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Pharmacological and neuroanatomical evidence for the involvement of the anterior pretectal nucleus in the antinociception induced by stimulation of the dorsal raphe nucleus in rats
Pain 74 (1998) 171–179
Pharmacological and neuroanatomical evidence for the involvement of the anterior pretectal nucleus in the antinociception induced by stimulation of the dorsal raphe nucleus in rats Maria Luiza N. Mamede Rosa a, Marina A. Oliveira a, Rodrigo B. Valente b, Norberto C. Coimbra b, Wiliam A. Prado a ,* a
Department of Pharmacology, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, 14049-900, Ribeira˜o Preto, SP, Brazil b Laboratory of Neuroanatomy and Neuropsychobiology of the Department of Morphology, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, 14049-900, Ribeira˜o Preto, SP, Brazil Received 7 July 1997; received in revised form 4 September 1997; accepted 29 September 1997
Abstract Several studies have shown that the anterior pretectal nucleus (APtN) is involved in descending inhibitory pathways that control noxious inputs to the spinal cord and that it may participate in the normal physiological response to noxious stimulation. Among other brain regions known to send inputs to the APtN, the dorsal column nuclei (DCN), pedunculopontine tegmental nucleus (PPTg), deep mesencephalon (DpMe), and dorsal raphe nucleus (DRN) are structures also known to be involved in antinociception. In the present study, the effects of stimulating these structures on the latency of the tail withdrawal reflex from noxious heating of the skin (tail flick test) were examined in rats in which saline or hyperbaric lidocaine (5%) was previously microinjected into the APtN. Brief stimulation of the PPTg, DpMe or DRN, but not the DCN, strongly depressed the tail flick reflex. The antinociceptive effect of stimulating the DRN, but not the PPTg or DpMe was significantly reduced, but not abolished, by the prior administration of the local anaesthetic into the APtN. The antinociception induced by stimulation of the PPTg or DpMe, therefore, is unlikely to depend on connections between these structures and the APtN. Similar inhibition of the effect of stimulating the DRN was obtained from rats previously microinjected with naloxone (2.7 nmol) or methysergide (2 nmol) into the APtN. Strongly labelled cells were identified in the DRN following microinjection of the fluorescent tracer Fast Blue into the APtN. These results indicate that the APtN may participate as a relay station through which the DRN partly modulates spinal nociceptive messages. In addition, they also indicate that endogenous opioid and serotonin can participate as neuromodulators of the DRN-APtN connection. 1998 International Association for the Study of Pain. Published by Elsevier Science B.V. Keywords: Antinociception; Anterior pretectal nucleus; Dorsal raphe nucleus; Tail-flick test
1. Introduction There are a number of regions of the brain where electrical stimulation causes antinociception. In most cases this effect is at least partly due to the activation of descending pathways that act to inhibit spinal sensory neurones (Besson and Chaouch, 1987). Much attention has been given to the involvement of the mesencephalic periaqueductal gray matter (PAG) including the dorsal raphe nucleus (DRN), and
* Corresponding author. Tel.: +55 016 6333035, ext. 3038; fax: +55 016 6332301; e-mail: [email protected]
the medullar nuclei raphe magnus (NRM), gigantocellularis, pars a, and paragigantocellularis (NRPG) in such a pain-inhibitory system. However, interest in the involvement of other brain structures in pain-control mechanisms has grown significantly. The anterior pretectal nucleus (APtN) is a midbrain structure known to be involved with the descending pain-inhibitory system (Rees and Roberts, 1993). At this nucleus, brief electrical stimulation produces long-lasting antinociception in rodents as evaluated by the tail-flick (Prado and Roberts, 1985; Wilson et al., 1991), paw pressure and formalin (Wilson et al., 1991), and jawopening tests (Chiang et al., 1989). Also, autotomy produced in rats by peripheral nerve section occurs earlier in
0304-3959/98/$19.00 1998 International Association for the Study of Pain. Published by Elsevier Science B.V. PII S0304-3959 (97 )0 0175-9
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APtN-lesioned rats (Rees et al., 1995). In contrast to the effects from other midbrain and brain stem structures (Rees and Roberts, 1993), the antinociception evoked from the APtN occurred without apparent aversive effects (Prado and Roberts, 1985). Stimulation of the APtN inhibits deep multireceptive neurones, but not superficial high threshold cells in the spinal dorsal horn (Rees and Roberts, 1987), thus differing from the effectiveness of stimulating the PAG or NRM against both cell types (Fields et al., 1977; Duggan and Griersmith, 1979; Duggan and Morton, 1983). Electrophysiological studies indicate that the descending pathways from the APtN relay in the pontine parabrachial region and the ventrolateral medulla to reach the spinal cord via dorsolateral funiculus (DLF) (Rees and Roberts, 1987; Terenzi et al., 1991, 1992). The APtN can also participate in the normal physiological response to noxious stimulation. Noxious stimuli increase the metabolic activity of the APtN (Porro et al., 1991) whereas stimulation of this nucleus reduces the aversiveness of medial hypothalamic stimulation (Branda˜o et al., 1991). High intensity cutaneous stimuli in the noxious range exciting APtN neurones via DLF fibres have also been demonstrated (Rees and Roberts, 1989a). Finally, the antinociceptive effects of dorsal column stimulation seem to depend on the integrity of the APtN (Rees et al., 1995). These findings led to the hypothesis that the antinociceptive effects of dorsal column stimulation are mediated via an ascending pathway that relays somewhere in the brain to activate a descending inhibition via APtN (Rees and Roberts, 1993). Somatosensory inputs to the APtN originate from several structures (Rees and Roberts, 1993), and include the somatosensory and motor cortices, deep mesencephalon, pontine parabrachial region, inferior olive, trigeminal sensory nuclei, dorsal column nuclei and superficial layers of the spinal dorsal horn. Afferents from most of these structures to the APtN are uncrossed, but a group of cells from the gracile nucleus projects to the contralateral APtN (Yoshida et al., 1992). Acetylcholinesterase fibres of the dorsal tegmental pathway originating from the pedunculopontine and laterodorsal tegmental nuclei passing through, below and lateral to the inferior pole of the APtN have also been identified (Wilson, 1985). Ascending serotonergic fibres from the dorsal raphe nucleus to the APtN have been histochemically demonstrated (Aghajanian et al., 1973). Although serotonergic innervation has been shown in the pretectal region, specific tract-tracers did not identify projections from the DRN to the APtN (Foster et al., 1989a; Terenzi et al., 1995; Zagon et al., 1995). On the other hand, microinjection of serotonin or morphine into the APtN evokes antinociception in rats (Prado, 1989). The present study was therefore undertaken to examine whether electrical stimulation of dorsal column nuclei (DCN), pedunculopontine tegmental nucleus (PPTg), deep mesencephalic nucleus (DpMe) or dorsal raphe nucleus (DRN) evokes antinociception in rats involving at least in
part connections with the APtN. It is shown that among these structures, the DRN is the only site at which the effects of the electrical stimulation are significantly reduced by pharmacological manipulation of the APtN. In addition, the connection DRN-APtN was histochemically confirmed using the fluorescent tract-tracer Fast Blue.
