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Ventrolateral periaqueductal gray matter and the control of tonic immobility
Brain Research Bulletin, Vol. 50, No. 3, pp. 201–208, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/99/$–see front matter
PII S0361-9230(99)00192-6
Ventrolateral periaqueductal gray matter and the control of tonic immobility Claudia Regina Monassi, Christie Ramos Andrade Leite-Panissi and Leda Menescal-de-Oliveira* Department of Physiology, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Brazil [Received 2 December 1998; Revised 12 July 1999; Accepted 20 July 1999] ABSTRACT: Tonic immobility is an inborn defensive behavior characterized by a temporary state of profound and reversible motor inhibition elicited by some forms of physical restraint. The periaqueductal gray matter (PAG) contains neural circuits involved in descending pain modulation, as well as in the modulation of TI. We have reported previously that the cholinergic stimulation of the ventrolateral PAG increases the duration of TI in guinea pigs. In the present study, we attempted to characterize further the modulation of TI by pharmacological alteration of the neurochemistry of the ventrolateral PAG circuitry. We observed that both cholinergic (carbachol, 5.4 nmol/0.2 l) and opioidergic stimulations (morphine, 4.48 nmol/0.2 l) of the ventrolateral PAG increase the duration of TI and that these effects can be reversed by pre-treatment with naloxone (2.74 nmol/0.2 l). Our results also showed that microinjection of the GABAergic agonist muscimol (1, 0.5, and 0.26 nmol/0.2 l) decreased the duration of TI episodes, while microinjection of the GABAergic antagonist bicuculline (1 nmol/l) increased it. Moreover, we observed that preadministration of muscimol (0.13 nmol/0.2 l) at a dose that had no effect per se at this site antagonized the potentiating effect of morphine. Our results suggest that this modulation of TI from the ventrolateral PAG circuitry is accomplished by a complex interaction of cholinergic, opioidergic, and GABAergic mechanisms, similar to that proposed for descending antinociceptive circuits. © 1999 Elsevier Science Inc.
acids [1,3,61]. For example, strong or moderate immobility, characterized by a period of profound inactivity, is observed after stimulating the ventrolateral part of the caudal one third of the PAG of the cat, while a strong flight response is observed after stimulating the dorsomedial PAG [61]. Neuroanatomical studies [12,13,26,38,39] have shown that the PAG has widespread connections with a large number of structures in the pontine and medullary tegmentum, as well as ascending efferent projections to the rostral midbrain, diencephalon, and telencephalon. The major descending projections of the PAG consist of predominantly ipsilateral directed at the mesencephalic and pontine tegment and terminating in the cuneiform nucleus, parabrachial nucleus, solitary tract nucleus, and lateral pontine tegment [28]. In contrast the ventrolateral portion of the PAG has preferential projections toward the nucleus raphe magnus, rostroventrolateral reticular nucleus and lateral paragigantocellular nucleus [13]. According to Holstege [28], there are also projections to the nucleus raphe pallidus and to the nucleus raphe obscurus and to the lateral region of the bulbar reticular formation. In addition, the neurons of the ventrolateral portion of the PAG send fibers to the nucleus raphe pallidus, which in turn send projections mainly to groups of somatic and autonomic of the ventral horn of the spinal cord [29,30]. The PAG has few direct projections to the spinal cord [8]. However, Mouton and Holstege [44] showed that the ventrolateral and lateral PAG also has direct projections to premotor interneurons in lamina VIII and in the medial part of lamina VII throughout the length of the spinal, suggesting that the PAG-spinal pathway is involved in the control of neck and back movements. The PAGspinal pathways are well-known to be multiply relayed descending projections [2] composed of a wide variety of fibers and synaptic profiles, and numerous kinds of neurotransmitters are involved, contributing to different functions [6]. The PAG has a large number of established and putative neurotransmitters present in its perikarya and afferent nerve terminals, including the amines acetylcholine, noradrenaline and 5-hydroxytryptamine [18], as well as enkephalin [43], neurotensin [56], glycine, glutamate, and GABA [10,17]. Basbaum and Fields [7] were the first to propose the presence of neuronal circuits inside the PAG comprising encephalinergic, GABAergic, and neurotensinergic neurons involved in the coordination of the activity of descending antinociceptive pathways. However, it is known that these circuits probably also participate in the regulation of the
KEY WORDS: Periaqueductal gray matter, Tonic immobility, Defensive behavior, Guinea pig.
