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Abstract

Little is known about trigeminal nociception-induced cerebral activity and involvement of cerebral structures in pathogenesis of trigeminovascular headaches such as migraine. Neuroimaging has demonstrated cortical, hypothalamic and brainstem activation during the attack and after abolition with sumatriptan. This has led to the conclusion that the dorsal raphe and locus coeruleus may initiate events that generate migraneous headache. Using a conscious rat model of trigeminal nociception and cerebral Fos expression as histochemical markers of neuronal activity, we characterized the pattern of brain activity after noxious trigeminal stimulation with capsaicin (250 and 1000 nm). A significantly increased Fos immunoreactivity was found in the trigeminal nucleus caudalis (layers I and II), the area postrema, the nucleus of the solitary tract, the parvicellular reticular nucleus, the locus coeruleus, the parabrachial nucleus and the raphe nuclei. In addition, the ventrolateral periaqueductal grey, the intralaminar thalamic and various hypothalamic areas, showed an enhanced Fos expression after the intracisternal administration of capsaicin. Other responding areas were the amygdala, the upper lip and forelimb regions of the primary somatosensory cortex, and the insula. Many of these areas participate in (anti)-nociception, although we cannot exclude the possibility that in conscious animals the pain-associated physiological and behavioural responses that are an intrinsic and necessary part of coping with pain have generated the increased Fos expression. Trigeminal stimulation-induced locus coeruleus, dorsal raphe and hypothalamic activation are opposed to a suggested pathogenic role of these nuclei in migraine and cluster headache, respectively.

Keywords

Capsaicin cluster headache migraine pain proto-oncogene protein c-Fos trigeminal

Introduction

The so-called trigemino-vascular system is thought to be the anatomical substrate for (neuro-)vascular headaches such as migraine and cluster headache (1, 2). Little is known about patterns of cerebral activity that are generated by the trigemino-vascular headaches. Essential data in this respect were collected with regional cerebral bloodflow (rCBF) measurement during a spontaneous migraine attack in patients (3). The attack was characterized by activation of several cortical areas and a few brainstem regions that coincided with the location of the dorsal raphe nucleus (DR) and locus coeruleus (LC). As the DR and LC activation remained after abolition of the migraine attack with sumatriptan, it was concluded that these regions might participate in the initiation of the attack. Subcutaneous injection of the irritant capsaicin into the forehead could not induce activation of the DR and LC (4). However, migraine is a diffuse, badly localized, deep, intracranial pain, whereas the experimental pain caused by subcutaneous capsaicin is superficial, sharp, well localized, and extracranial. It has been described in animal models that superficial pain and deep pain elicit different patterns of activation in the brain (5). Of course, it is ethically and technically difficult to induce intracranial pain in humans, but in animal models, this is quite commonly performed. Chemical (6), electrical (7, 8) and mechanical (9) stimulation of the intracranial trigeminal nerves has been used to mimic vascular headaches. Trigeminal afferent stimulation activates the cells in the outer layers (I and II) of the trigeminal nucleus caudalis (TNC I-II), the primary target of the intracranial trigeminal nociceptive afferents. The activation of the TNC I-II often has been revealed immunohistochemically by means of expression of the proto-oncogene protein Fos. A few of these studies described the Fos expression patterns in the brainstem and spinal cord (6, 8) in anaesthetized animals. The aim of the present investigation is to characterize the pattern of cerebral Fos expression after noxious trigeminal stimulation in conscious rats, using intracisternal administration of different concentrations of the irritant capsaicin (10). Behavioural changes generated by the noxious stimulation were monitored and analysed, and served as measures of pain intensity. Time spent on immobilization, and grooming and scratching of the head, correlated with the dosage and the Fos expression in the TNC I-II (10). The pattern of cerebral Fos expression that was induced by the intracranial noxious stimuli is discussed in light of a possible role of the affected regions in (anti)-nociception and pain behaviour, and the brain activity changes that were observed with PET-scan imaging in migraine and cluster headache patients.

Methods

Animals

Male Wistar rats weighing 310 ± 8 g were used. All rats were group housed (three rats/cage) on a light/dark regime (L/D: 08.00/20.00 h), and after the implantation of the cisterna magna cannula, they were isolated. Food and water were provided ad libitum. The experiments were approved by the committee for Animal Bio-Ethics of the University of Groningen (FDC 1051, FDC 1191) and performed according to the ethical guidelines for investigations of experimental pain in conscious animals (11).

