Occipital afferent activation of second order neurons in the trigeminocervical complex in rat.

Stimulation of the greater occipital nerve produces excitation of second order neurons in the trigeminocervical complex. Given that neck pain is very common in primary headache disorders, this convergent excitation may play a role in pain referral from cervical structures. While previous studies have demonstrated a physiological model for this convergence, this study sought an anatomical approach to examine the distribution of second order neurons in the trigeminocervical complex receiving greater occipital nerve input. In addition, the role of glutamatergic NMDA receptor activation within the trigeminocervical complex in response to cervical afferents was studied. Noxious stimulation of the occipital muscle in rat using mustard oil and mineral oil produced significantly altered Fos expression in the trigeminocervical complex compared with the surgical control (H(4)=31.3, P<0.001, Kruskal-Wallis). Baseline expression was 11 (median, range 4, 17) fos positive cells in the trigeminocervical complex, occipital muscle treated with mustard oil produced 23 (17, 33) and mineral oil a smaller effect of 19 (15, 25) fos positive cells, respectively (P=0.046). The effects of both mustard and mineral oil were reversed by the NMDA-receptor antagonist MK801. This study introduces a model for examining trigeminocervical complex activity after occipital afferent stimulation in the rat that has good anatomical resolution and demonstrates involvement of glutamatergic NMDA receptors at this important synapse.

Inhibition of nociceptive dural input in the trigeminal nucleus caudalis by somatostatin receptor blockade in the posterior hypothalamus.

Somatostatin is a neuromodulator in the central nervous system and is involved in the regulation of metabolic and neuroendocrine functions. Recent experimental and clinical findings point to a role for somatostatin in the central processing of nociception. We studied the effects of somatostatin receptor modulation in the posterior hypothalamic area (PH) of the rat on dural nociceptive input. Somatostatin (10 microg/microl) and the somatostatin antagonist cyclo-somatostatin (50 microg/microl) were microinjected into the PH and the effects on responses of neurons in the trigeminal subnucleus caudalis studied. Injection of somatostatin (n=11) did not affect A- and C-fibre responses to dural electrical stimulation, nor was spontaneous activity altered (P>0.05). Injection of cyclo-somatostatin (n=10) into the PH reduced A-(-35.5+/-5.8%) and C-fibre (-43.1+/-7.5%) responses to dural stimulation and resulted in decreased spontaneous activity (-38.1+/-7.3%, P<0.05). Responses to facial thermal stimulation were decreased by 51.2+/-5.8% (n=5). Control injections had no significant effect (n=9). Blockade of somatostatin receptors in the PH has an anti-nociceptive effect on dural and facial input, probably mediated via GABAergic mechanisms. As somatostatin is also involved in hypothalamic regulation of metabolic, neuroendocrine and autonomic functions, somatostatin receptor mechanisms in the PH may play a role in the pathophysiology of primary headache disorders, such as migraine or cluster headache.

Activation of 5-HT(1B/1D) receptor in the periaqueductal gray inhibits nociception.

It is considered that the site of action of the abortive antimigraine compounds acting at serotonin, 5-HT(1B/1D,) receptors (triptans) is the trigeminovascular system. We tested whether there is a non-trigeminal site of action. The 5-HT(1B/1D) agonist, naratriptan, was microinjected into the ventrolateral periaqueductal gray (vlPAG), and activity in the trigeminal nucleus caudalis (TNC) was monitored. Recordings were made from 20 nociceptive neurons in the dorsal horn of the TNC that received convergent input from the dura mater and face. Responses of neurons to dural, facial cutaneous and corneal stimulation were studied before and after injection of naratriptan. Naratriptan decreased the excitability to electrical stimulation of the dura mater as the A-fiber response decreased by 24 +/- 4.1% (p < 0.001) and the C-fiber response decreased by 42 +/- 8.2% (p < 0.001). Spontaneous activity was decreased by 38 +/- 7.5% (p < 0.001). After injection, the mechanical thresholds of the dura mater increased from (n = 14, p < 0.01). Responses to stimulation of the face and cornea were not altered by injection of naratriptan. These results suggest that 5-HT(1B/1D) receptor activation in the vlPAG activates descending pain-modulating pathways that inhibit dural, but not facial and corneal nociceptive input. These findings have implications for the understanding of the action of triptans in migraine and cluster headache, suggesting that brain loci other than the trigeminal nucleus may play a role in the clinical action of triptans.

Differential modulation of nociceptive dural input to [hypocretin] orexin A and B receptor activation in the posterior hypothalamic area.