2. Materials and methods 2.1. Subjects and surgery Male Wistar rats (140–160 g) were used in this study, and antinociception was assessed by analysis of changes in the latency for the tail-flick escape from noxious heating of the skin (tail flick test). Each animal was anaesthetised with thiopental sodium (40 mg/kg, i.p.) and prepared for microinjection of drugs into the APtN and electrical stimulation of the ipsilateral DCN, PPTg, DpMe or DRN. A 12-mm length of a 23-gauge stainless-steel guide cannula was stereotaxically implanted into the skull until its tip lay 4 mm above the APtN to allow microinjection into this nucleus. A monopolar teflon-insulated steel electrode (bare diameter tip of 0.125 mm) was also implanted stereotaxically to permit electrical stimulation of the contralateral DCN (gracile or cuneate), and the ipsilateral PPTg, DpMe or DRN. The guide cannula and electrode were fixed to the skull with two steel screws and dental cement. One of the screws was used as indifferent electrode during the stimulation procedures. In our experimental conditions the co-ordinates were AP +4.0; L 1.8; H 1.0 for APtN, AP −6.0; L 0.2; H −8.7 for the gracile nucleus, AP −5.2; L 1.6; H −8.3 for the cuneate nucleus, AP +0.6; L 1.0; H −7.5 for the PPTg, AP +2.0; L 1.8; H −7.1 for the DpMe, and AP 0; L 0.3; H −6.1 for the DRN, using the zero planes and incisor bar positions described by Paxinos and Watson (1986). All ventral co-ordinates referred to the skull surface. Each guide cannula was kept patent with a sterile obturator. After receiving penicillin (50 mg/kg, i.m.), the animal was allowed to recover for at least 1 week before the experiments. When indicated in Section 3, stimulation of the APtN was performed using a monopolar teflon-insulated electrode prepared according to Prado and Roberts (1985). The electrode was initially introduced into a dummy guide cannula and the wire was cut so as to project 4.0 mm from the guide cannula tip. The assembly was then introduced into the guide cannula implanted into the animal skull immediately before brain stimulation. After the end of the stimulation period the electrode was withdrawn to allow the introduction of the microinjection assembly into the guide cannula when necessary. 2.2. Tail flick test The rat was placed in a ventilated glass tube for periods of up to 20 s, with the tail laid across a nichrome wire coil
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maintained at room temperature (23 ± 2°C). The coil temperature was then raised by the passage of electric current, which was adjusted to ensure a tail withdrawal reflex within 2.5–3.5 s. A cut-off time of 6 s was established to minimise the probability of skin damage. Animals were tested every 5 min until a stable baseline tail-flick latency (BTFL) was obtained over three consecutive trials. Only rats showing stable BTFL after six trials were used in each experiment. After this procedure, microinjection of drug or saline was made into the APtN and tail-flick latency was measured at 5-min intervals for up to 20 min. Intracerebral electrical stimulation was applied and the algesimetric test was repeated again at 5-min intervals for up to 30 min. 2.3. Microinjection procedures Drugs or saline were microinjected into the APtN using a glass needle (70–80 mm o.d.) protected by a system of telescoping steel tubes as described elsewhere (Azami et al., 1980). The assembly was inserted into the guide cannula immediately before microinjection and the needle advanced to protrude 4 mm beyond the guide cannula tip. The volume of injection was 0.5 ml delivered at a constant rate over a period of 3 min, and the needle was removed 20 s after completion of this procedure. 2.4. Electrical stimulation Electrical stimulation (AC, 60 Hz) of 15-s duration and current intensity of 35 mA r.m.s. was applied to the brain structures of freely moving rats placed inside a glass-walled cage of 30 × 30 × 40 cm. At this intensity and duration, electrical stimulation of the APtN, PAG, NRM or NRPG has been shown to inhibit effectively the tail flick reflex in rats (Prado and Roberts, 1985). Each animal was stimulated on only one occasion. During the stimulation the voltage drop across a 100-Q resistor in series with the electrode was monitored on an oscilloscope and continually adjusted to 35 mA r.m.s. Neurological tests (flexion, grasping and righting reflexes and placing reactions) were performed before and after drug administration as well as after brain stimulation. 2.5. Histology At the end of the experiment Fast Green (0.5 ml) dye was microinjected to label the site of injection. The animal was then killed with an overdose of thiopental sodium and perfused through the heart with saline followed by buffered formalin-saline. Dye spots and electrode tract were localised from 50-mm serial coronal sections stained with Neutral Red, and identified on diagrams from the atlas of Paxinos and Watson (1986). Rats showing dye spots in the APtN and electrode tract tip inside the DCN, PPTg, DpMe or DRN were considered for further analysis. In the experiments with lidocaine and antagonists, however, rats showing dye
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spots near to the APtN were used to control the effects of drug diffusion. 2.6. Tracing study of the DRN-APtN connection Eight animals were anaesthetised with thiopental sodium (40 mg/kg, i.p.) and placed into a stereotaxic apparatus. Fast Blue tracer dye (75 nl of a 1% solution of Fast Blue in 0.01 M phosphate buffer, pH 7.4, containing 0.9% NaCl) was microinjected with a thin glass needle (o.d. 50 mm) into the APtN at the co-ordinates described in Section 2.1. The tracer solution was slowly and completely injected over a 5min period and left in place for a further 5-min period before being withdrawn. After 8 days of total survival time, the animals were anaesthetised with thiopental sodium (40 mg/kg, i.p.) and perfused through the heart. The blood was washed out with cold, oxygen enriched, Ca2+-free Tyrode buffer (40 ml at 4°C) followed by 200-ml ice-cold 4% (w/v) paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, for 15 min at a pressure of 50 mmHg. The brains were quickly removed and soaked for 4 h in fresh fixative at 4°C. After fixation, the brains were sectioned, disconnecting the mesencephalon and pons. The midbrains were rinsed in 10% and 20% sucrose dissolved in 0.1 M sodium phosphate buffer (pH 7.4) at 4°C for at least 12 h, in each solution. Tissue pieces were immersed in 2-methylbutane (Sigma), frozen on carbonic ice for 20 s, and cut on a cryostat in a mixture of 10.24 w/w polyvinyl alcohol and 4.26 w/w polyethylene glycol (Tissue Tek O.C.T.) at −25°C to bind tissue to the specimen block and to surround and cover the specimen. The slices were subsequently mounted with mineral oil and viewed with a fluorescence microscope (Axiophot, Zeiss) equipped with filter systems providing light of 360 hm wavelength. 2.7. Drugs Lidocaine chloride 5% (Xylocaine, hyperbaric solution) was purchased from Merrell Lepetit, Brazil, Fast Blue was from Sigma, and naloxone hydrochloride and methysergide maleate were from Research Biochemicals International (RBI), Natick, MA, USA. Except for lidocaine, which was not diluted, the remaining drugs were diluted in saline. 2.8. Statistics The effects of manipulating the APtN on the changes in tail flick latency produced by brain stimulation were analysed statistically by multivariate analysis of variance (MANOVA) with repeated measures to compare the group over all times. The factors analysed were treatments, time and treatment × time interactions. In the case of significant treatment × time interactions, unpaired Student’s ttest or one-way ANOVA followed by Duncan’s test was performed at each time. The analysis was performed with
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the statistical software package SPSS/PC+, version 3.0, and the level of significance was set at P , 0.05.
3. Results
lowed by immobility were observed during stimulation of the PPTg. Neurological deficits, however, were not noticed before or after stimulation procedures. The animals walked normally and responded to innocuous stimuli throughout the period of observation. Lidocaine, alone, had no significant effect on tail flick latency.
3.1. Stimulation of the APtN 3.4. Stimulation of the DpMe A group of four rats with guide cannula positioned to lie 4 mm above the APtN was used in this experiment. After BTFL determination, a removable monopolar electrode was introduced into the guide cannula and electrical stimulation was applied to the APtN. The tail flick latency was measured within 20 s after the end of the stimulation period and thereafter repeated at 10-min intervals during 60 min. All animals showed a strong and long-lasting increase in the tail flick latency which remained above the BTFL for up to 45 min, a result similar to that reported elsewhere (Prado and Roberts, 1985; Roberts and Rees, 1986). Two hours later, a new BTFL was determined and lidocaine (0.5 ml) was microinjected into the APtN, the microinjection needle was removed, and the monopolar electrode reinserted into the guide cannula. Ten minutes after the microinjection, electrical stimulation was again applied to the APtN and the tail flick latency measured within 20 s and then at 10min intervals during 30 min. Five minutes later (i.e., 45 min after lidocaine), the stimulation of the APtN was repeated and the tail-flick latency measured again within 20 s. No animal displayed tail flick latency significantly different from the BTFL in all measures conducted after lidocaine (not shown in figures). These results are indicative that the inhibitory effect of microinjecting lidocaine into the APtN lasts at least 45 min. We decided, therefore, to use this property of lidocaine to control the effectiveness of the local anaesthetic in the further experiments.
