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Nociceptive stimulation activates locus coeruleus neurones projecting to the somatosensory thalamus in the rat
http://www.100md.com 《生理学报》 2005年第15期
     Nociceptive stimulation activates locus coeruleus neurones projecting to the somatosensory thalamus in the rat

    D. L Voisin, N Guy, M Chalus and R Dallel

    1 Inserm E216 Neurobiologie de la douleur trigeminale, Faculte de Chirurgie Dentaire, 11 boulevard Charles de Gaulle, 63000 Clermont-Ferrand, France; Universite Clermont1, 63000 Clermont-Ferrand, France

    Abstract

, http://www.100md.com     In the thalamus, noradrenergic output from the pontine nucleus locus coeruleus (LC) may actively shape the response properties of various sensory networks en route to the cortex. Little is known, however, about the involvement of ascending noradrenergic innervation of the somatosensory thalamus in the processing of nociceptive information. To address this question, we combined the study of Fos expression upon nociceptive tooth pulp stimulation in the anaesthetized rat, with the detection of retrogradely traced neurones from the somatosensory thalamus. Cell bodies labelled retrogradely from the left thalamus were observed on both sides of the LC, with an ipsilateral predominance (n = 8). Electrical stimulation of the right incisor pulp (n = 4) provoked a significantly stronger Fos expression (around twice) than sham surgery (n = 4), in both the ipsi- and contralateral LC. Significantly larger numbers of double labelled neurones were counted in the LC of tooth-pulp-stimulated animals (representing around 30% of retrogradely labelled cells in LC) than in the LC of sham animals. They were found bilaterally, but with a clear, significant, ipsilateral (i.e. left) predominance. The present data offer an anatomical framework to understand how the LC is involved in the sensory processing of nociceptive information in the thalamus. For the first time, it is shown that nociceptive stimulation activates LC neurones projecting to the somatosensory thalamus. This suggests a new role for LC in modulating nociception within the thalamus.
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    Introduction

    Pain is a subjective experience that is generated within the brain and has both exteroceptive and interoceptive roles (Craig, 2003). The exteroceptive function allows extracting information about events in the environment in order to execute behaviours, such as escape and avoidance, that protect the organism from external threats. The interoceptive function is accompanied by homeostatic behaviours and autonomic responses that promote recuperation and healing (Wall, 1979). Adaptive responses and behaviours to painful stimuli require that these are analysed in terms of nature, intensity, location and duration (Price et al. 2003). The main pathway for such sensory processing of pain goes from the central projections of dorsal horn nociceptive neurones to the thalamic ventroposterior complex, that send axons off to the somatosensory cortex (Price et al. 2003).
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    Pain, however, does not simply result from the passive transfer of nociceptive information from the periphery to pain centres. At all levels of processing, the nociceptive message is modulated, amplified or inhibited, according to the physiological, emotional and cognitive state of the subject and to all other sensory messages perceived in the environment (Price et al. 2003). It is well known that the thalamus, through which sensory information reaches the cerebral cortex, differentially relays this input depending on the state of arousal and attention (Price, 1995). These functions depend on input from the monoaminergic transmitter systems (Hurley et al. 2004). The noradrenaline pathway arising in the locus coeruleus (LC), a brainstem nucleus, is the best understood of these systems (Aston-Jones et al. 1995). In the thalamus, noradrenaline may actively shape the response properties of various sensory networks en route to the cortex (Berridge & Waterhouse, 2003; Hurley et al. 2004). Little is known, however, about the involvement of ascending noradrenergic innervation of the somatosensory thalamus (i.e. the ventroposterior complex) in pain processing. Although anatomical studies have provided evidence that the ventroposterior thalamic complex receives projections from the LC (Simpson et al. 1997; Simpson et al. 1999) and that these projections are organized in relationship with functionally related efferent targets (Simpson et al. 1997), whether LC neurones projecting to the somatosensory thalamus are activated by nociceptive stimuli remains currently unknown. To address this question, we combined the study of Fos expression upon nociceptive tooth pulp stimulation in the anaesthetized rat, with the detection of retrogradely traced neurones from the corresponding somatosensory thalamus, which comprises the ventroposteromedial (VPM) and posterior (Po) nuclei of the thalamus.
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    Methods