Surgical procedures

All rats were anaesthetized with 0.4 mL/kg i.m. hypnorm (fentanyl 0.3 mg/mL and fluanisone 10 mg/mL; Janssen, Beerse, Belgium) and sodiumpentobarbital (24 mg/kg i.p.). A cisterna magna (CM) cannula was implanted under semisterile conditions 3 days before the start of the experiment. The cannula was made from a stainless steel needle (0.6 × 25 mm, 23 G×1 inch; Braun, Melsungen, Germany). We placed two small screws in the skull for the fixation of the cannula on the head. To facilitate the implantation of the cannula into the cisterna magna, the rats were fixated in a stereotaxic frame with the incisor bar at −7 mm (10). The correct position of the cannula in the cisterna magna was confirmed with extraction of cerebrospinal fluid, and then it was secured on the skull with dental cement (Kemdemt, Purton Swindon, UK) and closed with a polyethylene cap.

Drugs

Capsaicin stock solution—3.05 mg capsaicin per 1 mL of vehicle stock (saline-ethanol-Tween 80, 8:1:1)—was diluted 1:10 (1000 nm) or 1:40 (250 nm) with saline, containing 0.2% Evan's Blue (Merck, Darmstadt, Germany). Vehicle stock was diluted 1:10 in saline.

Experimental procedures

Intracisternal administration of the solutions

Rats were connected to a micro-injection pump (CMA100, Carnegie Medicin, Stockholm, Sweden) and then placed in an observation cage (30 × 30 × 30 cm). After a recovery of 10 min, 100 μL capsaicin (250 nm or 1000 nm) or vehicle solution was infused into the cisterna magna at a speed of 50 μL/min.

Behaviour

The behaviour of the rats was recorded on videotape from 5 min before until 10 min after the administration of the solutions into the cisterna magna. An analysis of the behavioural responses has been reported in previous publications (10, 12, 13). In brief, all animals receiving the capsaicin solution showed increased immobilization, and scratching and grooming of the head. The animals in the 1000 nm capsaicin group also showed escape behaviour and for ethical reasons the number of animals in this group was limited to four. After approximately 30 min, the animals resumed the natural nesting, grooming and eating behaviour.

Perfusion and immunocytochemistry

Two hours after capsaicin or vehicle administration, the rats were euthanized with sodium pentobarbital (60 mg/kg i.p.). They were transcardially perfused with 0.9% saline for 1 min, and 4% paraformaldehyde (PF) solution in 0.1 m phosphate-buffered saline (pH 7.4) for 20 min. Evans Blue staining of the epidural space demarcated the distribution of the administered solutions in the skull (10). No animals were removed from the study because of inadequate cannula placement or intracerebral drug distribution. The brains were post-fixed in 4% PF during 24 h, and cryoprotected overnight in 30% sucrose in 0.1 m phosphate buffer (pH 7.4). Coronal serial sections (40 μm thick) were prepared on a cryostat microtome at −15°C and collected in 0.2 m potassium phosphate-buffered saline (KPBS, pH 7.4) with sodiumazide(0.1%).

Fos protein expression was immunocytochemically characterized in free-floating sections according to the following protocol. In brief: the sections were rinsed 3 × 10 min in KPBS, pre-treated with 0.3% H₂O₂ in KPBS for 10 min, rinsed 3 × 10 min in KPBS and pre-incubated in 2% bovine serum albumin (BSA; Merck), 2% normal serum (NS, normal rabbit serum, Sigma Chemie, Bornem, Belgium) in KPBS for 4 h at room temperature. Subsequently, the sections were transferred to a KPBS solution containing 2% BSA, 2% NS, sheep-anti-c-Fos (1:2000; Cambridge Research Chemicals, Northwich, UK), and 0.5% triton X-100 (KPBS-T; Bayer, Deventer, Netherlands). After incubation overnight at room temperature, sections were rinsed and placed in a KPBS solution containing biotinylated rabbit-α-sheep IgG (1:200; Pierce, Rockford, USA) for 2 h. After 3 × 10 min washes in KPBS, the sections were incubated in avidine-biotine-peroxidase complex (Vector Labs, Burlingame, USA) in KPBS-T for 2 h at room temperature. After thorough washing in 0.1 m sodium acetate buffer (pH 6.0), the presence of peroxidase was revealed with 3.3′-diaminobenzidine tetrahydrochloride (0.05%) and ammonium chloride (0.04%) dissolved in 1/2 v distilled water and 1/2 v NAS solution consisting of 5% nickel-ammonium sulphate dissolved in 4/5 v 0.2 m sodium acetate and 1/5 v distilled water. The staining was started with 0.3% H₂O₂ and stopped after 20 min. Then the sections were washed, mounted on gelatin-coated slides, air dried, dehydrated in graded ethanols and xylol and cover-slipped with DEPEX. All staining procedures were with gentle agitation.