Electrical stimulation of the DpMe in animals previously microinjected with saline (control) or lidocaine into the ipsilateral APtN produced an immediate and short-lasting increase in tail flick latency (Fig. 2). The effect obtained for control rats was longer-lasting but the curves did not differ significantly regarding treatments (F1,8 = 1.90; P = 0.205) or had significant treatment × time interactions (F11,88 = 0.75; P = 0.69). Stimulation of the APtN conducted 45 min after the local anaesthetic produced tail flick latency not different from the BTFL in all animals of the test group (not shown in figures). Slow circling behaviour was displayed by some rats during DpMe stimulation. In a few rats this motor reaction was very strong. However, no motor change or other neurological deficit was observed throughout the remaining period of observation. 3.5. Stimulation of the DRN Electrical stimulation of the DRN in animals previously
3.2. Stimulation of the DCN Electrical stimulation of the gracile nucleus or cuneate nucleus did not significantly change the tail-flick latency (not shown in the figures). Thus, no further studies on the participation of the DCN-APtN in the pain control mechanism were conducted. 3.3. Stimulation of the PPTg Electrical stimulation of the PPTg in animals previously microinjected with saline (control) or lidocaine into the ipsilateral APtN produced immediate and long-lasting increase in tail flick latency (Fig. 1). The curves obtained did not differ significantly regarding treatments (F1,7 = 0.20; P = 0.67) or had significant treatment × time interactions (F11,77 = 0.23; P = 0.99). Stimulation of the APtN conducted 45 min after the local anaesthetic produced tail flick latency not different from the BTFL in all animals of the test group (not shown in figures). Motor reactions fol-
Fig. 1. Effect of 0.5 ml saline or hyperbaric lidocaine (5%) microinjected into the anterior pretectal nucleus (arrow 1) on the antinociception induced by stimulating the pedunculopontine tegmental nucleus of the rat (arrow 2). Points are mean (±SEM). Inset shows the anatomical location of the sites of microinjection or electrical stimulation of control and test groups at the AP levels indicated. The sections are taken from Paxinos and Watson (1986). APtN, anterior pretectal nucleus; PAG, periaqueductal gray; PPTg, pedunculopontine tegmental nucleus.
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microinjected with saline (control) or lidocaine into the ipsilateral APtN evoked an immediate increase in tail-flick latency (Fig. 3). Previous microinjection of naloxone (2.7 nmol) or methysergide (2 nmol) into the ipsilateral APtN also significantly reduced the antinociceptive effect of stimulating the DRN (Figs. 4 and 5, respectively). Since only one control group was used for the experiments stimulating the DRN, the curves in Figs. 3, 4 and 5 were statistically analysed altogether. The MANOVA revealed that these curves differ significantly regarding treatments (F3,22 = 3.31; P = 0.039) and had significant treatment × time interactions (F33,242 = 3.98; P = 0.001). ANOVA followed by Duncan’s test indicated that lidocaine-treated animals were significantly different from control at times 0–15 min. The naloxone-treated animals were significantly different from the control at times 0–15, and 25 min. Differences between control- and methysergide-treated animals were found at times 0–20 min. Stimulation of the APtN conducted 45 min after the local anaesthetic produced tail flick latency not different from the BTFL in all animals of the test group (not shown in figures). No motor changes or signs of aversiveness were observed during DRN stimulation. Also, no neurological deficit was detected throughout the period of observation. Neither lidocaine, naloxone nor methysergide, alone, had a significant effect on the tail flick latency. Fourteen rats were stimulated in the DRN but lidocaine, naloxone or methysergide were not microinjected in the APtN. In these cases, the dye spots were found in sites localised lateral, medial, below or above the APtN at dis-
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Fig. 3. Effect of 0.5 ml saline or hyperbaric lidocaine (5%) microinjected into the anterior pretectal nucleus (arrow 1) on the antinociception induced by stimulation of the dorsal raphe nucleus of the rat (arrow 2). Points are mean (±SEM). *Significantly different from saline-treated rats (P , 0.05). Inset as in Fig. 1. APtN, anterior pretectal nucleus; DRN, dorsal raphe nucleus.
tances of less than 0.5 mm from the APtN borders. The effects of stimulating the DRN in these rats did not differ from the control rats (not shown in figures), thus indicating that the effects of lidocaine or antagonists in the test groups were restricted to the APtN. 3.6. DRN-APtN connection In all animal utilised in this experiment, the microinjection sites were situated in the APtN. Eight days after the central microinjection of the fluorescent neurotracer, deeply labelled cells were identified in the DRN (Fig. 6). Some of these cells showed characteristics of neurones, such as short dendritic ramification and major prolongations suggesting axonal projections. Fluorescent glial nuclei were not present around the retrogradely labelled neurones. Fig. 6A shows deeply labelled neurones, whose details can be better analysed in Fig. 6B.
4. Discussion
Fig. 2. Effect of 0.5 ml saline or hyperbaric lidocaine (5%) microinjected into the anterior pretectal nucleus (arrow 1) on the antinociception induced by stimulation of the deep mesencephalic nucleus of the rat (arrow 2). Points are mean (±SEM). Inset as in Fig. 1. APtN, anterior pretectal nucleus; DpMe, deep mesencephalic nucleus; PAG, periaqueductal gray; RN, red nucleus.
The present study demonstrates that brief electrical stimulation of the PPTg, DpMe or DRN evokes antinociception in awake rats, as revealed by the increase of the tail flick latency to noxious heating of the skin. In addition we showed that administration of a local anaesthetic into the APtN significantly inhibited the antinociceptive effects of DRN stimulation but not the antinociceptive effects of PPTg
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Fig. 4. Effect of saline (0.5 ml) or naloxone (2.7 nmol/0.5 ml) microinjected into the anterior pretectal nucleus (arrow 1) on the antinociception induced by stimulation of the dorsal raphe nucleus of the rat (arrow 2). Points are mean (±SEM). *Significantly different from saline-treated rats (P , 0.05). Inset shows the anatomical location of the sites of microinjection or electrical stimulation of naloxone-treated animals at the AP levels indicated. Abbreviations as in Fig. 1.