    Adult male Sprague-Dawley rats were obtained from Charles River (L'Abresle, France) and maintained in a light- and temperature-controlled environment (lights on 07.00–21.00, 22°C) with food and water freely available. 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 and the European Communities Council directive of 24 November 1986 (86/609/EEC).
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    Fluorogold injection and tooth pulp electrical stimulation

    Animals (280–300 g) were anaesthetized with chloral hydrate (7%, 400 mg (kg body wt)–1 i.p). A soon as the depth of the anaesthesia was judged adequate (lack of motor reaction, hair raising and changes in breathing frequency to the manual pinch of the distal hindpaw), they were placed in a stereotaxic frame where rectal temperature was maintained at 37°C by a thermostatically controlled electric blanket. If the depth of the anaesthesia was judged inadequate, additional doses of 40 mg (kg body wt)–1 were administered until an adequate anaesthesia was achieved. Through a craniotomy, a glass micropipette containing the retrograde fluorescent tracer Fluorogold dissolved at 2% in a cacodylate vehicle (Schmued & Heimer, 1990) was inserted stereotaxically so that its tip (30–40 μm diameter) was located within the left ventroposterior thalamic complex (AP +4.5 mm to +5.8 mm, ML 2 mm to 3.2 mm, P 5.2 mm to 6.8 mm according to (Paxinos & Watson, 1997). Electrophoretic application of Fluorogold was made by 10 s pulses of positive direct current (3–5 μA) applied every 20 s for a period of 10–15 min. The microelectrode was left in situ for a further 5 min before withdrawal from the brain. One single application was made in the thalamus per animal. Following surgery, rats were individually kept in home-made recovery chambers until they recovered fully. One week later, rats were anaesthetized deeply with urethane (1.3 g (kg body wt)–1 i.p). Twenty minutes after the injection, the depth of the anaesthesia was tested, and the pulp of the right inferior incisive tooth was exposed through a drill hole and electrically stimulated using bipolar wire electrodes (10 mA, 2 ms, 1 Hz, 10 min). Such intensities recruit nociceptive C-fibres consistently. Sham animals underwent tooth surgery and insertion of electrodes only. In order to perform the surgery and the electrical stimulation without motor pain reaction from the animal, the dose of urethane used in this work was higher than the one used in previous studies of Fos expression by nociceptive facial stimulation in the rat (see for instance Strassman & Vos, 1993). If the depth of the anaesthesia was judged inadequate, additional doses of 0.1 g (kg body wt)–1 were administered until an adequate anaesthesia was achieved. Urethane produced a stable, prolonged anaesthesia that continued for the duration of the survival period. Two hours later, rats were perfused transcardially with warm (37°C) heparinized saline (25 IU heparin ml–1) followed by cold (10°C) phosphate-buffered solution (0.1 M, pH 7.6) containing 4% paraformaldehyde and 0.03% picric acid for 15 min. Thalamus and brainstem were then removed and transferred in the same paraformaldehyde–picric acid solution containing 30% sucrose at 4°C and left overnight. Coronal sections were cut on a freezing microtome and collected in a 0.05 M Tris-buffered saline (TBS). Thalamic sections (40 μm thick) were collected in two sets. Sections of the first set were mounted on gelatin-coated slides, covered using Vectashield (Vector, Burlingame, CA, USA) and viewed using a fluorescent microscope to check the location of Fluorogold deposit. Sections of the second set were further processed for immunostaining. Only brainstem sections (30 μm thick) of animals in which the injection site was located in the thalamus were processed further. A few selected sections were mounted separately and slightly counterstained with neutral red to help delineat the limits of the anatomical structures.
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    Fluorogold and Fos immunocytochemistry

    Free-floating thalamus and brainstem sections were placed in 1% normal goat serum for 1 h before incubation at 4°C in a rabbit antibody directed against Fluorogold (1: 6000, Chemicon, Temecula, CA, USA) for 48 h. Sections were then washed in TBS and placed in biotinylated goat anti-rabbit antibody (1: 200, Jackson Immunoresearch, West Grove, PA, USA) for 2 h at room temperature followed by incubation in Vector avidin–biotin–peroxidase complex (Vector ABC, 90 min at room temperature). Immunoreactivity for Fluorogold was visualized in sections using 3,3'-diaminobenzidene tetrahydrochloride (DAB). Brainstem sections underwent free-floating sequential immunostaining using primary rabbit antibodies specific Fos (1: 1000, 48 h, Oncogene Science), and a secondary goat anti-rabbit antibody (1: 400). Immunoreactivity for Fos was visualized using nickel–DAB. Thalamus sections underwent immunostaining for Fluorogold only. All sections were rinsed in TBS and transferred to gelatinized slides before being covered using DPX. All immunolabels were diluted in TBS containing 0.25% bovine serum albumin and 0.3% Triton X-100. Specificity controls consisted of the omission of the primary antibody and incubation of sections in inappropriate secondary antibodies. In all these control experiments, no specific staining was evident.
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    Data analysis

