sp5C and Sp5I. The small and large calibre primary afferent fibres from the vessels are indicated with thin and thick lines, respectively. The projections from the superficial layers of the ventrolateral C1/C2 DH to different areas in the brainstem and thalamus are indicated to the right. The branching line does not mean nerve bifurcation. Uncertainties regarding a projection are indicated with a question mark. Ipsi: ipsilateral; contra: contralateral. Other abbreviations are defined in the text.

The LPB is known to serve a central role in processing somatic and visceral noxious information (80). The LPB receives abundant inputs from spinal and trigeminal lamina I neurons driven by Aδ- and/or C-fibres. After the application of capsaicin in the cisterna magna, c-Fos immunoreactivity is significantly increased in the LPB (68). Together with the tracing data, this finding indicates important roles for the LPB in primary headaches, possibly including as a ‘relay’ for nociceptive signals directed to the thalamus as well as centres involved in homeostatic mechanisms (82,83).

A putative role of the CnF in nociception was reported two to three decades ago, and has now received additional attention (70,71,88). In man, fMRI has demonstrated significant activation in the CnF when visceral or somatic pain is induced by repeated electrical stimulation (70,71,89). Studies support that the CnF receives afferent inputs from presumed nociceptive spinal and medullary lamina I dorsal horn neurons (77). The CnF also receives massive input from the PAG (90,91). The available evidence supports the CnF being involved in mechanisms associated with pain, including those related to headaches.

The posterior thalamic nuclear group has been shown to receive massive spinal and trigeminal projections from both superficial and deep dorsal horn neurons in different species (77,81,92). In the PoT, a caudal extension of Po located between the medial portion of the medial geniculate nucleus and the APT, a large proportion of the neurons respond to noxious stimulation (93). PoT neurons convey nociceptive signals from dorsal horn neurons to the second somatosensory and insular cortices (93). We observed inputs to the PoT from the craniovascular-receptive zone in the cervical dorsal horn; thus, signals relayed through the PoT towards the cerebral cortex are likely to include those from nociceptors in cranial vessels.

The VPM relays sensory signals from the trigeminal system to the cerebral cortex. In rats, trigeminal projections to the VPM include those from the superficial laminae in the Sp5C (81). Considering the close relationship between the Sp5C and rostral-most level of the spinal dorsal horn, lamina I input from the latter region may target the VPM rather than the VPL, as indicated by the C1-C2 (74) and Sp5C injection findings (75). Thus, the VPM may serve as a thalamocortical relay for nociceptive signals originating in cranial blood vessels.

Conclusions

The terminations of the sensory perivascular nerves in the STA, SSS and MMA are located mainly in the ventrolateral C1-C3 spinal dorsal horns, Sp5C and Sp5I. The projections from the STA and MMA are ipsilateral, whereas those from the SSS are bilateral. The sensory nerve fibres in the examined vessels originate both in the TG and the spinal segment C2 DRG. The TG neurons supplying the vessels are primarily located in the part of the TG giving rise to the ophthalmic nerve branch. In the spinal dorsal horn, thin primary afferent fibres (C-fibres) from the cranial blood vessels terminate predominantly in the superficial layers (laminae I-II), whereas large calibre craniovascular afferents (Aδ-fibres) terminate in the deep layers (laminae III-IV). Thus, in addition to thin primary afferents likely to conduct nociceptive signals, the craniovascular nerves also contain large myelinated afferents that presumably serve other functions. The craniovascular sensory nerve centre is located in a region extending from the rostral cervical spinal dorsal horn to the Sp5I. Neurons in the ventrolateral portion of the C1/C2 spinal dorsal horn receive dense sensory nerve projections from cranial vessels and project to multiple nuclei in the brainstem and thalamus. Several of these nuclei were previously implicated in nociceptive functions, suggesting that they may also serve such functions in relation to primary headaches.

Footnotes

Funding

This work was supported by a grant from the Swedish Research Council (grant number 5958).

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Tracing neural connections to pain pathways with relevance to primary headaches

Abstract

Keywords

Introduction

Innervation of the cranial vessels

Primary afferent fibres in the cranial vasculature

Central projections of sensory nerves in the superficial temporal artery

Central projections of sensory nerves in the superior sagittal sinus

Central projections of sensory nerves in the middle meningeal artery

Retrograde labelling in the TG and C2 DRG

The central terminations of cranial vascular sensory fibres

Possible significance of primary afferent fibres with different diameters

Higher central nervous centres involved in headache pain processing

Projections from the ventrolateral C1-C2 dorsal horn

Projections from the medullary trigeminal nucleus caudalis (Sp5C)

Projections from the ventrolateral C1/C2 dorsal horn

Nuclei in the brainstem and thalamus possibly involved in primary headaches

Conclusions

Footnotes

Funding

References