or DpMe stimulation. Stimulation of the APtN conducted 45 min after the local anaesthetic produced tail flick latency not different from the BTFL. Thus, the failure of lidocaine microinjected into the APtN to attenuate the stimulationproduced antinociception from the PPTg or DpMe is not due to incomplete or absent effect of the local anaesthetic. Electrical stimulation of the DCN, gracile or cuneate, did not produce significant changes in tail flick latency. The tail-flick escape from noxious heat is a spinal reflex (Mayer and Liebeskind, 1974) and its inhibition by intracerebral stimulation is indicative that the stimulation somehow depresses spinal mechanisms. In the present study the stimulated animals showed no grossly abnormal behaviour after the stimulation. They walked normally and responded normally to innocuous stimuli. Motor reactions followed by immobility were observed during the stimulation of the PPTg and circling behaviour was observed during the stimulation of the DpMe. The antinociceptive effectiveness of stimulating the DpMe, APtN or ventral PAG (including the DRN) accompanied by no clear change in the animal performance of the animals in the rota-rod paradigm have already been demonstrated (Roberts and Rees, 1986). Therefore, motor deficit is unlike to be the reason for the inhibition of the tail flick reflex induced by stimulation of DpMe or DRN. The DCN, PPTg and DpMe are brain structures known to send inputs to the APtN and, therefore, may act as relay stations in ascending pathways to excite cells in this nucleus
(see Section 1). Antinociception following bilateral stimulation of the DCN in awake rats has been demonstrated in both the tail immersion and formalin tests (Saade´ et al., 1986). In that study, however, a much longer (10 min) and stronger stimulus (100 Hz, 0.05–0.5 mA) had to be used to show the effect of stimulating the DCN. Thirtysecond stimulation of the DCN in anaesthetised rats at intensities that only activate large-diameter afferents (50 Hz, 0.2 V) potently excites cells in the ipsilateral APtN and inhibits multireceptive spinal cell responses to cutaneous noxious stimuli (Rees and Roberts, 1989b). The different patterns of stimulation and methodologies may be the reason for the ineffectiveness of stimulating the DCN in our experiments. Therefore, the involvement of the DCN as a relay station in an ascending nociceptive pathway to excite cells in the APtN cannot be ruled out by the present study. The participation of the PPTg in descending pain control mechanisms has been proposed. Microinjection of carbachol (Brodie and Proudfit, 1984; Katayama et al., 1984) or nicotine (Iwamoto, 1989) into this nucleus produces strong antinociceptive effects in rats and cats. According to Iwamoto (1991), there is a tonically active cholinergic pathway that originates in the PPTg and terminates in the NRM and modulates nociception by activating descending pain inhibitory systems relaying within the NRM. The stimulationproduced antinociception from the PPTg shown here, therefore, may depend on the activation of a central pathway that probably does not involve the participation of the APtN.
Fig. 5. Effect of saline (0.5 ml) or methysergide (2 nmol/0.5 ml) microinjected into the anterior pretectal nucleus (arrow 1) on the antinociception induced by stimulation of the dorsal raphe nucleus of the rat (arrow 2). Points are mean (±SEM). *Significantly different from saline-treated rats (P , 0.05). Inset shows the anatomical location of the sites of microinjection or electrical stimulation of methysergide-treated animals at the AP levels indicated. Abbreviations as in Fig.1.
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The stimulation-produced antinociception from the DpMe has been demonstrated (Prado et al., 1984; Wang et al., 1992). Anatomical studies have shown a reciprocal connection between the DpMe and APtN (Foster et al., 1989a). Microinjection of GABA into the DpMe reduces the antinociceptive effects of stimulating the APtN, thus indicating that the DpMe can also act as a relay for the descending inhibitory effects of APtN stimulation (Wang et al., 1992).
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Microinjection of tetracaine into the APtN blocked the antinociceptive effects of stimulating the DpMe in anaesthetised rats, but the antinociceptive effects of stimulating the APtN were not changed by prior administration of tetracaine into the DpMe (Foster et al., 1989b). The utilisation of anaesthetised rats in the study of Foster et al. (1989b) may account in part for the differences. The present demonstration that microinjection of lidocaine into the APtN does not
Fig. 6. Photomicrographs of the coronal sections of the rat dorsal raphe nucleus after microinjection of neuronal tracer into the APtN. Note the cells labelled by the fluorescent tracer, with cytoplasm prolongations characteristic of neuronal cells. Magnification bar: 42 mm in (A) and 25 mm in (B).
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change the antinociceptive effects of stimulating the DpMe is indicative that the APtN is not involved in the antinociceptive mechanism activated by the DpMe. The DRN is another brain structure widely accepted as playing an important role in pain modulation (Wang and Nakai, 1994). In addition to the ventrolateral part of the PAG, the DRN is the only midbrain region where electrical stimulation causes antinociceptive effects accompanied by no aversive behavioural side-effects (Fardin et al., 1984), a property that was confirmed by the present results. The literature has provided evidence for the participation of the DRN in a descending pathway that acts to inhibit nociceptive cells in the spinal cord (Griffith and Gatipon, 1981; Yu et al., 1988). This nucleus projects few direct fibres to the spinal cord and many fibres to the NRM, which is a very important relay station in the descending pain-control system (Wang and Nakai, 1994). Endogenous opioids and 5hydroxytryptamine (5-HT) seem to be the neurotransmitters involved in the modulation of the DRN-NRM-spinal cord pathway. However, there are reports suggesting that, besides a pain-control function, the DRN apparently has a pain-perception function. Electrical activity of DRN cells is changed when noxious stimuli are applied peripherally (Sanders et al., 1980). Moreover, metabolic activity (Wang et al., 1988; Porro et al., 1991) and the c-fos expression (Daı´ et al., 1992) within the DRN are both increased by noxious stimulation. Finally, lesion of the NRM partially reverses the antinociceptive effect of DRN stimulation (Yu et al., 1988). It was then hypothesised that inhibition of reflexes to noxious stimulation by DRN stimulation might be partly achieved by a control of spinal dorsal horn cells via a descending pathway relaying in the NRM and partly by direct modulation of the DRN by ascending nociceptive messages (Besson and Chaouch, 1987; Wang and Nakai, 1994). Evidence for ascending pain-modulation projections from the DRN have appeared more recently in the literature. The electrical stimulation of, or microinjection of morphine into the DRN inhibits the response of cells to noxious stimulation in several brain structures, including the parafascicular nucleus of the thalamus, the nucleus ventralis posterolateralis of the thalamus, and the substantia nigra (Wang and Nakai, 1994). On the other hand, tracer studies on the efferent from the DRN (Wang and Nakai, 1994) or on the afferents to the APtN (Rees and Roberts, 1993) have shown no connections between these structures. The only available evidence came from a report by Aghajanian et al. (1973), who found ascending serotonergic fibres from the DRN projecting to the APtN. In this study we have confirmed that the APtN receives ascending projections from the DRN. After injection of the fluorescent neurotracer Fast Blue into the APtN, a dense collection of labelled cells was found in the DRN some of which showing characteristics of neurones, such as short dendritic ramification and major prolongations suggesting axonal projections. Fluorescent glial nuclei were not found around the retro-
gradely labelled neurones, thus indicating that migration of tracer due to its uptake by fibres of passage is unlikely to be the reason for the labelling of DRN neurones (Kuypers et al., 1980). We have further demonstrated that application of lidocaine into the APtN partially reverses the antinociceptive effects of stimulating the DRN. The efficacy of lidocaine for the production of functional neural block has already been documented (Aimone et al., 1988). According to Sandku¨hler and Gebhart (1984), lidocaine (0.5 ml, 4%) produced a functional neural block having a radius of approximately 0.5 mm. The antinociception induced by stimulating the DRN was not changed when lidocaine was microinjected into areas less than 0.5 mm from the APtN. Thus, the effect of the local anaesthetic seems to be restricted to the APtN. The result is, therefore, indicative that the APtN may be another relay through which the DRN partly modulates spinal nociceptive messages. This alternative is reinforced by the demonstration that prior microinjection of methysergide or naloxone into the APtN also partially reduces the antinociceptive effects of stimulating the DRN. The effects of the antagonists also seem to be restricted to the APtN since the DRN stimulation-induced antinociception was not changed by microinjecting the antagonists into areas less than 0.5 mm from the APtN. The microinjection of morphine or 5-HT (Prado, 1989), and m- or 5-HT1B agonists (Rosa and Prado, 1997) into the APtN has earlier been shown to mimic the antinociceptive effects of stimulating this nucleus electrically. Altogether, these results confirm that APtN integrity is at least in part necessary for the antinociceptive effects of stimulating the DRN. In addition, they indicate that at least endogenous opioids and 5-HT can participate as neuromodulators in the DRN-APtN connection.
Acknowledgements This work was supported by FAPESP. M.L.N.M.R. was the recipient of CAPES and CNPq fellowships. R.B.V. was the recipient of a CNPq fellowship. We thank Mr. P.R. Castania for technical assistance.