    Computer-assisted bright-field images of injection sites and representative labelling were obtained using a CCD colour video camera (Sony DXC-950P) connected to a Nikon Optiphot-2 microscope. In Fig. 3, the photomicrograph at x 20 magnification was obtained through an Axiophot Zeiss microscope using a Leica DFC 300 FX colour camera. Images were exported to Adobe PhotoShop (v 5.5) to adjust brightness and contrast before adjusting the image scale.
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    A, digitized photomicrographs of a coronal section through the LC showing examples of double labelled neurones (arrows). B, schematic diagrams illustrating the rostro-caudal distribution of double-labelled neurones in the LC of a single animal. Numbers show the anteroposterior distance from the coronal plane passing through the interaural line.

    Each injection site was analysed using coronal sections processed with DAB. The delineation of the VPM and Po was identified using the Paxinos and Watson atlas (Paxinos & Watson, 1997). Representations of the sites of injection were grouped on standard drawings of the thalamus.
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    Retrogradely labelled cell bodies and Fos-like immunopositive nuclei were counted according to their location along five different rostrocaudal planes within the LC. The delineation of the LC was based upon the Paxinos and Watson atlas (Paxinos & Watson, 1997) and our own myeloarchitectural atlas. Brainstem sections were categorized according to their approximate rostrocaudal location from the interaural line (IA). Data are expressed as the sum of the total number of labelled cells counted in the structure from all five sections which were analysed (mean per rat ± S.D.). They were analysed using two-way ANOVAs followed by appropriate post hoc test. The level of significance was set at P < 0.05. For illustrations, representative examples of the distribution of retrogradely labelled cell bodies were grouped on standard drawings of the LC.
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    Results

    Location of injection sites and distribution of retrograde labelling in LC

    The present results are based on eight experiments (selected from 18 experiments), in which animals received an injection of Fluorogold that appeared as a dense core of intense fluorescence confined within the boundaries of the left VPM and Po. This was confirmed, using DAB, by the visualization of focal immunoreactivity for Fluorogold surrounded by strongly immunoreactive neurones in a halo of staining and more lightly stained neurones. The location and maximal dorso-lateral extent of sites are illustrated in Fig. 1A. Four animals were sham and the four other ones were electrically stimulated. In the experiments that were not selected, the location or the extent of the site of injection was outside the VPM–Po limits.
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    A, standard drawings of the thalamus showing the level at which tracer deposit was maximal in 8 animals (VPM, ventroposteromedial; Po, posterior nuclei of the thalamus). B, digitized photomicrographs of coronal sections through the LC showing examples of retrograde labelling following an injection of retrograde tracer into the ventroposterior thalamic complex. Approximate level is –0.9 mm from IA. C, schematic diagrams illustrating the rostro-caudal distribution of retrogradely labelled neurones in the LC of a single animal. In A and C, numbers show the anteroposterior distance from the coronal plane passing through the interaural line.
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    Retrogradely labelled cells were evident by the presence of a brown staining in the cell soma and main dendritic processes (Fig. 1B). They were found bilaterally, but with a significant, ipsilateral (i.e. left) predominance (Fig. 1C, Table 1). They were distributed throughout the rostrocaudal extent of the LC, but with a slightly caudal concentration (Fig. 1C). There was no difference in the numbers and distribution of retrogradely labelled neurones in the LC of sham and electrically stimulated animals (Table 1).
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    Fos expression in LC following tooth pulp stimulation

    Anaesthetized, sham animals as well as electrically stimulated rats displayed bilateral Fos expression in the LC (n = 4 per group, Table 1, Fig. 2A). The average counts of Fos-immunoreactive nuclei in electrically stimulated rats, however, were significantly larger (around twice) than counts in sham rats (Table 1). Fos-immunoreactive nuclei were distributed throughout the rostrocaudal extent of the LC, and equally both ispsilateral and contralateral to the stimulus (Fig. 2B, Table 1).
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    A, digitized photomicrographs of coronal sections through the LC showing examples of Fos immunoreactivity in the LC of 2 different animals. Approximate levels are –0.8 mm (top) and –1.0 mm (bottom) from IA. B, schematic diagrams illustrating the rostro-caudal distribution of Fos-like immunoreactive nuclei in the LC of a single stimulated animal. Numbers show the anteroposterior distance from the coronal plane passing through the interaural line.