The novel neuropeptides orexin A and B are selectively synthesised in the lateral and posterior hypothalamus and are involved in hypothalamic regulation of autonomic and neuroendocrine functions. Recent findings point also to a role in nociception. As the posterior hypothalamus is involved in the central modulation of nociception we studied the effects of hypocretin/orexin receptor activation in the posterior hypothalamic area (PH) of the rat on dural nociceptive input. Orexins were microinjected into the PH and the effects on responses of neurones in the caudal trigeminal nucleus studied. Injection of orexin A decreased the A- and C-fibre responses to dural electrical stimulation as well as spontaneous activity. Responses to noxious thermal stimulation of the facial skin were also decreased by orexin A. Injection of orexin B into the PH, however, elicited increased responses to dural stimulation in A- and C-fibre responses and resulted in increased spontaneous activity. Responses to facial thermal stimulation were also increased by orexin B. Control injection of saline into the PH had no significant effect. The results show a differential modulation of dural nociceptive input by orexin A and B receptor activation in the PH. The results support the role of the PH in the nociceptive processing of meningeal input. As both peptides are also involved in hypothalamic regulation of neuroendocrine and autonomic functions, orexinergic mechanisms in the PH may provide a link for endocrine and autonomic changes as well as nociceptive phenomena seen in primary headache disorders.

Effect of IL-1beta microinjection into the posterior hypothalamic area on trigeminal nociception in the rat.

The hypothalamus has been implicated in the pathophysiology of the most disabling forms of primary headache, namely migraine and cluster headache. Interleukin-1beta (IL-1beta) is highly expressed in the hypothalamus. We recorded from the trigeminal nucleus caudalis of rats using extracellular electrophysiological methods from neurons responding to electrical stimulation of the peri-middle meningeal artery dura mater and having receptive fields in the ophthalmic division of the trigeminal nerve. Data were collected from fifteen clusters of wide-dynamic range neurons with stable baseline firing responses between 97 and 101% ( n=3 for each unit) to stimulation. Microinjection of IL-1beta into the posterior hypothalamus of 9 animals resulted in a modest inhibition of evoked trigeminal responses in three units, no effect in six and no overall effect for the entire cohort studied. The mean maximum response was a non-significant reduction in firing to 83+/-7% ( n=9) at 30 minutes post-injection of IL-1beta. There was some variation of effect dependent on site of injection with central posterior hypothalamus being the predominant area that resulted in inhibition. There was no inhibition in the six animals injected with vehicle (saline). If there is an important effect for IL-1beta in the posterior hypothalamus it is likely to be highly somatotopically restricted.

The periaqueductal grey matter modulates trigeminovascular input: a role in migraine?

The periaqueductal grey (PAG) region of the brainstem is a known modulator of somatic pain transmission. Migraine is likely to be due to episodic brain dysfunction in pathways involved in the control of pain and other sensory modalities, such as light and sound. To investigate the influence of the PAG on pain transmission from intracranial structures, we examined spinal trigeminal neuronal activity in response to PAG stimulation in a model of trigeminovascular nociception in the cat. Evoked trigeminal neuronal activity in the spinal cord was reversibly inhibited by stimulation of the PAG. The effect was robust with a mean reduction in evoked activity of -61+/-21%. This effect could be seen both ipsilateral and contralateral to the side of PAG stimulation and was well localised to the ventrolateral PAG. These data demonstrate that a role of the PAG is to inhibit afferent trigeminal nociceptive traffic. Considered with neurosurgical and human functional imaging studies, these data support the notion that brainstem dysfunction might lead to disinhibition of trigeminal afferents and be important in the pain process of migraines.

The NMDA receptor antagonist MK-801 reduces Fos-like immunoreactivity within the trigeminocervical complex following superior sagittal sinus stimulation in the cat.

Expression of Fos protein is an indicator of neuronal perturbation and is readily observed in the caudal medulla and the spinal cord following trigeminovascular nociceptive activation by electrical stimulation of the superior sagittal sinus (SSS) in the cat. It has been shown in the rat that N-methyl-D-aspartate (NMDA) receptor blockade causes a reduction in Fos protein expression after generalised meningeal irritation. We wished to examine if the same relationship was true in the cat, using the same non-competitive NMDA receptor antagonist MK-801, and a trigeminovascular-specific stimulus. A group of experimental animals underwent stimulation following blinded administration of MK-801 (4 mg/kg i.v.); control animals underwent stimulation minus MK-801, and a non-stimulated control animal underwent surgery alone. The regions examined for Fos-like immunoreactivity were the trigeminal nucleus caudalis (TNC) and its caudal extension into the C(1) and C(2) levels of the upper cervical spinal cord. The Fos-positive cell counts for the three regions (TNC, C(1) and C(2)) were grouped together for analysis. In the control stimulated group a median of 78 (56-99, quartile range, n=4) cells were Fos-positive. In the group treated with MK-801 the median number of Fos-positive cells was reduced to 40 (30-48; P<0.03, n=7). The large reduction that was observed in SSS stimulation-evoked Fos protein expression following the administration of MK-801, taken together with electrophysiological data, indicates a role for glutamate in neurotransmission within the trigeminocervical complex. Understanding glutamatergic mechanisms in the trigeminocervical complex offers mechanistic insight and therapeutic possibilities for primary neurovascular headaches, such as migraine.