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Pharmacological and neuroanatomical evidence for the involvement of the anterior pretectal nucleus in the antinociception induced by stimulation of the dorsal raphe nucleus in rats Maria Luiza N. Mamede Rosa a, Marina A. Oliveira a, Rodrigo B. Valente b, Norberto C. Coimbra b, Wiliam A. Prado a ,* a
Department of Pharmacology, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, 14049-900, Ribeira˜o Preto, SP, Brazil b Laboratory of Neuroanatomy and Neuropsychobiology of the Department of Morphology, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, 14049-900, Ribeira˜o Preto, SP, Brazil Received 7 July 1997; received in revised form 4 September 1997; accepted 29 September 1997
Abstract Several studies have shown that the anterior pretectal nucleus (APtN) is involved in descending inhibitory pathways that control noxious inputs to the spinal cord and that it may participate in the normal physiological response to noxious stimulation. Among other brain regions known to send inputs to the APtN, the dorsal column nuclei (DCN), pedunculopontine tegmental nucleus (PPTg), deep mesencephalon (DpMe), and dorsal raphe nucleus (DRN) are structures also known to be involved in antinociception. In the present study, the effects of stimulating these structures on the latency of the tail withdrawal reflex from noxious heating of the skin (tail flick test) were examined in rats in which saline or hyperbaric lidocaine (5%) was previously microinjected into the APtN. Brief stimulation of the PPTg, DpMe or DRN, but not the DCN, strongly depressed the tail flick reflex. The antinociceptive effect of stimulating the DRN, but not the PPTg or DpMe was significantly reduced, but not abolished, by the prior administration of the local anaesthetic into the APtN. The antinociception induced by stimulation of the PPTg or DpMe, therefore, is unlikely to depend on connections between these structures and the APtN. Similar inhibition of the effect of stimulating the DRN was obtained from rats previously microinjected with naloxone (2.7 nmol) or methysergide (2 nmol) into the APtN. Strongly labelled cells were identified in the DRN following microinjection of the fluorescent tracer Fast Blue into the APtN. These results indicate that the APtN may participate as a relay station through which the DRN partly modulates spinal nociceptive messages. In addition, they also indicate that endogenous opioid and serotonin can participate as neuromodulators of the DRN-APtN connection. 1998 International Association for the Study of Pain. Published by Elsevier Science B.V. Keywords: Antinociception; Anterior pretectal nucleus; Dorsal raphe nucleus; Tail-flick test
1. Introduction There are a number of regions of the brain where electrical stimulation causes antinociception. In most cases this effect is at least partly due to the activation of descending pathways that act to inhibit spinal sensory neurones (Besson and Chaouch, 1987). Much attention has been given to the involvement of the mesencephalic periaqueductal gray matter (PAG) including the dorsal raphe nucleus (DRN), and
* Corresponding author. Tel.: +55 016 6333035, ext. 3038; fax: +55 016 6332301; e-mail: [email protected]
the medullar nuclei raphe magnus (NRM), gigantocellularis, pars a, and paragigantocellularis (NRPG) in such a pain-inhibitory system. However, interest in the involvement of other brain structures in pain-control mechanisms has grown significantly. The anterior pretectal nucleus (APtN) is a midbrain structure known to be involved with the descending pain-inhibitory system (Rees and Roberts, 1993). At this nucleus, brief electrical stimulation produces long-lasting antinociception in rodents as evaluated by the tail-flick (Prado and Roberts, 1985; Wilson et al., 1991), paw pressure and formalin (Wilson et al., 1991), and jawopening tests (Chiang et al., 1989). Also, autotomy produced in rats by peripheral nerve section occurs earlier in
0304-3959/98/$19.00 1998 International Association for the Study of Pain. Published by Elsevier Science B.V. PII S0304-3959 (97 )0 0175-9
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APtN-lesioned rats (Rees et al., 1995). In contrast to the effects from other midbrain and brain stem structures (Rees and Roberts, 1993), the antinociception evoked from the APtN occurred without apparent aversive effects (Prado and Roberts, 1985). Stimulation of the APtN inhibits deep multireceptive neurones, but not superficial high threshold cells in the spinal dorsal horn (Rees and Roberts, 1987), thus differing from the effectiveness of stimulating the PAG or NRM against both cell types (Fields et al., 1977; Duggan and Griersmith, 1979; Duggan and Morton, 1983). Electrophysiological studies indicate that the descending pathways from the APtN relay in the pontine parabrachial region and the ventrolateral medulla to reach the spinal cord via dorsolateral funiculus (DLF) (Rees and Roberts, 1987; Terenzi et al., 1991, 1992). The APtN can also participate in the normal physiological response to noxious stimulation. Noxious stimuli increase the metabolic activity of the APtN (Porro et al., 1991) whereas stimulation of this nucleus reduces the aversiveness of medial hypothalamic stimulation (Branda˜o et al., 1991). High intensity cutaneous stimuli in the noxious range exciting APtN neurones via DLF fibres have also been demonstrated (Rees and Roberts, 1989a). Finally, the antinociceptive effects of dorsal column stimulation seem to depend on the integrity of the APtN (Rees et al., 1995). These findings led to the hypothesis that the antinociceptive effects of dorsal column stimulation are mediated via an ascending pathway that relays somewhere in the brain to activate a descending inhibition via APtN (Rees and Roberts, 1993). Somatosensory inputs to the APtN originate from several structures (Rees and Roberts, 1993), and include the somatosensory and motor cortices, deep mesencephalon, pontine parabrachial region, inferior olive, trigeminal sensory nuclei, dorsal column nuclei and superficial layers of the spinal dorsal horn. Afferents from most of these structures to the APtN are uncrossed, but a group of cells from the gracile nucleus projects to the contralateral APtN (Yoshida et al., 1992). Acetylcholinesterase fibres of the dorsal tegmental pathway originating from the pedunculopontine and laterodorsal tegmental nuclei passing through, below and lateral to the inferior pole of the APtN have also been identified (Wilson, 1985). Ascending serotonergic fibres from the dorsal raphe nucleus to the APtN have been histochemically demonstrated (Aghajanian et al., 1973). Although serotonergic innervation has been shown in the pretectal region, specific tract-tracers did not identify projections from the DRN to the APtN (Foster et al., 1989a; Terenzi et al., 1995; Zagon et al., 1995). On the other hand, microinjection of serotonin or morphine into the APtN evokes antinociception in rats (Prado, 1989). The present study was therefore undertaken to examine whether electrical stimulation of dorsal column nuclei (DCN), pedunculopontine tegmental nucleus (PPTg), deep mesencephalic nucleus (DpMe) or dorsal raphe nucleus (DRN) evokes antinociception in rats involving at least in
part connections with the APtN. It is shown that among these structures, the DRN is the only site at which the effects of the electrical stimulation are significantly reduced by pharmacological manipulation of the APtN. In addition, the connection DRN-APtN was histochemically confirmed using the fluorescent tract-tracer Fast Blue.