Quantification

TNC layer I-II

All Fos-ir cells in the TNC I-II were counted from −1 to −6 mm caudal from obex. For the −1 mm count, we averaged the total number of cells in sections located between −0.5 to −1.5 mm because lateralization of the Fos expression was not found in the TNC I-II after intracisternal administration of capsaicin.

Higher order Fos expression

The cytoarchitecture of the brain as defined by Paxinos and Watson (14) was used to delineate the regions of interest. The atlas of Swanson (15) defined the laminar organization of the cortex. The Fos expression in all regions that showed increased Fos-staining in one or more of the experimental groups in a pre-quantification screening of the material was quantified by an observer blinded from the treatments. Areas that according to the literature are considered relevant for pain perception and behavioural responses also were quantified. For small-size areas such as the locus coeruleus we used two sections and for the larger structures four–five sections to calculate the average number of Fos-ir cells.

Statistics

The one-way anova with Student-Newman-Keuls test as multiple comparison method (pairwise) was used to test differences between the three groups. In cases of non-normal distribution or unequal variance, the Kruskall-Wallis anova on ranks with Dunn's test as multiple comparison method (pairwise) was performed. P < 0.05 was considered significant.

Results

TNC Fos-expression pattern

The capsaicin-induced pattern of Fos expression in the caudal medulla oblongata and TNC is shown in Fig. 1. Control rats, receiving solvent in the cisterna magna, showed a small number of Fos-positive cells in the caudal brainstem, in the parvocellular reticular formation (PCRt), the peri-ambiguus area (Amb), the caudal ventrolateral reticular formation (CVL), and the lateral reticular nucleus (LRt). Fos-positive cells were located in the deep parts of the TNC, the nucleus of the solitary tract (NTS), and layers 3 and 4 of the upper cervical spinal segments. The pattern of capsaicin-induced Fos expression was significantly different, with a dense labelling in layers I and II of the TNC from the most rostral to the caudal level. Also, layers I and II of the upper cervical spinal segments showed a considerable number of Fos-expressing cells. The lateral part of cervical layer V showed a few Fos-positive cells in these animals. As in the control condition, we found Fos expression in the PCRt, the Amb, the CVL and the NTS. Significantly, increased numbers of labelled cells were found in the NTS and the area postrema (AP). The capsaicin-induced expression in layer I/II of the spinal segments was homogenous, whereas in the TNC the Fos labelling was in clusters that mainly were located in the dorsal and ventral parts of the TNC.

Figure 1

Schematic representation of the distribution of the Fos expression in the spinal trigeminal system, from the rostral (+ 1.1 mm from obex) to the caudal level (− 5.4 mm from obex), after infusion of solvent and 1000 nm capsaicin solution into the cisterna magna of conscious rats. The labelling was bilateral but for reasons of clarity and inclusion of anatomical labels, we illustrated the right side.

Fos expression in the CNS

The effect of intracisternal administration of vehicle (control, n = 5), 250 (C250, n = 8) and 1000 nm capsaicin (C1000, n = 4) on cerebral Fos expression is shown in Table 1.

TABLE 1

Numbers of Fos immunoreactive cells per section as found in various nuclei after intracisternal infusion of vehicle (control, n = 5), and 250 nm (C250, n = 8) or 1000 nm capsaicin (C1000, n = 4). Data expressed as mean± SEM.