    Distribution of double-labelled neurones in LC
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    Double labelled neurones were apparent as cells displaying both brown cytoplasmic (Fluorogold) and black nucleus (Fos) staining (Fig. 3A). While small numbers of double labelled neurones were found in the LC of sham animals (representing around 10% of all retrogradely labelled cells in LC), significantly larger numbers were counted in the LC of tooth-pulp-stimulated animals (representing around 30% of retrogradely labelled cells in LC; Table 1). They were found bilaterally, but with a clear, significant, ipsilateral (i.e. left) predominance (Fig. 3C, Table 1). They were also distributed with a caudal concentration (Fig. 1C).
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    Discussion

    The present data offer an anatomical framework to understand how the LC is involved in the sensory processing of nociceptive information in the thalamus. For the first time, it is shown that nociceptive stimulation activates LC neurones projecting to the somatosensory thalamus. This suggests that LC output may shape the responses of the sensory network processing nociceptive information in the relay nuclei of the thalamus.
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    First, immunocytochemical detection of the protein product (Fos) of the c-fos immediate early gene was used to study neuronal activation in the LC of urethane-anaesthetized rats following electrical tooth pulp stimulation. The electrical stimulation of the incisor pulp is a common experimental nociceptive stimulus. Indeed, the current intensity used in this work is known to produce intense nociceptive behaviour in freely moving rats (Dallel et al. 1989) and to induce Fos protein in the medullary dorsal horn and first segment of the spinal cord (Iwata et al. 1998). Since anaesthesia and incisor tooth movement are effective in evoking Fos expression in the LC (Magdalena et al. 2004), we were careful to select an appropriate control group to determine what exact event was directly responsible for provoking expression of the gene. The increased Fos expression following electrical stimulation could thus be due to the activation of nociceptive pulpal nerves by the electrical stimuli. Indeed, some authors believed it is possible, using bipolar stimulating electrodes, to stimulate pulpal nerve fibres only (Toda et al. 1981; Myslinski & Matthews, 1987). Others, however, believed that current spread cannot be avoided (Hayashi, 1980; Engstrand et al. 1983). Thus, increased Fos expression could also be due to activation of periodontal nerves by the same stimuli. However, non-nociceptive mechanical or proprioceptive peripheral stimulation appears to be ineffective in inducing central Fos expression (Bullitt, 1990). It is likely therefore that increased Fos expression following electrical stimulation in the LC was due to the activation of nociceptive fibres by the electrical stimuli.
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    Given the general agreement that increased Fos expression is a consequence of increased neuronal activity (Harris, 1998), the finding that electrical stimulation of the tooth pulp evoked increased neuronal activity in the LC is consistent with earlier results showing that this nucleus is an integrator of pain signals (Jones, 1991). Accordingly, previous studies have shown that a significant increased Fos immmunoreactivity is found in the LC after nociceptive stimulations of various kinds such as subcutaneous formalin (Baulmann et al. 2000), colorectal distension (Traub et al. 1996) or trigeminal stimulation with intracisternal capsaicin (Ter Horst et al. 2001). Furthermore, in the present work, Fos-immunoreactive nuclei were distributed bilaterally, with no lateral predominance. This is consistent with earlier reports showing that unilateral hindpaw inflammation (Palkovits et al. 1999; Tsuruoka et al. 2003) as well as peripheral mono-neuropathy (Mao et al. 1993) induce bilateral activation of the LC in the rat. Multiple pathways may be involved in LC activation by nociceptive stimuli (Van Bockstaele & Aston-Jones, 1995). Tract tracing and microphysiology studies have revealed, however, that major afferents to LC appear to be limited to two rostral medullar areas, the nucleus paragigantocellularis lateralis (LPGi) and the nucleus prepositus hypoglossi (Aston-Jones et al. 1995). It was demonstrated that nociceptive responses of LC neurones to foot-shock stimuli are mediated through the LPGi (see Aston-Jones et al. 1995). Input from tooth pulp to LC neurones as well involves multiple synapses, but their detailed description is not yet known (Igarashi et al. 1979). Inputs to the pericoerulear region may also supply nociceptive information to the LC through extranuclear LC dendrites (see Aston-Jones et al. 1995).