INTRODUCTION The periaqueductal gray matter (PAG) refers to the region of the midbrain that surrounds the cerebral aqueduct. This neural substrate plays a crucial role in integrating the somatic and autonomic responses characteristic of the defense reactions [1]. According to Bandler and Carrive [1], the “defence reaction” is often referred to as the pattern of behavioural and cardiovascular change characteristic of an animal’s reaction to threatening or stressful stimuli. The PAG controls a wide range of functions, including the modulation of pain [7,9], analgesia [50], autonomic responses [37], vocalization [32] and different patterns of behavior, such as threat display, fight, flight, and immobility responses [1,3,41]. Each of these patterns of behavior can be elicited in specific parts of the PAG by both electrical stimulation and stimulation with excitatory amino
* Address for corresponding author: Prof. Leda Menescal-de-Oliveira, Department of Physiology, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, 14049-900 Ribeira˜o Preto, Sa˜o Paulo, Brazil. Fax: ⫹55-16-633-0017; E-mail: [email protected]
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various functions attributed to the PAG in addition to the modulation of antinociception. The GABAergic neuronal elements, mostly consisting of local interneurons, are integral components of the intrinsic neuronal circuitry of the PAG. Approximately half of the PAG neurons can be excited by GABA antagonists [9]. This implies that much of the GABAergic inhibitory control in the PAG is tonically active and inhibition of this network is an important mechanism for activation of PAG output neurons. Tonic immobility is a temporary state of profound motor inhibition elicited experimentally by some types of stimulation and restraint. The maneuvers used in the laboratory to induce TI are an attempt to simulate the physical restraints that can result in the immobility of a prey when caught and/or carried by a predator. Within a natural context, this behavior is a component of antipredatory behavior and the last resource used by the subject to reduce the probability of continued attack by the predator, thus being of adaptive value. Little is known about the regions involved in TI organization. The use of the transection technique has permitted to define the levels of the nervous system which participate in TI modulation in toads and rabbits [36]. Klemm [36] proposed that TI may be considered to be a reflex whose expression appears to be related to the activation of a pool of interneurons in the reticular formation of the brain stem that may influence descending neurons which inhibit the motor neurons of the spinal medulla. Some studies [14,35] have shown that the integrity of midbrain regions is essential for the expression of this behavior, although other brain regions may also modulate the immobility response. Thus, localized lesions in limited regions of the limbic system of rabbits [60] altered the duration of TI, indicating that other structures may participate in TI modulation. Studies carried out in our laboratory [40,41,46] have shown that some central structures known to be involved in the regulation of nociceptive responses, such as the parabrachial region [33,34], lateral hypothalamus [20] and PAG [7,50], also participate in the modulation of the TI response. Some studies have shown that during TI, and therefore already in the absence of restraining maneuvers, there is activation of pain-suppressing mechanisms [15,16,23]. The parallel occurrence of analgesia during TI suggests the existence of neural substrates shared by the two systems that could be activated simultaneously by the same environmental stimuli. A recent study from our laboratory [41] showed that different regions of the PAG and the cholinergic system are involved in the modulation of tonic immobility (TI) in guinea pigs. The cholinergic stimulation of the dorsal PAG decreases the duration of TI episodes, while stimulation of the ventrolateral region increases it. Further, it has been postulated that this effect is mediated by muscarine receptors. Thus, the study of the role of other neural transmitters may be relevant to the understanding of the intrinsic neural circuits of the PAG involved in the modulation of this behavior. On this basis, the objective of the present study was to investigate the possibility that the ventrolateral PAG modulates TI episodes by an interaction of cholinergic, opioidergic and GABAergic mechanisms. For this purpose, we microinjected carbachol, morphine, naloxone, muscimol and bicuculline into the ventrolateral PAG of different groups of guinea pigs and observed the effects of these drugs on TI duration. MATERIALS AND METHODS Animals Adult male guinea pigs (Cavia porcellus, n ⫽ 59), weighing 450 –500 g from the animal care facility of the Faculty of Medicine of Ribeira˜o Preto were used in the present study. The animals were kept under controlled temperature (24 ⫾ 1°C) and illumination