Area (distance to bregma in mm)	Control	C250	C1000
Hindbrain
Cervical spinal cord
Level 3, layer X	4 ± 1	5 ± 1	7 ± 2
Level 1, layer X	4 ± 1	7 ± 1	10 ± 2
Spinal trigeminal nucleus
Caudal part, layer I, II (−17.68<>−19.68)	14 ± 3	118 ± 25 ∗	722 ± 63 ∗ †
(−14.6<>−16.6)	7 ± 2	89 ± 20 ∗	821 ± 42 ∗ †
Caudal, layer V (−16.6)	5 ± 1	19 ± 7	32 ± 6
Interpolar (−13.3)	33 ± 15	49 ± 13	111 ± 40
Oral (−10.5)	4 ± 2	5 ± 2	5 ± 2
Area postrema (−13.6)	4 ± 3	39 ± 23	281 ± 96 ∗ †
Nucleus of the solitary tract
Caudal
Lateral (−13.6)	2 ± 1	11 ± 3 ∗	22 ± 2 ∗ †
Medial	4 ± 2	69 ± 38	482 ± 94 ∗ †
Rostral
Lateral (−12.3)	2 ± 0	8 ± 2	9 ± 3
Medial	3 ± 1	13 ± 4	28 ± 9 ∗ †
Parvicellular reticular nucleus (−12.3)	8 ± 3	36 ± 7	54 ± 3 ∗ †
Raphe magnus (−11.3<>−10.3)	6 ± 2	13 ± 3 ∗	31 ± 6 ∗ †
Lateral parabrachial nucleus
Lateral (−9.3)	21 ± 5	117 ± 22 ∗	272 ± 23 ∗ †
Medial	3 ± 1	23 ± 5 ∗	34 ± 5 ∗
Median raphe (−8.0)	2 ± 0	5 ± 1	14 ± 2 ∗ †
Locus coeruleus (−9.3<>−10.3)	5 ± 1	28 ± 8 ∗	71 ± 4 ∗ †
Midbrain
Dorsal raphe nucleus
Dorsal (−7.8)	7 ± 4	4 ± 1	17 ± 10
Ventral	33 ± 10	32 ± 8	95 ± 20 ∗ †
Ventrolateral	89 ±10	115 ± 22	197 ± 28 ∗ †
Periaqueductal gray
Dorsolateral (−7.8)	14 ± 1	26 ± 5	36 ± 24
Dorsomedial	60 ± 19	74 ± 9	59 ± 21
Lateral	126 ± 24	112 ± 15	147 ± 31
Ventrolateral	100 ± 18	135 ± 23	257 ± 39 ∗ †
Periaqueductal gray
Dorsolateral (−6.8)	19 ± 3	14 ± 3	11 ± 2
Dorsomedial	45 ± 8	31 ± 7	27 ± 5
Lateral	129 ± 24	85 ± 8	90 ± 16
Forebrain, subcortical
Amygdaloid nucleus
Basolateral (−2.8)	26 ± 2	94 ± 19 ∗	44 ± 3 †
Central	24 ± 3	137 ± 23 ∗	281 ± 101 ∗ †
Medial	79 ± 16	127 ± 18	321 ± 59 ∗ †
Thalamic nucleus
Centrolateral (−2.8)	21 ± 5	41 ± 5	42 ± 5
Centromedial	74 ± 14	117 ± 18	201 ± 51 ∗ †
Mediodorsal	26 ± 7	67 ± 17	52 ± 13
Paracentral	10 ± 3	13 ± 2	23 ± 8
Paraventricular	100 ± 12	251 ± 56	151 ± 24
Reuniens	5 ± 1	17 ± 5 ∗	8 ± 1
Rhomboid	14±5	61 ± 15 ∗	19 ± 2 †
Submedius	0 ± 0	2 ± 1	2 ± 1
Posterior thalamic group	0 ± 0	5 ± 3	1 ± 1
Ventral posteromedial/lateral group	2 ± 1	6 ± 2	4 ± 2
Hypothalamus
Dorsomedial (−2.8)	144 ± 16	245 ± 32 ∗	331 ± 45 ∗
Lateral	25 ± 7	40 ± 8	32 ± 8
Ventromedial	22 ± 3	59 ± 12	105 ± 39 ∗
Paraventricular (−1.4<>−1.8)	59 ± 9	306 ± 76 ∗	396 ± 56 ∗
Supraoptic (−1.4<>−1.8)	9 ± 2	88 ± 24 ∗	167 ± 26 ∗ †
Zona incerta (−2.8)	28 ± 9	46 ± 6	30 ± 7
Hippocampus (−2.8)	63 ± 9	113 ± 9 ∗	53 ± 10 †
Accumbens nucleus
Core (1.6)	122 ± 24	95 ± 6	125 ± 35
Shell	230 ± 10	361 ± 58	422 ± 76
Forebrain, cortical
Motor cortex
Primary (1.2)	154 ± 27	384 ± 89	240 ± 39
Secondary	517 ± 60	699 ± 93	396 ± 99
Primary somatosensory cortex, forelimb area
Layer 1, 2, 3 (1.2)	26 ± 5	32 ± 6	37 ± 12
Layer 4	38 ± 9	57 ± 10	56 ± 15
Layer 5	7 ± 1	20 ± 4	41 ± 17 ∗
Layer 6	40 ± 3	84 ± 13 ∗	71 ± 10
Primary somatosensory cortex, jaw area
Layer 1, 2, 3 (1.2)	75 ± 11	147 ± 27	164 ± 37
Layer 4	156 ± 33	386 ± 71	310 ± 96
Layer 5	20 ± 4	73 ± 20	119 ± 57
Layer 6	145 ± 16	411 ± 85	385 ± 88
Jaw area, oral surface
Layer 1, 2, 3 (1.2)	20 ± 3	99 ± 35	67 ± 7
Layer 4	15 ± 5	290 ± 122	136 ± 32
Layer 5	6 ± 2	46 ± 20	32 ± 8
Layer 6	38 ± 7	249 ± 82	140 ± 26
Upper lip area
Layer 1, 2, 3 (1.2)	13 ± 1	61 ± 10 ∗	60 ± 8 ∗
Layer 4	8 ± 2	160 ± 36 ∗	134 ± 22 ∗
Layer 5	7 ± 2	44 ± 10 ∗	43 ± 8 ∗
Layer 6	31 ± 3	273 ± 57 ∗	174 ± 21
Agranular insular cortex (1.2)	44 ± 8	79 ± 11	73 ± 32
Granular/dysgranular insular cortex (1.2)	34 ± 6	115 ± 18 ∗	131 ± 42 ∗
Cingulate cortex
Area 1 (1.2)	167 ± 12	227 ± 27	144 ± 21
Area 2	94 ± 7	192 ± 29	160 ± 30
Cingulate cortex, area 1 (3.7)	144 ± 23	195 ± 42	247 ± 48
Prelimbic cortex (3.7)	251 ± 45	287 ± 67	315 ± 55