4991W93 inhibits release of calcitonin gene-related peptide in the cat but only at doses with 5HT(1B/1D) receptor agonist activity?

Calcitonin gene-related peptide (CGRP) is a marker for trigeminovascular activation and is released during the headache phase of migraine and cluster headache. CGRP may have a role in migraine through its potent cranial vasodilator effects, or by an action on trigeminal nerve activity, both of which are targeted by 5HT(1B/1D) agonist drugs. CP122,288, a conformationally restricted analogue of sumatriptan that is a potent inhibitor of neurogenic plasma protein extravasation (PPE), was ineffective at inhibiting CGRP release at a single low dose; and is also ineffective as an acute anti-migraine compound. However, it remained unclear as to whether, as a class, the conformationally-restricted triptan analogues could have inhibitory effects on CGRP in higher doses. 4991W93, a conformationally restricted analogue of zolmitriptan, is also a potent inhibitor of PPE at doses without 5HT(1B/1D)-mediated effects, that was developed as an anti-migraine drug, and thus was suitable to test whether higher doses of such conformationally restricted triptan analogues could inhibit trigeminal-evoked CGRP release. The superior sagittal sinus (SSS) was stimulated in 14 anaesthetised cats and external jugular vein blood samples were analysed by radioimmunoassay for CGRP levels before, 1 min after SSS stimulation, and 1 min after SSS stimulation in the presence of 4991W93. Stimulation of the SSS resulted in release of CGRP from the external jugular vein. 4991W93 at a dose of 0.1 and 10 microg/kg, selected for maximal PPE blocking effects in rodents, was ineffective at inhibiting CGRP release, with an SSS stimulation level of 78+/-4 pmol/l compared to a post-4991W93 level of 79+/-3 pmol/l (n=4). In comparison CGRP release was inhibited after a dose of 100 microg/kg 4991W93 from 64+/-6 to 36+/-3 pmol/l (n=5). Given that 4991W93 is inactive clinically at non-vascular doses, it seems clear that the 5HT(1B/1D) agonist effects of the compound are necessary for blockade of CGRP release and thus any anti-migraine action. Taken with the clinical results, these data emphasise the importance of CGRP release in migraine, and suggest that other non-5HT-based pharmacological targets may account for PPE blockade in animal studies.

Vasodilator agents and supracollicular transection fail to inhibit cortical spreading depression in the cat.

It remains an open question as to whether cortical spreading depression (CSD) is the pathophysiological correlate of the neurological symptoms in migraine with aura. In the experimental animal, CSD is an electrophysiological phenomenon mainly mediated via NMDA receptors. However, according to case reports in humans, visual aura in migraine can be alleviated by vasodilator substances, such as amyl nitrite and isoprenaline. There is also circumstantial evidence that brainstem nuclei (dorsal raphe nucleus and locus coeruleus) may play a pivotal role in the initiation of aura. In this study, CSD was elicited in alpha-chloralose anesthetized cats by cortical needle stab injury and monitored by means of laser Doppler flowmetry. Topical application of isoprenaline (0.1-1%) and amyl nitrite (0.05%) onto the exposed cortex had no effect on the elicitation or propagation of CSD. Also, after supracollicular transection, subsequent CSDs showed no differences in the speed of propagation and associated flow changes. We conclude from these data that--given CSD probably exists in humans during migraine--spreading neurological deficits during migraine aura are independent of brainstem influence and have a primarily neuronal rather than vascular mechanism of generation.

Blockade of calcitonin gene-related peptide release after superior sagittal sinus stimulation in cat: a comparison of avitriptan and CP122,288.