(12-h cycle) in Plexiglas-wall cages (56 ⫻ 17 ⫻ 39 cm, five animals per cage) with free access to water and food. Conditions of animal housing and all experimental procedures obeyed institutional guidelines, and were in accordance with National Institutes of Health guidelines for animal care. Experimental Procedure Each animal was submitted to five control maneuvers of TI induction and the duration of the episodes was recorded. Tonic immobility induction consisted of sudden inversion, which is characterized by gravitational and tactile stimulation, and manual restraint of the animal. The experimenter held the animal around the thorax with her hands, quickly inverted it and pressed it on its back in a V-shaped plywood box covered with a thick foam layer (25 cm long ⫻ 15 cm high) until struggling ceased. The pressure applied by the hands of the experimenter was proportional to the resistance offered by the animal to the restraining maneuver. The restraint and postural inversion maneuvers used in the laboratory are an attempt to simulate the preying condition. When the animal stopped moving, the experimenter’s hand was slowly withdrawn and a chronometer started to time the duration (in seconds) of the response, which ended when the animal resumed the upright position. When TI occurs, it persists in the absence of restraint for periods varying from a few seconds to several minutes. If the animal did not reach immobility within 60 s, the episode was recorded as having zero duration. A guide cannula (14 mm in length and 0.6 mm in outer diameter, prepared from a hypodermic needle) was implanted unilaterally into the ventrolateral PAG under anesthesia (40 mg/kg pentobarbital sodium, intraperitoneal [i.p.]). The animal was attached to the Kopf stereotaxic apparatus, with the mouthpiece 21.4 mm below the interauricular line. According to the Ro¨ssner atlas for guinea pigs [52], the stereotaxic coordinates for the guide placement were 9.4 mm caudal to the bregma, 0.8 mm lateral to the midline, and 2.8 mm above the intraural line. The guide cannula was lowered to a depth of 1 mm above the target region and fixed to the skull by means of auto-polymerizing resin and an additional anchoring screw. All animals were allowed to recover from surgery for 5– 6 days. After recovery from surgery, the intracerebral injections were performed with a thin dental needle (0.3 mm outside diameter [O.D.] and 1 mm longer than the guide cannula) introduced through the guide cannula until its tip was 1 mm below the cannula end. A volume of 0.2 l was injected over a period of 60 s using a 10 l Hamilton microsyringe (US), and the dental needle was left in place for an additional 60 s to avoid reflux. The movement of an air bubble inside the PE 10 polyethylene tubing connecting the microsyringe to the dental needle confirmed drug flow. The microinjections were made on consecutive days and 1 min after each treatment five TI episodes were successively induced and their durations recorded. The animals were divided into six experimental groups. A group of 12 animals (group 1) received carbachol (CCh, 5.4 nmol) on the first day of the experiment and naloxone (NAL, 2.74 nmol) followed 10 min later by CCh (at the same dose) on the next day. The animals of group 2 (n ⫽ 12) were microinjected with morphine sulphate (MS, 4.48 nmol) on the first day and 24 h later, with MS preceded (10 min) by naloxone. Animals in groups 3, 4 and 5 were microinjected with muscimol and 24 h later with bicuculline at different doses. In group 3 (n ⫽ 12), the animals were microinjected with 1 nmol of muscimol and 0.022 nmol of bicuculline on the next day. In group 4 (n ⫽ 8), the animals were microinjected with 0.5 nmol of muscimol and 24 hours later, with 0.2 nmol of bicuculline. In group 5
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FIG. 1. Schematic drawing of frontal sections obtained at representative levels of the guinea pig periaqueductal gray matter (PAG). The open circles (E) represent the sites where carbachol (CCh) and naloxone followed by CCh were microinjected. The filled circles (F) depict the sites of microinjection of morphine and naloxone followed by morphine. The filled triangles () represent the sites where muscimol and bicuculline were microinjected. The open squares (䊐) indicate the sites of microinjection of morphine and muscimol followed by morphine. All microinjections were made on the right side, but bicuculline and muscimol sites as well as muscimol followed by morphine sites are illustrated on the left for clarity. Abbreviations: A, aqueductus Sylvii; IC, inferior colliculus; SC, superior colliculus.
(n ⫽ 7), the animals were microinjected with 0.26 nmol of muscimol and with 1 nmol of bicuculline on the next day. In group 6 (n ⫽ 8), the animals were microinjected with muscimol (0.13 nmol) and 24 h later, with MS (4.8 nmol) and on the next day with muscimol (0.13 nmol) followed 10 min later by MS. All drugs were administered in a volume of 0.2 l.
group and by one-way ANOVA for each treatment assessed. The degree of freedom of the repeated measure (treatment) was corrected by the Huynh-Feld parameter. A post hoc Duncan’s Multiple Range test ( p ⬍ 0.05) was run for multiple comparisons.