∗

Significantly different from control.

†

Significantly different from C250

Hindbrain

From the rostral to the caudal level, Fos-ir in the TNC I-II (Fig. 2) was significantly increased in the C250 and C1000 group (C250: 103 ± 22; C1000: 772 ± 52; control: 10 ± 2). Two other brainstem areas in the C1000 group showed robust Fos induction, namely the caudomedial nucleus of the solitary tract (cmNTS; control: 4 ± 2, C250: 69 ± 38, C1000: 482 ± 94) and the area postrema (AP; control: 4 ± 3, C250: 39 ± 23, C1000: 281 ± 96). Although capsaicin administration increased the Fos expression in layer V of the TNC, the difference between the groups was not significant (P = 0.056). Layer X of the spinal cord, and the trigeminal nucleus oralis (TNO) and interpolaris (TNI), were not affected by the intracisternal capsaicin administration. The caudal lateral NTS (control: 2 ± 1, C250: 11 ± 3), the parvicellular reticular nucleus (PCRt; control: 8 ± 3, C250: 36 ± 7), the locus coeruleus (control: 5 ± 1, C250: 28 ± 8; Fig. 2), and the lateral (lPBA; control: 21 ± 5, C250: 117 ± 22) and medial parabrachial nuclei (mPBA; control: 3 ± 1, C250: 23 ± 5) showed a significantly increased Fos-ir in the C250 group, and in the C1000 group, with the exception of the mPBA (caudal lateral NTS: 22 ± 2, PCRt: 54 ± 3, LC: 71 ± 4, lPBA: 274 ± 23; Fig. 3). The median raphe (MR) and raphe magnus (RMg) Fos expression also was enhanced in the C1000 group (MR: control: 2 ± 0, C1000: 14 ± 2; RMg: control: 6 ± 2, C1000: 31 ± 6).

Figure 2

Photomicrographs of the Fos expression in the TNC I-II after intracranial noxious stimulation in conscious rats. A: Low-power magnification of TNC Fos-ir after 1000 nm capsaicin; B: high-power magnification of A; C: TNC Fos expression after intracranial vehicle administration.

Figure 3

Photomicrographs showing enhanced Fos expression in the dorsal raphe nucleus (bottom) and locus coeruleus (top) after intracranial infusion of vehicle (left) and 1000 nm capsaicin (right).

Midbrain

In the C1000 group, a significantly increased Fos expression was found in the ventral (control: 33 ± 10, C1000: 95 ± 20) and ventrolateral (control: 89 ± 10, C1000: 197 ± 28) dorsal raphe nuclei (Fig. 3), and in the ventrolateral periaqueductal grey (vlPAG; control: 100 ± 18, C1000: 257 ± 39). Other subdivisions of the PAG and the midbrain reticular formation did not show significant changes of Fos-ir after the intracisternal capsaicin administration.

Forebrain, subcortical areas

All the intralaminar thalamic nuclei showed an increased Fos expression in the C250 and C1000 groups but the effect was significant only in the C1000 central medial thalamic nucleus (CM; control: 74 ± 14, C1000: 201 ± 51). The reuniens and rhomboid thalamic nuclei exhibited a significantly increased Fos expression only in the C250 group (control: 5 ± 1, C250: 17 ± 5; control: 14 ± 5, C250: 61 ± 15, respectively). Much to our surprise, in all groups the Fos expression was very low in the nucleus submedius, and the ventrobasal thalamic nuclei (posterior (Po), ventral posterolateral (VPL), ventral posteromedial (VPM)).