2. Materials and methods 2.1. Subjects and surgery Male Wistar rats (140–160 g) were used in this study, and antinociception was assessed by analysis of changes in the latency for the tail-flick escape from noxious heating of the skin (tail flick test). Each animal was anaesthetised with thiopental sodium (40 mg/kg, i.p.) and prepared for microinjection of drugs into the APtN and electrical stimulation of the ipsilateral DCN, PPTg, DpMe or DRN. A 12-mm length of a 23-gauge stainless-steel guide cannula was stereotaxically implanted into the skull until its tip lay 4 mm above the APtN to allow microinjection into this nucleus. A monopolar teflon-insulated steel electrode (bare diameter tip of 0.125 mm) was also implanted stereotaxically to permit electrical stimulation of the contralateral DCN (gracile or cuneate), and the ipsilateral PPTg, DpMe or DRN. The guide cannula and electrode were fixed to the skull with two steel screws and dental cement. One of the screws was used as indifferent electrode during the stimulation procedures. In our experimental conditions the co-ordinates were AP +4.0; L 1.8; H 1.0 for APtN, AP −6.0; L 0.2; H −8.7 for the gracile nucleus, AP −5.2; L 1.6; H −8.3 for the cuneate nucleus, AP +0.6; L 1.0; H −7.5 for the PPTg, AP +2.0; L 1.8; H −7.1 for the DpMe, and AP 0; L 0.3; H −6.1 for the DRN, using the zero planes and incisor bar positions described by Paxinos and Watson (1986). All ventral co-ordinates referred to the skull surface. Each guide cannula was kept patent with a sterile obturator. After receiving penicillin (50 mg/kg, i.m.), the animal was allowed to recover for at least 1 week before the experiments. When indicated in Section 3, stimulation of the APtN was performed using a monopolar teflon-insulated electrode prepared according to Prado and Roberts (1985). The electrode was initially introduced into a dummy guide cannula and the wire was cut so as to project 4.0 mm from the guide cannula tip. The assembly was then introduced into the guide cannula implanted into the animal skull immediately before brain stimulation. After the end of the stimulation period the electrode was withdrawn to allow the introduction of the microinjection assembly into the guide cannula when necessary. 2.2. Tail flick test The rat was placed in a ventilated glass tube for periods of up to 20 s, with the tail laid across a nichrome wire coil
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maintained at room temperature (23 ± 2°C). The coil temperature was then raised by the passage of electric current, which was adjusted to ensure a tail withdrawal reflex within 2.5–3.5 s. A cut-off time of 6 s was established to minimise the probability of skin damage. Animals were tested every 5 min until a stable baseline tail-flick latency (BTFL) was obtained over three consecutive trials. Only rats showing stable BTFL after six trials were used in each experiment. After this procedure, microinjection of drug or saline was made into the APtN and tail-flick latency was measured at 5-min intervals for up to 20 min. Intracerebral electrical stimulation was applied and the algesimetric test was repeated again at 5-min intervals for up to 30 min. 2.3. Microinjection procedures Drugs or saline were microinjected into the APtN using a glass needle (70–80 mm o.d.) protected by a system of telescoping steel tubes as described elsewhere (Azami et al., 1980). The assembly was inserted into the guide cannula immediately before microinjection and the needle advanced to protrude 4 mm beyond the guide cannula tip. The volume of injection was 0.5 ml delivered at a constant rate over a period of 3 min, and the needle was removed 20 s after completion of this procedure. 2.4. Electrical stimulation Electrical stimulation (AC, 60 Hz) of 15-s duration and current intensity of 35 mA r.m.s. was applied to the brain structures of freely moving rats placed inside a glass-walled cage of 30 × 30 × 40 cm. At this intensity and duration, electrical stimulation of the APtN, PAG, NRM or NRPG has been shown to inhibit effectively the tail flick reflex in rats (Prado and Roberts, 1985). Each animal was stimulated on only one occasion. During the stimulation the voltage drop across a 100-Q resistor in series with the electrode was monitored on an oscilloscope and continually adjusted to 35 mA r.m.s. Neurological tests (flexion, grasping and righting reflexes and placing reactions) were performed before and after drug administration as well as after brain stimulation. 2.5. Histology At the end of the experiment Fast Green (0.5 ml) dye was microinjected to label the site of injection. The animal was then killed with an overdose of thiopental sodium and perfused through the heart with saline followed by buffered formalin-saline. Dye spots and electrode tract were localised from 50-mm serial coronal sections stained with Neutral Red, and identified on diagrams from the atlas of Paxinos and Watson (1986). Rats showing dye spots in the APtN and electrode tract tip inside the DCN, PPTg, DpMe or DRN were considered for further analysis. In the experiments with lidocaine and antagonists, however, rats showing dye
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spots near to the APtN were used to control the effects of drug diffusion. 2.6. Tracing study of the DRN-APtN connection Eight animals were anaesthetised with thiopental sodium (40 mg/kg, i.p.) and placed into a stereotaxic apparatus. Fast Blue tracer dye (75 nl of a 1% solution of Fast Blue in 0.01 M phosphate buffer, pH 7.4, containing 0.9% NaCl) was microinjected with a thin glass needle (o.d. 50 mm) into the APtN at the co-ordinates described in Section 2.1. The tracer solution was slowly and completely injected over a 5min period and left in place for a further 5-min period before being withdrawn. After 8 days of total survival time, the animals were anaesthetised with thiopental sodium (40 mg/kg, i.p.) and perfused through the heart. The blood was washed out with cold, oxygen enriched, Ca2+-free Tyrode buffer (40 ml at 4°C) followed by 200-ml ice-cold 4% (w/v) paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, for 15 min at a pressure of 50 mmHg. The brains were quickly removed and soaked for 4 h in fresh fixative at 4°C. After fixation, the brains were sectioned, disconnecting the mesencephalon and pons. The midbrains were rinsed in 10% and 20% sucrose dissolved in 0.1 M sodium phosphate buffer (pH 7.4) at 4°C for at least 12 h, in each solution. Tissue pieces were immersed in 2-methylbutane (Sigma), frozen on carbonic ice for 20 s, and cut on a cryostat in a mixture of 10.24 w/w polyvinyl alcohol and 4.26 w/w polyethylene glycol (Tissue Tek O.C.T.) at −25°C to bind tissue to the specimen block and to surround and cover the specimen. The slices were subsequently mounted with mineral oil and viewed with a fluorescence microscope (Axiophot, Zeiss) equipped with filter systems providing light of 360 hm wavelength. 2.7. Drugs Lidocaine chloride 5% (Xylocaine, hyperbaric solution) was purchased from Merrell Lepetit, Brazil, Fast Blue was from Sigma, and naloxone hydrochloride and methysergide maleate were from Research Biochemicals International (RBI), Natick, MA, USA. Except for lidocaine, which was not diluted, the remaining drugs were diluted in saline. 2.8. Statistics The effects of manipulating the APtN on the changes in tail flick latency produced by brain stimulation were analysed statistically by multivariate analysis of variance (MANOVA) with repeated measures to compare the group over all times. The factors analysed were treatments, time and treatment × time interactions. In the case of significant treatment × time interactions, unpaired Student’s ttest or one-way ANOVA followed by Duncan’s test was performed at each time. The analysis was performed with
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the statistical software package SPSS/PC+, version 3.0, and the level of significance was set at P , 0.05.
3. Results
lowed by immobility were observed during stimulation of the PPTg. Neurological deficits, however, were not noticed before or after stimulation procedures. The animals walked normally and responded to innocuous stimuli throughout the period of observation. Lidocaine, alone, had no significant effect on tail flick latency.
3.1. Stimulation of the APtN 3.4. Stimulation of the DpMe A group of four rats with guide cannula positioned to lie 4 mm above the APtN was used in this experiment. After BTFL determination, a removable monopolar electrode was introduced into the guide cannula and electrical stimulation was applied to the APtN. The tail flick latency was measured within 20 s after the end of the stimulation period and thereafter repeated at 10-min intervals during 60 min. All animals showed a strong and long-lasting increase in the tail flick latency which remained above the BTFL for up to 45 min, a result similar to that reported elsewhere (Prado and Roberts, 1985; Roberts and Rees, 1986). Two hours later, a new BTFL was determined and lidocaine (0.5 ml) was microinjected into the APtN, the microinjection needle was removed, and the monopolar electrode reinserted into the guide cannula. Ten minutes after the microinjection, electrical stimulation was again applied to the APtN and the tail flick latency measured within 20 s and then at 10min intervals during 30 min. Five minutes later (i.e., 45 min after lidocaine), the stimulation of the APtN was repeated and the tail-flick latency measured again within 20 s. No animal displayed tail flick latency significantly different from the BTFL in all measures conducted after lidocaine (not shown in figures). These results are indicative that the inhibitory effect of microinjecting lidocaine into the APtN lasts at least 45 min. We decided, therefore, to use this property of lidocaine to control the effectiveness of the local anaesthetic in the further experiments.