The pathophysiological basis for the pain of migraine has been the subject of substantial attention and must include activation of elements of the trigeminal innervation of the cranial vessels, the trigeminovascular system. Recently, consideration of trigeminal-evoked neurogenic plasma protein extravasation (PPE) as a model for the pain has driven the search for compounds with specific anti-extravasation properties. Calcitonin gene-related peptide (CGRP) is a marker for trigeminovascular activation and is released during the headache phase of migraine and cluster headache. CGRP may have a role in migraine through its potent cranial vasodilator effects or by an action on trigeminal nerve activity, both of which are targeted by 5HT(1B/1D)agonist drugs but does not itself produce PPE. It has been suggested that 5HT(1B/1D)agonists may have an anti-migraine effect via inhibition of PPE in the dura mater. Avitriptan and CP122,288 both have strong binding affinities for 5HT(1B/1D)receptors, but only CP122,288 is a potent inhibitor of PPE. In this study we sought to compare the effects of CP122,288 and avitriptan on jugular vein CGRP release after stimulation of the superior sagittal sinus (SSS) in the cat. In eleven anaesthetized cats external jugular vein blood samples were analyzed by radioimmunoassay for CGRP levels in three settings: a) control, b) 1 min after SSS stimulation and c) 1 min after SSS stimulation in presence of drug. Stimulation of the SSS resulted in release of CGRP from the external jugular vein (77+/-1 pmol/L). At a PPE-inhibitory dose in rat (100 ng/kg intravenously) CP122, 288 had no effect on CGRP release (77+/-6 pmol/L) whereas at a clinically relevant dose (50 microgram/kg intravenously) avitriptan blocked CGRP release. This study demonstrates that the potent inhibitor of PPE, CP122, 288, which has been shown in clinical trials to be ineffective in treating acute migraine attacks, had no effect on CGRP release, whereas the effective anti-migraine drug and relatively impotent inhibitor of PPE, avitriptan, blocked CGRP release. These data emphasize the importance of CGRP release and its possible independence from PPE in migraine and more importantly suggest that other non-5HT-based pharmacological targets may account for PPE blockade in animal studies.

Substance P blockade with the potent and centrally acting antagonist GR205171 does not effect central trigeminal activity with superior sagittal sinus stimulation.

The development and use of serotonin-1B/1D agonists to treat the acute attack of migraine has been a significant advance, but their vasoconstrictor effects have lead to a search for non-vasoconstrictor approaches to the management of the acute attack of migraine. One such suggested approach has been substance P (neurokinin-1) antagonists, since substance P is involved in mediating neurogenic plasma protein extravasation and has long been held to have a role in pain transmission. In this study, one such candidate compound, GR205171, a highly lipophilic potent neurokinin-1 antagonist, has been tested in a model of trigeminovascular nociception with considerable predictive value for anti-migraine activity. The superior sagittal sinus was isolated in the alpha-chloralose (60 mg/kg, i.p., and 20 mg/kg, i.v., supplemented every 2 h)-anaesthetized cat. The sinus was stimulated electrically (100 V, 250 micros duration, 0.3 Hz) and neurons in the dorsal C2 spinal cord monitored using electrophysiological methods. In separate experiments, the animals were prepared for stimulation and then maintained for 24 h before stimulation and perfusion for Fos immunohistochemistry. Stimulation of the superior sagittal sinus resulted in activation of cells in the dorsal horn of C2. Cells fired with a probability of 0.7 +/- 0.1 at a latency of 10.7 +/- 0.2 ms. Administration of GR205171 (100 microg/kg, i.v.) had no effect on probability of firing or latency. Stimulation of the sinus in separate cats resulted in increased expression over control levels in the superficial laminae of the trigeminal nucleus caudalis and C1/2 dorsal horns. GR205171 in the same dose had no effect upon Fos expression. Inhibition of substance P by the potent, selective and brain penetrant neurokinin-1 antagonist GR205171 had no effect upon either cell firing or Fos expression in the central trigeminal cells activated by stimulation of the superior sagittal sinus. These data and the published clinical data for other compounds suggest that neurokinin-1 blockade alone will not be an effective anti-migraine strategy. Further data will be required to assess whether neurokinin-1 antagonists will have any more general value in pain.

Stimulation of the greater occipital nerve increases metabolic activity in the trigeminal nucleus caudalis and cervical dorsal horn of the cat.