The C1000 group showed a significantly increased Fos expression in the central (CeA; control: 24 ± 3, C1000: 281 ± 101) and medial amygdala (MeA; control: 79 ± 16, C1000: 321 ± 59), and the C250 group in the central (C250: 137 ± 23) and basolateral nucleus (BLA; control: 26 ± 2; C250: 94 ± 19). In both treatment groups, most subnuclei of the hypothalamus exhibited an enhanced Fos expression (dorsomedial (DMH): control: 144 ± 16, C250: 245 ± 32, C1000: 331 ± 45; paraventricular (PVH), Fig. 4: control: 59 ± 9, C250: 306 ± 76, C1000: 396 ± 56; supraoptic (SO), Fig. 4: control: 9 ± 2, C250: 88 ± 24, C1000: 167 ± 26). The ventromedial hypothalamic nucleus (VMH) was increased only in the C1000 group (control: 22 ± 3, C1000: 105 ± 39). The Fos-ir in the lateral hypothalamic area was not altered by intracisternal capsaicin administration.

Figure 4

Photomicrographs showing the paraventricular (bottom) and supraoptic (top) hypothalamic Fos-ir after intracranial infusion of vehicle (left) and 1000 nm capsaicin (right).

Forebrain, cortical

Capsaicin-treated animals showed a higher Fos expression in all areas of the cortex. Significant effects of the intracranial nociceptive stimulation were found in the forelimb region of the primary somatosensory cortex (SI) (layer 5: control: 7 ± 1, C1000: 41 ± 17; layer 6: control: 40 ± 3, C250: 84 ± 13), all layers of the upper lip region of the SI, the granular and dysgranular insular cortex (control: 34 ± 6; C250: 115 ± 18; C1000: 131 ± 42). Fos expression in all other cortical areas (cingulate cortex, prelimbic cortex, agranular insular cortex, motor cortex and SI, jaw region, oral surface), although higher, was not significantly different between the vehicle and capsaicin groups.

Discussion

Intracisternal infusion of capsaicin in conscious rats induced a distinctive pattern of Fos expression in the brain, in the trigeminal system and sensory cortex, and in nuclei participating in pain reduction (LC, DR, RMg) and regulation of autonomic functions (NTS, PBA, CeA, vlPAG, PVH). Notably, little Fos-ir was observed in the ventrobasal thalamic nuclei, although this is an important relay in the trigemino-thalamo-cortical pathway. The agranular insular, cingulate and prelimbic cortex showed no significant effect of the treatment.

Trigeminal nociceptive stimuli, and the physiological and behavioural responses elicited by the painful stimulation, contribute to the induction of the Fos expression in un-anaesthetized animals. This could be considered as a limitation of the approach but the behavioural and physiological ‘pain’ responses are an intrinsic and necessary part of coping with pain in animals as well as in humans. The cerebral activation patterns revealed with Fos immunocytochemistry in this study thus most closely will resemble the pattern of brain activation in patients suffering from a headache. Unfortunately, we cannot identify with this method the regions that are selectively activated by the trigeminal pain. To discern nociception-induced activation from physiological and/or behaviour-induced regional brain activation, other experimental set-ups should be used (16). Moreover, Fos immunocytochemistry has other limitations; Fos is a protein marker of neuronal activation (17) and does not identify inhibition of neuronal activity as part of the trigeminal pain transmission and response process. Histological markers of neuronal inhibition are not available yet and thus we characterized the trigeminal nociception-induced activity changes in the Fos positive regions.

Hindbrain

The ventrolateral, mediolateral and dorsomedial part of the TNC are innervated by, respectively, the ophthalmic, maxillary and mandibular branches of the trigeminal nerve (18). All tree branches carry fibres that innervate the meninges and meningeal vasculature (19, 20), which is illustrated in the present study by the increased Fos expression in all dorsoventral parts of the TNC I-II after the intracranial trigminovascular stimulation.

The largest proportion of the intracranial (6, 9, 20, 21) and extracranial (23–27) trigeminal nociceptive afferents terminate in layers I and II₀ of the TNC (9, 28) and other targets are layer V of the TNC (9, 24, 29), layer I-II and X of the upper cervical dorsal horn (7, 22, 26), and the TNO (23, 27, 30, 31). In addition to the TNC I-II, only layer I-II of the upper cervical spinal cord showed a significantly increased Fos expression after intracisternal capsaicin administration.

Trigeminal afferents innervate the ventrolateral NTS (32, 33) and accordingly a dose-dependent enhancement of Fos expression was observed in the lateral NTS, at the level of the AP, after trigeminal stimulation with capsaicin. The medial NTS (mNTS) showed a larger Fos expression increase that may be triggered by vagal afferent activation as part of physiological adaptations to pain. The mNTS and the AP are both innervated by vagal afferents (32) and electrical stimulation of the vagus nerve can induce sensation of headache in patients (34).

The PCRt, the LC and the lPBA showed a dose-dependent Fos-ir response. The PCRt receives trigeminal input (35) but it is unclear whether this is of nociceptive origin, as capsaicin-induced grooming and scratching of the head (10, 12) may have elicited trigeminal mechanoreceptor activation.