Electrical stimulation of the DpMe in animals previously microinjected with saline (control) or lidocaine into the ipsilateral APtN produced an immediate and short-lasting increase in tail flick latency (Fig. 2). The effect obtained for control rats was longer-lasting but the curves did not differ significantly regarding treatments (F1,8 = 1.90; P = 0.205) or had significant treatment × time interactions (F11,88 = 0.75; P = 0.69). Stimulation of the APtN conducted 45 min after the local anaesthetic produced tail flick latency not different from the BTFL in all animals of the test group (not shown in figures). Slow circling behaviour was displayed by some rats during DpMe stimulation. In a few rats this motor reaction was very strong. However, no motor change or other neurological deficit was observed throughout the remaining period of observation. 3.5. Stimulation of the DRN Electrical stimulation of the DRN in animals previously
3.2. Stimulation of the DCN Electrical stimulation of the gracile nucleus or cuneate nucleus did not significantly change the tail-flick latency (not shown in the figures). Thus, no further studies on the participation of the DCN-APtN in the pain control mechanism were conducted. 3.3. Stimulation of the PPTg Electrical stimulation of the PPTg in animals previously microinjected with saline (control) or lidocaine into the ipsilateral APtN produced immediate and long-lasting increase in tail flick latency (Fig. 1). The curves obtained did not differ significantly regarding treatments (F1,7 = 0.20; P = 0.67) or had significant treatment × time interactions (F11,77 = 0.23; P = 0.99). Stimulation of the APtN conducted 45 min after the local anaesthetic produced tail flick latency not different from the BTFL in all animals of the test group (not shown in figures). Motor reactions fol-
Fig. 1. Effect of 0.5 ml saline or hyperbaric lidocaine (5%) microinjected into the anterior pretectal nucleus (arrow 1) on the antinociception induced by stimulating the pedunculopontine tegmental nucleus of the rat (arrow 2). Points are mean (±SEM). Inset shows the anatomical location of the sites of microinjection or electrical stimulation of control and test groups at the AP levels indicated. The sections are taken from Paxinos and Watson (1986). APtN, anterior pretectal nucleus; PAG, periaqueductal gray; PPTg, pedunculopontine tegmental nucleus.
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microinjected with saline (control) or lidocaine into the ipsilateral APtN evoked an immediate increase in tail-flick latency (Fig. 3). Previous microinjection of naloxone (2.7 nmol) or methysergide (2 nmol) into the ipsilateral APtN also significantly reduced the antinociceptive effect of stimulating the DRN (Figs. 4 and 5, respectively). Since only one control group was used for the experiments stimulating the DRN, the curves in Figs. 3, 4 and 5 were statistically analysed altogether. The MANOVA revealed that these curves differ significantly regarding treatments (F3,22 = 3.31; P = 0.039) and had significant treatment × time interactions (F33,242 = 3.98; P = 0.001). ANOVA followed by Duncan’s test indicated that lidocaine-treated animals were significantly different from control at times 0–15 min. The naloxone-treated animals were significantly different from the control at times 0–15, and 25 min. Differences between control- and methysergide-treated animals were found at times 0–20 min. Stimulation of the APtN conducted 45 min after the local anaesthetic produced tail flick latency not different from the BTFL in all animals of the test group (not shown in figures). No motor changes or signs of aversiveness were observed during DRN stimulation. Also, no neurological deficit was detected throughout the period of observation. Neither lidocaine, naloxone nor methysergide, alone, had a significant effect on the tail flick latency. Fourteen rats were stimulated in the DRN but lidocaine, naloxone or methysergide were not microinjected in the APtN. In these cases, the dye spots were found in sites localised lateral, medial, below or above the APtN at dis-
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Fig. 3. Effect of 0.5 ml saline or hyperbaric lidocaine (5%) microinjected into the anterior pretectal nucleus (arrow 1) on the antinociception induced by stimulation of the dorsal raphe nucleus of the rat (arrow 2). Points are mean (±SEM). *Significantly different from saline-treated rats (P , 0.05). Inset as in Fig. 1. APtN, anterior pretectal nucleus; DRN, dorsal raphe nucleus.
tances of less than 0.5 mm from the APtN borders. The effects of stimulating the DRN in these rats did not differ from the control rats (not shown in figures), thus indicating that the effects of lidocaine or antagonists in the test groups were restricted to the APtN. 3.6. DRN-APtN connection In all animal utilised in this experiment, the microinjection sites were situated in the APtN. Eight days after the central microinjection of the fluorescent neurotracer, deeply labelled cells were identified in the DRN (Fig. 6). Some of these cells showed characteristics of neurones, such as short dendritic ramification and major prolongations suggesting axonal projections. Fluorescent glial nuclei were not present around the retrogradely labelled neurones. Fig. 6A shows deeply labelled neurones, whose details can be better analysed in Fig. 6B.
4. Discussion
Fig. 2. Effect of 0.5 ml saline or hyperbaric lidocaine (5%) microinjected into the anterior pretectal nucleus (arrow 1) on the antinociception induced by stimulation of the deep mesencephalic nucleus of the rat (arrow 2). Points are mean (±SEM). Inset as in Fig. 1. APtN, anterior pretectal nucleus; DpMe, deep mesencephalic nucleus; PAG, periaqueductal gray; RN, red nucleus.
The present study demonstrates that brief electrical stimulation of the PPTg, DpMe or DRN evokes antinociception in awake rats, as revealed by the increase of the tail flick latency to noxious heating of the skin. In addition we showed that administration of a local anaesthetic into the APtN significantly inhibited the antinociceptive effects of DRN stimulation but not the antinociceptive effects of PPTg
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Fig. 4. Effect of saline (0.5 ml) or naloxone (2.7 nmol/0.5 ml) microinjected into the anterior pretectal nucleus (arrow 1) on the antinociception induced by stimulation of the dorsal raphe nucleus of the rat (arrow 2). Points are mean (±SEM). *Significantly different from saline-treated rats (P , 0.05). Inset shows the anatomical location of the sites of microinjection or electrical stimulation of naloxone-treated animals at the AP levels indicated. Abbreviations as in Fig. 1.