Patients with primary headache syndromes often describe a distribution of pain that involves both frontal and occipital parts of the head. Such a distribution of pain does not respect the cutaneous sensory innervation of the head which would divide it into anterior (trigeminally innervated) and posterior (spinal nerve root innervated) regions. Studies of pain-producing intracranial structures, such as the superior sagittal sinus, have demonstrated that second order neurons as caudal as C2 are activated after either electrical or mechanical stimulation. For this study cats were anaesthetised with halothane (during surgery) and alpha-chloralose (60 mg/kg, i.p., then 20 mg/kg intravenous maintenance), paralysed (gallamine 6 mg/kg) and ventilated. The greater occipital nerve was isolated bilaterally and stimulated unilaterally using hook electrodes with stimuli of 100 V at 0.3 Hz. Metabolic activity in the caudal brain stem and upper cervical cord was measured using 2-deoxyglucose autoradiography and quantitative densitometry. Stimulation of the greater occipital nerve increased metabolic activity by 220% ipsilateral to stimulation and by a lesser amount contralaterally. Increases in metabolic activity were seen in the dorsal horn at the level of C1 and C2 as might be predicted from the cervical origin of the nerve. Neuronal activation appeared contiguous with the trigeminal nucleus caudalis and was in the same distribution as is seen when trigeminally-innervated structures are stimulated. These data suggest that the well recognised clinical phenomenon of pain at the front and back of the head and in the upper neck are likely to be a consequence of overlap of processing of nociceptive information at the level of the second order neurons.

Direct evidence for central sites of action of zolmitriptan (311C90): an autoradiographic study in cat.

The trigeminovascular system consists of bipolar neurons which innervate pain-sensitive intracranial structures and projecting to neurons in the superficial laminae of the caudal trigeminal nucleus and of the dorsal horns of C1 and C2. The serotonin (5HT1B/D) agonist zolmitriptan (311C90) has been shown to be effective in the treatment of acute attacks of migraine and experimental data suggest that it may have both peripheral and central sites of action. This study sought to further investigate possible central effects of zolmitriptan (311C90) by examining its distribution in the central nervous system. Specific binding of [3H]-zolmitriptan was determined both ex vivo and in vitro in the cat brain. For the ex vivo studies, cats were anaesthetized with halothane and alpha-chloralose (60 mg/kg intraperitoneal). A femoral vein catheter was inserted for injection of the [3H]-zolmitriptan and then 1 h after injection the brain removed. For the in vitro studies fresh frozen brain slices were incubated with labelled and masking concentrations of zolmitriptan. The distribution of [3H]-zolmitriptan was determined using quantitative autoradiographic methods. The in vitro work demonstrated specific binding of [3H]-zolmitriptan in the superficial laminae of the trigeminal nucleus caudalis and dorsal horns of the C1 and C2 cervical spinal cord. The density of binding was 53 +/- 9 fmol/mg for the trigeminal nucleus caudalis, 47 +/- 7 fmol/mg for C1 and 50 +/- 6 fmol/mg for C2. The ex vivo work demonstrated binding in anatomically identical areas which was less dense than that seen with the in vitro method. These data confirm the existence of a population of receptors that specifically bind zolmitriptan following systemic administration. These receptors may, in part, be responsible for its clinical efficacy and reinforce the importance of central trigeminal neurons as a possible site of action of anti-migraine drugs.

Stimulation of an intracranial trigeminally-innervated structure selectively increases cerebral blood flow.

The cranial circulation, both extracerebral and cerebral, is innervated by fibers from the trigeminal nerve. This system is known as the trigeminovascular system. The large venous sinuses and dura mater are pain-sensitive and are innervated primarily by branches of the ophthalmic division of the trigeminal nerve. Studies were conducted in the alpha-chloralose anaesthetised cat to examine bulk carotid and cerebral blood flow responses to electrical stimulation of the trigeminal ganglion and superior sagittal sinus. Bulk carotid blood flow was measured using an ultrasonic flow probe and meter applied to the common carotid artery while cerebral blood flow was measured using laser Doppler flowmetry. Vascular resistance was calculated using simultaneously collected blood pressure data. Stimulation of the trigeminal ganglion resulted in a frequency-dependent reduction in both bulk carotid and cerebral vascular resistance. The mean maximal reduction was 39 +/- 5% at 20/s for the carotid bed and 37 +/- 6% at 20/s for the cerebral circulation. Stimulation of the superior sagittal sinus resulted in a frequency-dependent reduction in resistance that involved the cerebral circulation with little effect on bulk carotid resistance. The mean maximum reduction was 37 +/- 6% at 20/s for the cerebral circulation and 11 +/- 3% at 2/s for bulk carotid resistance. The more focused effects of superior sagittal sinus suggest a highly organised somatotopic arrangement of the trigeminal innervation of the cranial circulation. Such a physiological schema fits the known anatomy as reflected by the differential peptidergic innervation from the trigeminovascular system to cranial vessels and may be important in understanding the pathophysiology of migraine, cluster headache and subarachnoid haemorrhage.