Bilateral Descending Hypothalamic Projections to the Spinal Trigeminal Nucleus Caudalis in Rats

Luccarini P (2013) Bilateral Descending Hypothalamic Projections to the Spinal Trigeminal Nucleus Caudalis in Rats. PLoS ONE 8(8): e73022. doi:10.1371/journal.pone.0073022 Bilateral Descending Hypothalamic Projections to the Spinal Trigeminal Nucleus Caudalis in Rats Khaled Abdallah 0 Alain Artola 0 Lnaic Monconduit 0 Radhouane Dallel 0 Philippe Luccarini (RD) 0 Izumi Sugihara, Tokyo Medical and Dental University, Japan 0 Clermont Universite, Universite d'Auvergne, NEURO-DOL: Trigeminal Pain and Migraine , BP 10448, Clermont-Ferrand; Inserm, U1107, Clermont-Ferrand , France Several lines of evidence suggest that the hypothalamus is involved in trigeminal pain processing. However, the organization of descending hypothalamic projections to the spinal trigeminal nucleus caudalis (Sp5C) remains poorly understood. Microinjections of the retrograde tracer, fluorogold (FG), into the Sp5C, in rats, reveal that five hypothalamic nuclei project to the Sp5C: the paraventricular nucleus, the lateral hypothalamic area, the perifornical hypothalamic area, the A11 nucleus and the retrochiasmatic area. Descending hypothalamic projections to the Sp5C are bilateral, except those from the paraventricular nucleus which exhibit a clear ipsilateral predominance. Moreover, the density of retrogradely FG-labeled neurons in the hypothalamus varies according to the dorso-ventral localization of the Sp5C injection site. There are much more labeled neurons after injections into the ventrolateral part of the Sp5C (where ophthalmic afferents project) than after injections into its dorsomedial or intermediate parts (where mandibular and maxillary afferents, respectively, project). These results demonstrate that the organization of descending hypothalamic projections to the spinal dorsal horn and Sp5C are different. Whereas the former are ipsilateral, the latter are bilateral. Moreover, hypothalamic projections to the Sp5C display somatotopy, suggesting that these projections are preferentially involved in the processing of meningeal and cutaneous inputs from the ophthalmic branch of the trigeminal nerve in rats. Therefore, our results suggest that the control of trigeminal and spinal dorsal horn processing of nociceptive information by hypothalamic neurons is different and raise the question of the role of bilateral, rather than unilateral, hypothalamic control. - Pain is a complex experience that involves sensorydiscriminative, cognitive-evaluative, and affective-emotional components. Transmission of nociceptive messages is thus modulated by different central nervous system networks according to the nature of the painful stimulus and behavioral state of the individual [1]. For instance, descending pathways from brainstem and hypothalamus are known to either inhibit or facilitate transmission of nociceptive information at the level of the spinal dorsal horn and the spinal trigeminal nucleus caudalis (Sp5C). The hypothalamus integrates multiple functions including endocrine and autonomic control, thermoregulation, sleep, appetite, emotional behavior and arousal, and governs the rhythmicity and timing of many body functions [2]. Evidence from neuroimaging studies in man suggest that hypothalamus is also a key player in nociceptive processing, particularly in trigeminal pain syndromes such as migraine [3] and trigeminal autonomic cephalalgias [4] including cluster headache [5,6]. This prompted the use of deep-brain stimulations to modulate this region in patients with refractory chronic cluster headache [710]. Animal studies, using electrophysiological recordings in rats [1113] and cats [14] or Fos expression as a histochemical marker of neuronal activity [1517], suggest that the hypothalamus is activated following trigeminal stimulation. It is widely accepted that trigeminal sensory information can reach the hypothalamus via multisynaptic pathways through the brainstem, thalamus and cortex. Recently, however, anatomical [1821] and electrophysiological [22] studies showed that a substantial number of Sp5C neurons directly send their axons to hypothalamic regions. The hypothalamus modulates the perception of trigeminal pain [23]. Stimulation or lesion of the A11 nucleus decrease or increase, respectively, dural stimulation-evoked responses of Sp5C neurons [24]. This raises the question as to whether hypothalamic areas directly project to the Sp5C. To address this issue, we have carried out an anatomical study in the hypothalamus: we microinjected the retrograde tracer, Fluorogold (FG), into Sp5C and looked for retrogradely FG-labeled neurons in hypothalamic nuclei. Materials and Methods Adult male Sprague Dawley rats were obtained from Charles River laboratories (France) and maintained in a light- and temperature controlled environment (lights on 19.007.00 h, 22 C) with food and water ad libitum. All efforts were made to minimize the number of animals used. The experiments followed the ethical guidelines of the International Association for the Study of Pain [25] and ethical guidelines of the directive 2010/63/UE of the European Parliament and of the Council on the protection of animals used for scientific purposes. Protocols applied in this study have been approved by the local animal experimentation committee: CEMEAA Comit dEthique en Matire dExprimentation Animale Auvergne (n CE 28-12). Fluorogold injection Animals (250-300 g) were anesthetized with chloral hydrate (400 mg/kg body weight, intraperitoneally (i.p.) and placed in a stereotaxic frame. After surgical removing of the atlantooccipital membrane, glass micropipettes (3040 m diameter tips) filled with a 2% solution of Fluorogold (hydroxystilbamidine, Molecular Probes, Reference H22845), diluted in 0.1 M cacodylic acid were positioned at 12.4 mm caudal to the obex and inserted into the Sp5C, as lateral on the right as possible (about 2.7 mm), according to Paxinos and Watson [26] with an angle of 80 to the horizontal plane at various depth to reach areas where ophtalmic, maxillar or mandibullar primary afferents terminate. The actual position of the iontophoretic injection was verified by recording the extracellular neuronal response to cutaneous mechanical stimulation (brush) of the corresponding dermatome: ophtalmic, maxillar or mandibullar. Once the micropipette was in place, direct positive current (5 A) was applied for 30 s every 30 s for 1520 min. The microelectrode was left in situ for a further 5 min before withdrawal from the brain. A single injection into the Sp5C was performed in each animal. Following a postoperative survival period of one week, animals were deeply anaesthetized with urethane (1.5 g/kg i.p) and perfused transcardially over a 15 min period with warm (37 C) heparinized saline (25 IU heparin/mL) followed by cold (10 C) phosphate-buffered solution (0.1 M, pH 7.6) containing 4% paraformaldehyde and 0.03% picric acid. The brain and first cervical segment (C1) were removed and then cryoprotected in a buffered (...truncated)