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    Second, a retrograde tracing procedure was used to study projections from the LC to the somatosensory thalamus. It is well established that the medial division of the VPM and the Po provide the main relays of ascending trigeminal somatosensory information to the somatic sensory cortex (Price, 1995). As described by Simpson before (Simpson et al. 1997), we show here that they receive bilateral input from both LC nuclei, with the majority of LC efferents originating in the ipsilateral LC nucleus and with a slightly caudal concentration. Since virtually all neurones located within the compact LC nucleus are noradrenergic (Aston-Jones et al. 1995), the LC appears to provide the VPM and Po with noradrenergic input. Accordingly, in their comprehensive study of the noradrenergic innervation of the thalamus, Lindvall et al. (1974) found that the ventroposterior complex receives moderate to dense noradrenergic innervation. More recently, Westlund et al. (1991) have confirmed the presence of innervation by noradrenergic fibres in the ventral posterolateral nucleus of the thalamus of the macaque monkey by electron microscopy. Boutons containing markers for noradrenaline were observed contacting dendrites and somata in this region. The origins of these projections were found to be primarily in the LC. In addition, an extensive galanin-immunoreactive fibre innervation exists in the rodent trigeminosensory thalamus, much of which originates from LC neurones (Lechner et al. 1993; Simpson et al. 1999). Noradrenaline and galanin thus may be cotransmitters and comodulators of neuronal function in the VPM and Po.
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    Third, using a combination of retrograde tracing with immunocytochemical labelling of the Fos protein, we show that trigeminal nociceptive input activates a subpopulation of LC neurones that project onto the corresponding somatosensory thalamus. This suggests a new role for LC in modulating nociception within the thalamus. The effects of LC output on nociceptive-evoked responses of VPM and Po may be different from the well-described inhibitory role of LC in pain modulation through its descending projections (Jones, 1991; Fields & Basbaum, 1999). Dual effects on nociception have already been reported, facilitatory and inhibitory, at the medial thalamus level, through 1 and 2 receptors, respectively (Zhang et al. 1997). However, the effects of LC output on nociceptive-evoked responses in VPM and Po still remain to be studied. One might speculate that they are consistent with previous demonstrations of noradrenaline modulatory actions on central neurones (see Berridge & Waterhouse, 2003 for review). These actions can display complex, non-monotonic dose–response relationships and may include suppression of firing, increase in signal/noise ratio, augmentation of stimulus-induced changes in firing rate as well as gating of responses to otherwise subthreshold synaptic inputs (Berridge & Waterhouse, 2003). They are cell-specific and crucially determined by the receptor identity and effector systems of the target neurone (Berridge & Waterhouse, 2003). As a general rule, the noradrenergic system appears to modulate stimulus coding in mammalian sensory networks in a way that is contingent on behavioural state (Hurley et al. 2004). For instance, with respect to the sensory processing of whisker information, Devilbiss & Waterhouse (2004) have recently shown that in the VPM, at a given level of LC output, noradrenaline produces heterogenous modulatory effects on excitatory and inhibitory components of neural responses to trigeminal input. Sensory signal processing is thus continually altered over the range of tonic LC discharge frequencies that occur in the waking animal. Such changes may account for LC-mediated shifts in sensory network performance across multiple stages of arousal and attention. With respect to nociceptive information processing, in the sensory thalamus, the LC output may also shape the neural responses to nociceptive inputs according to the vegetative and emotional state of the subject. Indeed, the LC is closely linked with autonomic circuitry in the central nervous system and the LC system may serve as the cognitive limb of a global rapid response system, whereby LC and autonomic systems are co-activated in parallel to yield rapid adaptive responses to urgent stimuli (Aston-Jones et al. 1995).
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    In summary, nociceptive activation of LC neurones projecting to the related sensory thalamus may provide a means whereby the sensory encoding of nociceptive information is contingent on the vegetative and emotional processes set in motion by the nociceptive signal itself (Price, 2000).

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