Layer I cells of the TNC project to the LC in cat and monkey (36), suggesting that the outer layers of the TNC activated the LC in the conscious rats. The role of ascending and descending LC pathways in pain control is well documented (37–39). The LC projects to the TNC (40) and consequently TNC-induced LC activation may be part of a feedback loop for pain reduction.

The lateral PBA is innervated by TNC layer I cells (28), the NTS (especially the dorsomedial portion) and the AP (41). These connections provide anatomical relationships to regions that are activated by trigeminal- and vagus-mediated sensations. The IPBA is a relay area for the central autonomic/emotional control circuitry (42) that generates the behavioural and autonomic adaptations to stressful conditions such as headache.

The Raphe Magnus supplies a serotonergic input to the TNC I-II (43) and can inhibit nociception-induced TNC I-II responses in rats (44) and cats (45). This antinociception pathway most likely was activated in the C1000 group, which showed a small but significant increase of Fos-ir in the RMg compared with controls.

Midbrain nuclei

The dorsal raphe (DR), a region selectively activated during a migraine attack, even after abolishment of the attack with sumatriptan (3), showed enhanced Fos expression after intracranial capsaicin administration in the 1000 nm group. In the C250 group, the DR was not activated and thus it can be concluded that strong nociceptive trigeminal stimulation is necessary for DR activation. The DR participates in central antinociception circuitry, like the LC (46–48). The ventrolateral part of the DR projects to the TNC (49) and the LC (50), a source of a noradrenergic input to the TNC (40). Serotonergic and noradrenergic modulation of trigeminal nociception has been demonstrated (39) and the DR thus can influence the pain transmission in the TNC directly, as well as indirectly via the LC pathway.

The PAG is involved in antinociception (45, 51, 52) and especially the ventrolateral PAG (vlPAG) showed enhanced Fos expression after induction of deep somatic and visceral pain in the rat (16). Cutaneous noxious stimulation induced Fos-ir in the lateral and dorsolateral segments of the PAG (53). Activation of the vlPAG has been demonstrated after sagittal sinus stimulation in the anaesthetized cat (53). Enhanced Fos expression in the vlPAG after intracisternal administration of capsaicin in conscious animals corroborated these findings and illustrated that intracranial nociceptive trigemino-vascular stimulation is perceived as a deep noxious pain. Stimulation of the vlPAG in cats inhibited the nociceptive neurones in the TNC (45), which yielded evidence for a role in antinociception. However, the vlPAG also showed increased Fos expression after social (54) or restraint stress (55) and was associated with the quiescence response after a stressful encounter (56). Therefore, it may be concluded that vlPAG activation is part of a generalized stress response that induces immobilization. The intracisternal capsaicin infusion generated a long-lasting immobilization (10, 12, 13), in line with the role of the vlPAG. The vlPAG activity most likely is determined by the intensity of the stressor, as was demonstrated in studies using combined loose restraint stress and noxious colorectal distension (55).

Forebrain nuclei, subcortical

In the subcortical forebrain nuclei, intracisternal capsaicin administration induced Fos expression in the amygdala, the medial thalamic, and various hypothalamic nuclei. The basolateral, medial and central nuclei of the amygdala have been associated with analgesia (56–58). The CeA has an important general integrative role in autonomic functions related to pain, and receives input from the outer layers of the TNC via the lateral parabrachial area (28), and from the NTS (42, 59).

The intralaminar thalamic nuclei all showed enhanced Fos expression after intracisternal capsaicin treatment but the increase was significant only in the CM. The intralaminar nuclei, including the CM, receive input from the outer layers of the TNC and spinal cord via the lPBA (60, 61), a region activated by intracranial capsaicin administration involved in autonomic regulation. None of the ventrobasal thalamic nuclei, the VPM, VPL and Po, showed enhanced Fos expression after capsaicin treatment, albeit that the VPM and Po receive input from the TNC I-II (62). Enhanced local cerebral glucose utilization (63) and increased cell firing (64) was found in the VPM after trigeminal nociception in anaesthetized animals. This activation was not induced by the anaesthetic because thalamic Fos-ir was generated in the intralaminar but not in the ventrobasal nuclei after tooth movement in anaesthetized animals (65). Most likely, the ventrobasal thalamus neurones cannot induce Fos, which has been reported before (66).