Drugs
The localization of the sites in the ventrolateral PAG where drugs were microinjected is shown in Fig. 1. As shown in a previous report [41], the microinjections of carbachol into the ventrolateral PAG of guinea pig increased the duration of TI episodes when compared to control episodes. However, when the microinjection of this drug was preceded by naloxone the potentiating effect of CCh was blocked (Fig. 2A). Repeated measures ANOVA revealed a significant difference between treatments [F(1, 16) ⫽ 16.81, p ⬍ 0.001]. The Duncan post-hoc test showed a significant difference between CCh and control ( p ⬍ 0.05) and CCh preceded by naloxone ( p ⬍ 0.05). In group 2, the microinjection of morphine (MS) into the ventrolateral PAG also increased significantly the duration of TI episodes and this effect was reversed by previous naloxone microinjection (Fig. 2B). These findings were supported by repeated measures ANOVA, which revealed a significant difference between treatments [F(1, 15) ⫽ 12.12, p ⬍ 0.001]. The Duncan test demonstrated a significant difference between morphine and control ( p ⬍ 0.05) and the treatment with morphine preceded by naloxone ( p ⬍ 0.05), but not between the treatment preceded by naloxone and control. The effects of muscimol and bicuculline into the ventrolateral PAG are shown in Fig. 3. In groups 3, 4, and 5, the results showed that the microinjection of muscimol (at all doses used) into the ventrolateral PAG decreased the duration of TI episodes, while microinjection of bicuculline did not alter (groups 3 and 4) or increased the duration of TI episodes (1 nmol/0.2 l, group 5). A two-way ANOVA showed that there was a significant main effect of treatment [F(2, 46) ⫽ 41.23, p ⬍ 0.001] and a significant
Carbachol (Sigma), naloxone hydrochloride (Sigma), morphine sulfate (Sigma), muscimol (Sigma), and bicuculline methionide (Sigma) were dissolved in physiological saline (0.9% NaCl). Physiological saline also served as vehicle control. The criteria for selecting the above doses have been described in previous reports [31,40,41,46]. Histological Verification Upon completion of the experiments, the animals received an overdose of sodium pentobabital and was perfused intracardially with saline followed by 10% formalin. The brains were removed and fixed in 10% formalin. The routine histological procedures for sectioning were used and stained preparations were observed under the light microscope to determine the location of the stimulated sites according to the Ro¨ssner atlas [52]. Statistical Analysis The results are reported as the mean ⫾ SEM of the durations of the TI episodes for each treatment. Due to variability and skewness in the TI duration data, a natural logarithmic (ln) transformation was performed on all durations scores prior to statistical analysis. Then, for groups 1, 2, and 6 the transformed data were submitted to repeated measure analysis of variance (ANOVA). However, for groups 3, 4, and 5 the data were submitted to two-way factorial analysis of variance followed, when significant treatment ⫻ dose interactions were found, by a repeated measure ANOVA for each
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FIG. 2. Duration of tonic immobility episodes. (A) Mean TI durations before (CONT) and after carbachol (CCh, 5.4 nmol/0.2 l) and CCh preceded by naloxone (CCh, 5.4 nmol/0.2 l ⫹ NAL, 2.74 nmol/0.2 l) microinjection into the ventrolateral periaqueductal gray matter of guinea pigs (n ⫽ 12). (B) Mean TI durations before (CONT) and after morphine (MS, 4.48 nmol/0.2 l) microinjection, and after pretreatment with naloxone plus MS microinjection (NAL, 2.74 nmol/0.2 l ⫹ MS, 4.48 nmol/0.2 l) into the ventrolateral PAG (n ⫽ 12). *p ⬍ 0.05 compared with control (A, B), after microinjection of naloxone followed by CCh (A) and after naloxone followed by morphine (B). The vertical bars indicate the standard error of the mean.
interaction between treatment and dose [F(4, 46) ⫽ 3.41, p ⬍ 0.01]. The repeated measure ANOVA showed a significant difference between treatments in group 3, F(1, 15) ⫽ 11.88, p ⬍ 0.001; in group 4, F(2, 14) ⫽ 18.28, p ⬍ 0.001; and in group
FIG. 3. Duration of tonic immobility episodes. (A) Mean TI durations before (CONT) and after bicuculline (BIC, 0.022 nmol/0.2 l) and muscimol (MUSC, 1 nmol/0.2 l) microinjection into the ventrolateral PAG of guinea pigs (n ⫽ 12). (B) Mean durations of five TI episodes before (CONT) and after bicuculline (0.2 nmol/0.2 l) and after muscimol (0.5 nmol/0.2 l) microinjection (n ⫽ 8). (C) Mean TI durations before (CONT) and after bicuculline (1 nmol/0.2 l) and after muscimol (0.26 nmol/0.2 l) microinjection (n ⫽ 7). *p ⬍ 0.05 compared to control; ⫹p ⬍ 0.05 compared to bicuculline. The vertical bars indicate the SEM of the mean.
MONASSI ET AL.
FIG. 4. Mean TI durations before (CONT) and after muscimol (MUSC, 0.13 nmol/0.2 l) and after morphine (MS, 4.48 nmol/0.2 l) microinjection and after microinjection of muscimol followed by morphine (MUSC, 0.13 nmol/0.2 l ⫹ MS, 4.48 nmol/0.2 l) into the ventrolateral PAG of guinea pigs (n ⫽ 8). *p ⬍ 0.05 compared with control and ⫹p ⬍ 0.05 compared to morphine. The vertical bars indicate the standard error of the mean.