or DpMe stimulation. Stimulation of the APtN conducted 45 min after the local anaesthetic produced tail flick latency not different from the BTFL. Thus, the failure of lidocaine microinjected into the APtN to attenuate the stimulationproduced antinociception from the PPTg or DpMe is not due to incomplete or absent effect of the local anaesthetic. Electrical stimulation of the DCN, gracile or cuneate, did not produce significant changes in tail flick latency. The tail-flick escape from noxious heat is a spinal reflex (Mayer and Liebeskind, 1974) and its inhibition by intracerebral stimulation is indicative that the stimulation somehow depresses spinal mechanisms. In the present study the stimulated animals showed no grossly abnormal behaviour after the stimulation. They walked normally and responded normally to innocuous stimuli. Motor reactions followed by immobility were observed during the stimulation of the PPTg and circling behaviour was observed during the stimulation of the DpMe. The antinociceptive effectiveness of stimulating the DpMe, APtN or ventral PAG (including the DRN) accompanied by no clear change in the animal performance of the animals in the rota-rod paradigm have already been demonstrated (Roberts and Rees, 1986). Therefore, motor deficit is unlike to be the reason for the inhibition of the tail flick reflex induced by stimulation of DpMe or DRN. The DCN, PPTg and DpMe are brain structures known to send inputs to the APtN and, therefore, may act as relay stations in ascending pathways to excite cells in this nucleus
(see Section 1). Antinociception following bilateral stimulation of the DCN in awake rats has been demonstrated in both the tail immersion and formalin tests (Saade´ et al., 1986). In that study, however, a much longer (10 min) and stronger stimulus (100 Hz, 0.05–0.5 mA) had to be used to show the effect of stimulating the DCN. Thirtysecond stimulation of the DCN in anaesthetised rats at intensities that only activate large-diameter afferents (50 Hz, 0.2 V) potently excites cells in the ipsilateral APtN and inhibits multireceptive spinal cell responses to cutaneous noxious stimuli (Rees and Roberts, 1989b). The different patterns of stimulation and methodologies may be the reason for the ineffectiveness of stimulating the DCN in our experiments. Therefore, the involvement of the DCN as a relay station in an ascending nociceptive pathway to excite cells in the APtN cannot be ruled out by the present study. The participation of the PPTg in descending pain control mechanisms has been proposed. Microinjection of carbachol (Brodie and Proudfit, 1984; Katayama et al., 1984) or nicotine (Iwamoto, 1989) into this nucleus produces strong antinociceptive effects in rats and cats. According to Iwamoto (1991), there is a tonically active cholinergic pathway that originates in the PPTg and terminates in the NRM and modulates nociception by activating descending pain inhibitory systems relaying within the NRM. The stimulationproduced antinociception from the PPTg shown here, therefore, may depend on the activation of a central pathway that probably does not involve the participation of the APtN.
Fig. 5. Effect of saline (0.5 ml) or methysergide (2 nmol/0.5 ml) microinjected into the anterior pretectal nucleus (arrow 1) on the antinociception induced by stimulation of the dorsal raphe nucleus of the rat (arrow 2). Points are mean (±SEM). *Significantly different from saline-treated rats (P , 0.05). Inset shows the anatomical location of the sites of microinjection or electrical stimulation of methysergide-treated animals at the AP levels indicated. Abbreviations as in Fig.1.
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The stimulation-produced antinociception from the DpMe has been demonstrated (Prado et al., 1984; Wang et al., 1992). Anatomical studies have shown a reciprocal connection between the DpMe and APtN (Foster et al., 1989a). Microinjection of GABA into the DpMe reduces the antinociceptive effects of stimulating the APtN, thus indicating that the DpMe can also act as a relay for the descending inhibitory effects of APtN stimulation (Wang et al., 1992).
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Microinjection of tetracaine into the APtN blocked the antinociceptive effects of stimulating the DpMe in anaesthetised rats, but the antinociceptive effects of stimulating the APtN were not changed by prior administration of tetracaine into the DpMe (Foster et al., 1989b). The utilisation of anaesthetised rats in the study of Foster et al. (1989b) may account in part for the differences. The present demonstration that microinjection of lidocaine into the APtN does not
Fig. 6. Photomicrographs of the coronal sections of the rat dorsal raphe nucleus after microinjection of neuronal tracer into the APtN. Note the cells labelled by the fluorescent tracer, with cytoplasm prolongations characteristic of neuronal cells. Magnification bar: 42 mm in (A) and 25 mm in (B).
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change the antinociceptive effects of stimulating the DpMe is indicative that the APtN is not involved in the antinociceptive mechanism activated by the DpMe. The DRN is another brain structure widely accepted as playing an important role in pain modulation (Wang and Nakai, 1994). In addition to the ventrolateral part of the PAG, the DRN is the only midbrain region where electrical stimulation causes antinociceptive effects accompanied by no aversive behavioural side-effects (Fardin et al., 1984), a property that was confirmed by the present results. The literature has provided evidence for the participation of the DRN in a descending pathway that acts to inhibit nociceptive cells in the spinal cord (Griffith and Gatipon, 1981; Yu et al., 1988). This nucleus projects few direct fibres to the spinal cord and many fibres to the NRM, which is a very important relay station in the descending pain-control system (Wang and Nakai, 1994). Endogenous opioids and 5hydroxytryptamine (5-HT) seem to be the neurotransmitters involved in the modulation of the DRN-NRM-spinal cord pathway. However, there are reports suggesting that, besides a pain-control function, the DRN apparently has a pain-perception function. Electrical activity of DRN cells is changed when noxious stimuli are applied peripherally (Sanders et al., 1980). Moreover, metabolic activity (Wang et al., 1988; Porro et al., 1991) and the c-fos expression (Daı´ et al., 1992) within the DRN are both increased by noxious stimulation. Finally, lesion of the NRM partially reverses the antinociceptive effect of DRN stimulation (Yu et al., 1988). It was then hypothesised that inhibition of reflexes to noxious stimulation by DRN stimulation might be partly achieved by a control of spinal dorsal horn cells via a descending pathway relaying in the NRM and partly by direct modulation of the DRN by ascending nociceptive messages (Besson and Chaouch, 1987; Wang and Nakai, 1994). Evidence for ascending pain-modulation projections from the DRN have appeared more recently in the literature. The electrical stimulation of, or microinjection of morphine into the DRN inhibits the response of cells to noxious stimulation in several brain structures, including the parafascicular nucleus of the thalamus, the nucleus ventralis posterolateralis of the thalamus, and the substantia nigra (Wang and Nakai, 1994). On the other hand, tracer studies on the efferent from the DRN (Wang and Nakai, 1994) or on the afferents to the APtN (Rees and Roberts, 1993) have shown no connections between these structures. The only available evidence came from a report by Aghajanian et al. (1973), who found ascending serotonergic fibres from the DRN projecting to the APtN. In this study we have confirmed that the APtN receives ascending projections from the DRN. After injection of the fluorescent neurotracer Fast Blue into the APtN, a dense collection of labelled cells was found in the DRN some of which showing characteristics of neurones, such as short dendritic ramification and major prolongations suggesting axonal projections. Fluorescent glial nuclei were not found around the retro-
gradely labelled neurones, thus indicating that migration of tracer due to its uptake by fibres of passage is unlikely to be the reason for the labelling of DRN neurones (Kuypers et al., 1980). We have further demonstrated that application of lidocaine into the APtN partially reverses the antinociceptive effects of stimulating the DRN. The efficacy of lidocaine for the production of functional neural block has already been documented (Aimone et al., 1988). According to Sandku¨hler and Gebhart (1984), lidocaine (0.5 ml, 4%) produced a functional neural block having a radius of approximately 0.5 mm. The antinociception induced by stimulating the DRN was not changed when lidocaine was microinjected into areas less than 0.5 mm from the APtN. Thus, the effect of the local anaesthetic seems to be restricted to the APtN. The result is, therefore, indicative that the APtN may be another relay through which the DRN partly modulates spinal nociceptive messages. This alternative is reinforced by the demonstration that prior microinjection of methysergide or naloxone into the APtN also partially reduces the antinociceptive effects of stimulating the DRN. The effects of the antagonists also seem to be restricted to the APtN since the DRN stimulation-induced antinociception was not changed by microinjecting the antagonists into areas less than 0.5 mm from the APtN. The microinjection of morphine or 5-HT (Prado, 1989), and m- or 5-HT1B agonists (Rosa and Prado, 1997) into the APtN has earlier been shown to mimic the antinociceptive effects of stimulating this nucleus electrically. Altogether, these results confirm that APtN integrity is at least in part necessary for the antinociceptive effects of stimulating the DRN. In addition, they indicate that at least endogenous opioids and 5-HT can participate as neuromodulators in the DRN-APtN connection.
Acknowledgements This work was supported by FAPESP. M.L.N.M.R. was the recipient of CAPES and CNPq fellowships. R.B.V. was the recipient of a CNPq fellowship. We thank Mr. P.R. Castania for technical assistance.
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