All hypothalamic nuclei, except the LH, showed increased Fos expression after capsaicin treatment, an activation that may involve the trigemino-hypothalamic connection (67). Dorsomedial (DMH) and ventromedial hypothalamic (VMH) lesioned animals showed hyperalgesia after nociception (68) and restraint stress induced Fos expression in the DMH and the PVH, a region that controls the pituitary-adrenal system and receives input from the DMH (69). Fos expression in the PVH was enhanced in restraint animals undergoing simultaneous noxious visceral stimulation (55). Electrical stimulation of the PVH induced analgesia (70, 71) and PVH lesions not only significantly increased paw-licking scores in the formalin test (72) but it also decreased the cold-water swim stress-induced analgesia in the tail flick test (73). The latter suggests an important role for the PVH in stress-induced analgesia, but this may depend on the stressor, type, or location of the pain, as it was demonstrated that the PVH does not mediate stress-induced analgesia after restraint in the formalin test (72).

Forebrain nuclei, cortical

A relative large proportion of the somatosensory cortex of the rat (66%) consists of neurones that somatotopically represent the face (74) and especially the whiskers. Only the layers of the upper lip region of the SI showed a significantly enhanced Fos expression in capsaicin-treated rats. The ventrobasal thalamic nuclei (VPL and VPM) and the Po (75), the main relay areas in the trigemino-thalamic-cortical pain pathway, innervate the SI. Layers 3, 4 and 6 of the SI are innervated by the VPM (62) and layers 1 and 5a by the Po (18). Nociceptive responsive neurones are primarily located in layers 5 and 6 (76, 77). Increased Fos expression in layers 5 and 6 of the forelimb SI region may be associated with the head grooming and head scratching that is induced by the intracranial capsaicin (10, 12).

Regions of the cortex that are involved in autonomic and emotional control, like the insular, cingulate and prelimbic cortex, didn't show quantitative differences of Fos expression, neither in the capsaicin-treated rats, nor in the vehicle and capsaicin-treated animals. During the infusion of vehicle, the controls were put into a novel environment, which they most likely perceive as a mild stressor. Vehicle-treated rats therefore also were submitted to (a mild form of) stress, inducing a moderate Fos expression in circuitry for autonomic and emotional control. This may explain the small differences in the cingulate and prelimbic Fos expression between the various groups.

General discussion

This is the first report describing cerebral Fos expression patterns in conscious rats after intracranial nociceptive trigeminal stimulation. In the first half of this century, experimental intracranial nociceptive trigeminal stimulation was performed in humans to study trigeminovascular headaches such as migraine and cluster headache (78, 79) but nowadays this is not acceptable anymore. Intracranial nociceptive trigeminal stimulation in conscious animals may provide an alternative for such studies. PET scans have shown activation of brainstem and hypothalamic regions during migraine (3) and cluster headache (80), respectively. Next to cortical (audiovisual, cingulate) regions, brainstem areas coinciding with the LC and DR were active during the migraine attack. The activation of the LC and DR remained after sumatriptan treatment, leading the authors to conclude that these regions may be involved in the generation of the migraine attack (3).

The inferior hypothalamic grey has been associated with the pathophysiology of cluster headache and showed enhanced rCBF during the bout, but not outside the bout (80). In other headache types, such as migraine (3) or experimental head pain (4), the inferior hypothalamic grey is not activated. Robust intracranial nociception (1000 nm) increased Fos expression in the LC, the DR, the DMD, the VMH, the PVH and the SO in conscious rats, which implies that in response to the intracranial trigeminovascular nociception these areas become activated. Activated regions in cluster headache and migraine patients overlap with regions responding to intracranial trigeminal stimulation in rats. Our data thus yield evidence for hypothalamic and DR/LC activation in cluster headache and migraine in response to the pain of the headache. Pain-induced activation of the LC and the DR, both participating in antinociceptive mechanisms (39), may be necessary to completely block the pain of the migraine attack after administration of sumatriptan, which provides an alternative explanation for the LC and DR activation shown by Weiller (3). This pain-induced activation of central antinociceptive pathways may be an important aspect of the mode of action of sumatriptan and explain the lack of a prophylactic effect when it is taken in the aura phase (81).

Footnotes

Acknowledgements

The authors would like to thank M.B. Spoelstra for histological work and Glaxo-Wellcome, Zeist, the Netherlands, for their generous financial support.

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Trigeminal Nociception-Induced Cerebral Fos Expression in the Conscious Rat

Abstract

Keywords

Introduction

Methods

Animals

Surgical procedures

Drugs

Experimental procedures

Intracisternal administration of the solutions

Behaviour

Perfusion and immunocytochemistry

Quantification

TNC layer I-II

Higher order Fos expression

Statistics

Results

TNC Fos-expression pattern

Fos expression in the CNS

Hindbrain

Midbrain

Forebrain, subcortical areas

Forebrain, cortical

Discussion

Hindbrain

Midbrain nuclei

Forebrain nuclei, subcortical

Forebrain nuclei, cortical

General discussion

Footnotes

Acknowledgements

References