5, F(1, 10) ⫽ 13.42, p ⬍ 0.001. Duncan’s post-hoc comparisons between treatments in group 3 showed that the duration of TI episodes after microinjection of 0.022 nmol of bicuculline did not differ from control durations ( p ⬎ 0.05), while the dose of 1 nmol of muscimol significantly decreased the TI durations when compare to control ( p ⬍ 0.05) and to bicuculline ( p ⬍ 0.05) microinjection. In group 4, the Duncan test showed a significant difference between muscimol (0.5 nmol) and control ( p ⬍ 0.05) and bicuculline ( p ⬍ 0.05), but not between bicuculline (0.2 nmol) and control. In group 5, the microinjection of 0.26 nmol of muscimol also decreased the duration of TI episodes ( p ⬍ 0.05, Duncan), while the microinjection of 1 nmol of bicuculline significantly increased the durations of TI episodes when compare to control ( p ⬍ 0.05, Duncan) and to muscimol microinjection ( p ⬍ 0.05, Duncan). One-way ANOVA followed by the Duncan test applied to the data for each treatment showed that while the control durations did not differ between groups 3, 4, and 5, the effect of bicuculline and muscimol were dose-dependent. The two lowest doses (0.022 and 0.2 nmol) of bicuculline did not alter TI duration, but the highest dose (1 nmol) of bicuculline significantly increased TI duration, and its effect was different from that of the other two doses ( p ⬍ 0.05, Duncan test). The three doses of muscimol used decreased the duration of TI, and their effect were not significantly different. Curiously, the dose of 1 nmol of muscimol appeared to increase the variability of TI duration, although its effect was not significantly different from that of the other two doses. The results for group 6 are shown in Fig. 4. As shown for group 2, the microinjection of MS (4.48 nmol) increased the duration of TI episodes, but this effect was blocked by pretreatment with an ineffective dose of muscimol (0.13 nmol). These findings were supported by the repeated measures ANOVA, which showed a significant difference between treatments [F(3, 13) ⫽ 23.41, p ⬍ 0.01]. The Duncan test revealed that while the treatment with muscimol (0.13 nmol) did not differ from the control durations, there were significant difference between MS and control ( p ⬍ 0.05), and MS and muscimol ( p ⬍ 0.05), and also between MS
PERIAQUEDUCTAL GRAY MATTER AND TONIC IMMOBILITY and the treatment with muscimol followed by MS ( p ⬍ 0.05). Treatment with muscimol followed by MS did not differ from the control situation. DISCUSSION The results of the present study indicate that the ventrolateral PAG possibly modulates TI duration via neural circuits involving cholinergic, opioidergic, and GABAergic mechanisms. In a previous study [41], we demonstrated that cholinergic stimulation of the ventrolateral PAG increases the duration of TI episodes and this effect is blocked by atropine microinjected into the same site, indicating that the effect was mediated by muscarinic receptors. In the present study we observed that the effect of increased TI duration induced by carbachol microinjected into the ventrolateral PAG can also be blocked by previous injection of naloxone into the same site. These results suggest that the action of the cholinergic system inside the ventrolateral PAG seems to be opioiddependent. On this basis, we may propose that the cholinergic transmission may activate the liberation of endorphins/enkephalins from interneurons of the ventrolateral PAG, resulting in increased TI. Indeed, Vicent and Reiner [57] demonstrated that the lack of abundance of choline acetyltransferase-immunoreactive cells and nerve terminals in the cat PAG does not appear to match with the presence of a high density of muscarinic receptors [19,25]. Thus, we may assume that the neurons having opioid receptors in the ventrolateral PAG may also contain muscarinic receptors, because in the present study, the effect of carbachol was blocked both by atropine [41] and by naloxone. We also observed that microinjection of morphine into the ventrolateral PAG also increased the duration of TI episodes and this effect was antagonized by naloxone, indicating that this action is probably mediated by -opioid receptors. Morphine injected into the ventrolateral PAG [21,48] produces antinociception by activating structures of the rostral ventromedial medulla that liberate the centrifugal pain-inhibitory system. As proposed by Basbaum and Fields [7] and Depaulis et al. [21], opiate activation of the descending pain-inhibitory system may result from a disinhibition of PAG output neurons. Opioids are generally considered to exert an indirect effect by decreasing the tonic activity of GABA neurons [42], thus causing a disinhibition of the PAG output neurons. The disinhibition of PAG output neurons may be important not only for the antinociceptive effect but also for the action of the PAG when performing some of the diverse functions that are attributed to it. On the basis of these findings, we may raise the hypothesis that the increased TI induced by morphine occurs in the same way, i.e., by disinhibition of PAG output neurons and the consequent activation of the descending pathways involved in TI modulation. The descending projections are mainly directed at the mesencephalic and pontine tegment, terminating in the cuneiform nucleus, parabrachial nucleus, and lateral pontine tegment [28]. In addition, the neurons of the ventrolateral PAG send fibers to the nucleus raphe magnus and raphe pallidus, and the latter in turn sends projections mainly to groups of somatic and autonomic motor neurons of the ventral horn of the spinal cord [29,30]. During TI there is inhibition of the righting reflex, which is essentially regulated by motor neurons distributed along the ventral horn of the spinal cord. Thus, we may propose that these motor neurons involved in the righting reflex receive inhibitory influences from the descending pathways of the nucleus raphe pallidus originating from, or activated by, the ventrolateral PAG. As proposed by Klemm [36], TI may be considered to be a reflex whose expression appears to be related to the activation of a pool of interneurons in the reticular formation of the brain stem that may influence descending neurons which inhibit the motor neurons of the spinal medulla. Another possible explanation for
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these results is that the ventrolateral PAG may exert an inhibitory influence directly on premotor and/or motor neurons of the ventral horn of the spinal cord involved in the righting reflex. In fact, more recently Mouton and Holstege [44] demonstrated the existence of direct projections from ventrolateral PAG to premotor interneurons in laminae VIII and VII, which extend throughout the length of the spinal cord and are involved in the control of neck and back movements. There is evidence that at least part of the neural circuits and the neurotransmitters involved in TI organization also play an important role in the modulation of antinociceptive responses. A number of studies have demonstrated that systemic injection of morphine increased the duration of TI in chickens [49], guinea pigs [45], and rabbits [15]. Also, microinjection of CCh into the basal midbrain of awake toads, at the doses that increase TI, abolishes the motor reflex triggered by a noxious stimulus [27]. Other studies [11,24, 34] have demonstrated that cholinergic agonists have antinociceptive properties in addition to potentiating the antinociceptive effects triggered by opioids [47]. Rodgers and Randal [51] argued that many of the stimuli that induce analgesia in rodents also elicit defense reactions, suggesting that there may be an important relationship between specific defense reactions and specific forms of analgesia. According to Bandler and Carrive [1] the “defence reaction” is often referred to as the pattern of behavioural and cardiovascular change characteristic of an animal’s reaction to threatening or stressful stimuli. More specifically, Rogers and Randal [51] suggested that non-opioid analgesia is activated by flight-eliciting stimuli, whereas opioid analgesia is activated by stimuli that elicit the terminal defense reaction of TI. Similarly, some investigators observed that electrical stimulation or stimulation with excitatory amino acids of the ventrolateral PAG results in opioid-related antinociception, hypotension, and defensive freezing, whereas dorsomedial and dorsolateral PAG stimulation results in non-opioid hypoalgesia, hypertension, and avoidance escape or aggressive responses [4,22,37]. Since Reynolds’ report [50], a large number of behavioral, pharmacological, and electrophysiological studies have established an important role for the PAG in the modulation of pain. It is possible that disinhibition through inhibitory GABA transmission is involved in the activation of PAG output neurons and behavioral antinociception [42]. The microinjection of muscimol into this substrate antagonizes the analgesia produced by simultaneous morphine microinjection and, conversely, microinjection of bicuculline, a GABA antagonist, produces analgesia [42]. These data suggest that the GABAergic network is an important mechanism for the activation of PAG output neurons. The present results show that microinjection of muscimol into the ventrolateral PAG decreased the duration of TI episodes, suggesting that the GABAergic neurons of the ventrolateral PAG have an inhibitory influence on PAG-output neurons that are involved in the modulation of TI in guinea pigs. On the other hand, the increase duration of TI caused by bicuculline microinjection into the ventrolateral PAG suggests disinhibition of PAG output neurons that may possibly be tonically inhibited. Since the ventrolateral PAG has high densities of GABA receptors and GABA neurons [5] disinhibition of neurons by blockage of GABA receptors with bicuculline is a very powerful method for activation of descending pathways from the PAG [54,55]. Within this context there is the possibility that both morphine and bicuculline act by possibly activating the same pathways that may probably act on the depression of the righting reflex. Indeed, Roychowdhury and Fields [53] observed that the antinociceptive action of bicuculline injected into the ventrolateral PAG was partially reversed by naloxone, indicating that the release of endogenous opioids is necessary for this effect. Thus, on the basis of the above data,
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FIG. 5. Schematic diagram of the hypotheses for the interaction within the ventrolateral periaqueductal gray matter (PAG) of cholinergic, opioidergic, and GABAergic systems involved in TI modulation. Local carbachol injection may activate opioidergic neurons, which in turn may inhibit GABAergic neurons, disinhibiting PAG output neurons and promoting the activation of descending pathway that may act by increasing TI. Local morphine injection activating opioidergic neurons and bicuculline injection inhibiting GABAergic neurons also results in disinhibition of PAG output neurons with activation of descending pathways and with the same effect on TI. Muscimol may facilitate the tonic inhibitory action of GABA inhibiting ventrolateral PAG output neurons and decreasing TI. Œ, inhibitory transmission; ‚, represent excitatory transmission. Abbreviations: CCh, cholinergic termination; GABA, GABAergic neuron; OP, opioidergic neuron.
MONASSI ET AL. effect on TI may occur directly or indirectly on the motor neurons of the ventral horn of the spinal cord. In summary, the present experiments have demonstrated that the involvement of the ventrolateral PAG in the modulation of TI in guinea pigs possibly occurs via an interaction between cholinergic, opioidergic and GABAergic systems. The cholinergic and opioidergic stimulation of the ventrolateral PAG increases the duration of TI, and both effects can be reversed by pretreatment with naloxone. Thus, these results suggest an interaction between opioid and cholinergic systems into the ventrolateral PAG, causing an increase in TI duration. Furthermore, the effect of muscimol (at effective doses) also showed that the GABAergic neurons of the ventrolateral PAG have an inhibitory influence on PAG-output neurons involved in the modulation of TI, and this effect seems to occur tonically and is mediated by GABAA receptors. We also observed that muscimol at a dose that has no effect per se on TI, when injected before morphine at the same site blocks the facilitatory effect of morphine. This supports the suggestion that the opioid effects may occur via inhibition of GABA interneurons and also that the modulation of TI by the ventrolateral PAG might, in part, occur via a neural circuit similar to that proposed for the antinociceptive system. ACKNOWLEDGEMENTS
another hypothesis for explaining the effect of bicuculline may be via the liberation of endogenous opioids, although in the present study we did not use naloxone to reverse the effect of bicuculline. Many reports have suggested an interaction between opioid and GABAergic circuits inside the PAG in the modulation of nociception [7,21,42,58,59]. Since opioids exert an inhibitory effect on neural excitability and transmitter release, it has been proposed that opioids in the PAG act by releasing descending output neurons by GABAergic inhibition, which may in turn lead to a disinhibition of descending antinociceptive circuits. Activation of this system inhibits nociceptive neurons in the dorsal horn of the spinal cord. In the present study we observed that in the ventrolateral PAG pretreatment with muscimol at a dose that has no effect per se, blocks the action of morphine on the duration of TI. This result suggests that microinjection of muscimol may reinforce the tonic inhibitory influence of GABAergic neurons on ventrolateral PAGoutput neurons, perhaps at the same time preventing the effect of morphine. According to this hypothesis, it is possible that during TI, the opioids in the PAG act by inhibiting the GABA interneurons. These findings represent indirect experimental evidence that the circuits containing opioidergic and GABAergic neurons inside the ventrolateral PAG involved in TI modulation may be similar, in part, to the circuits proposed by Basbaum and Fields [7] for the modulation of antinociception. Figure 5 schematically shows the neural circuits inside the ventrolateral PAG that may possibly be involved in TI modulation. Although in a speculative manner, the present results may permit us to postulate that the ventrolateral PAG may function as an interface in the systems that modulate TI and analgesia. This region contains neurons involved in the organization of different stages of defensive behavior including TI, and neurons related to pain modulation. These neuronal groups may be mobilized simultaneously by the same environmental stimuli in a situation of confrontation, resulting in analgesia that may permit the execution of defensive behaviors. It is possible to propose descending pathways originating from the PAG and modulating both analgesia and TI. There is reason to believe that descending pathways originating from the PAG exert a net inhibitory effect on nociceptive transmission at the level of the dorsal horn, whereas the inhibitory
We would like to thank Dr. Pravin K. Mishira from DBTS/University of Illinois College of Medicine at Peoria, for constructive comments on the manuscript and Mrs Mariulza Rocha Brentegani and Mr Rubens Fernando de Melo for technical assistance and histological processing. This work was supported by FAPESP (Proc. 97/08787-5).
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PII S0361-9230(99)00192-6
Ventrolateral periaqueductal gray matter and the control of tonic immobility Claudia Regina Monassi, Christie Ramos Andrade Leite-Panissi and Leda Menescal-de-Oliveira* Department of Physiology, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Brazil [Received 2 December 1998; Revised 12 July 1999; Accepted 20 July 1999] ABSTRACT: Tonic immobility is an inborn defensive behavior characterized by a temporary state of profound and reversible motor inhibition elicited by some forms of physical restraint. The periaqueductal gray matter (PAG) contains neural circuits involved in descending pain modulation, as well as in the modulation of TI. We have reported previously that the cholinergic stimulation of the ventrolateral PAG increases the duration of TI in guinea pigs. In the present study, we attempted to characterize further the modulation of TI by pharmacological alteration of the neurochemistry of the ventrolateral PAG circuitry. We observed that both cholinergic (carbachol, 5.4 nmol/0.2 l) and opioidergic stimulations (morphine, 4.48 nmol/0.2 l) of the ventrolateral PAG increase the duration of TI and that these effects can be reversed by pre-treatment with naloxone (2.74 nmol/0.2 l). Our results also showed that microinjection of the GABAergic agonist muscimol (1, 0.5, and 0.26 nmol/0.2 l) decreased the duration of TI episodes, while microinjection of the GABAergic antagonist bicuculline (1 nmol/l) increased it. Moreover, we observed that preadministration of muscimol (0.13 nmol/0.2 l) at a dose that had no effect per se at this site antagonized the potentiating effect of morphine. Our results suggest that this modulation of TI from the ventrolateral PAG circuitry is accomplished by a complex interaction of cholinergic, opioidergic, and GABAergic mechanisms, similar to that proposed for descending antinociceptive circuits. © 1999 Elsevier Science Inc.
acids [1,3,61]. For example, strong or moderate immobility, characterized by a period of profound inactivity, is observed after stimulating the ventrolateral part of the caudal one third of the PAG of the cat, while a strong flight response is observed after stimulating the dorsomedial PAG [61]. Neuroanatomical studies [12,13,26,38,39] have shown that the PAG has widespread connections with a large number of structures in the pontine and medullary tegmentum, as well as ascending efferent projections to the rostral midbrain, diencephalon, and telencephalon. The major descending projections of the PAG consist of predominantly ipsilateral directed at the mesencephalic and pontine tegment and terminating in the cuneiform nucleus, parabrachial nucleus, solitary tract nucleus, and lateral pontine tegment [28]. In contrast the ventrolateral portion of the PAG has preferential projections toward the nucleus raphe magnus, rostroventrolateral reticular nucleus and lateral paragigantocellular nucleus [13]. According to Holstege [28], there are also projections to the nucleus raphe pallidus and to the nucleus raphe obscurus and to the lateral region of the bulbar reticular formation. In addition, the neurons of the ventrolateral portion of the PAG send fibers to the nucleus raphe pallidus, which in turn send projections mainly to groups of somatic and autonomic of the ventral horn of the spinal cord [29,30]. The PAG has few direct projections to the spinal cord [8]. However, Mouton and Holstege [44] showed that the ventrolateral and lateral PAG also has direct projections to premotor interneurons in lamina VIII and in the medial part of lamina VII throughout the length of the spinal, suggesting that the PAG-spinal pathway is involved in the control of neck and back movements. The PAGspinal pathways are well-known to be multiply relayed descending projections [2] composed of a wide variety of fibers and synaptic profiles, and numerous kinds of neurotransmitters are involved, contributing to different functions [6]. The PAG has a large number of established and putative neurotransmitters present in its perikarya and afferent nerve terminals, including the amines acetylcholine, noradrenaline and 5-hydroxytryptamine [18], as well as enkephalin [43], neurotensin [56], glycine, glutamate, and GABA [10,17]. Basbaum and Fields [7] were the first to propose the presence of neuronal circuits inside the PAG comprising encephalinergic, GABAergic, and neurotensinergic neurons involved in the coordination of the activity of descending antinociceptive pathways. However, it is known that these circuits probably also participate in the regulation of the
KEY WORDS: Periaqueductal gray matter, Tonic immobility, Defensive behavior, Guinea pig.
INTRODUCTION The periaqueductal gray matter (PAG) refers to the region of the midbrain that surrounds the cerebral aqueduct. This neural substrate plays a crucial role in integrating the somatic and autonomic responses characteristic of the defense reactions [1]. According to Bandler and Carrive [1], the “defence reaction” is often referred to as the pattern of behavioural and cardiovascular change characteristic of an animal’s reaction to threatening or stressful stimuli. The PAG controls a wide range of functions, including the modulation of pain [7,9], analgesia [50], autonomic responses [37], vocalization [32] and different patterns of behavior, such as threat display, fight, flight, and immobility responses [1,3,41]. Each of these patterns of behavior can be elicited in specific parts of the PAG by both electrical stimulation and stimulation with excitatory amino
* Address for corresponding author: Prof. Leda Menescal-de-Oliveira, Department of Physiology, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, 14049-900 Ribeira˜o Preto, Sa˜o Paulo, Brazil. Fax: ⫹55-16-633-0017; E-mail: [email protected]
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various functions attributed to the PAG in addition to the modulation of antinociception. The GABAergic neuronal elements, mostly consisting of local interneurons, are integral components of the intrinsic neuronal circuitry of the PAG. Approximately half of the PAG neurons can be excited by GABA antagonists [9]. This implies that much of the GABAergic inhibitory control in the PAG is tonically active and inhibition of this network is an important mechanism for activation of PAG output neurons. Tonic immobility is a temporary state of profound motor inhibition elicited experimentally by some types of stimulation and restraint. The maneuvers used in the laboratory to induce TI are an attempt to simulate the physical restraints that can result in the immobility of a prey when caught and/or carried by a predator. Within a natural context, this behavior is a component of antipredatory behavior and the last resource used by the subject to reduce the probability of continued attack by the predator, thus being of adaptive value. Little is known about the regions involved in TI organization. The use of the transection technique has permitted to define the levels of the nervous system which participate in TI modulation in toads and rabbits [36]. Klemm [36] proposed that TI may be considered to be a reflex whose expression appears to be related to the activation of a pool of interneurons in the reticular formation of the brain stem that may influence descending neurons which inhibit the motor neurons of the spinal medulla. Some studies [14,35] have shown that the integrity of midbrain regions is essential for the expression of this behavior, although other brain regions may also modulate the immobility response. Thus, localized lesions in limited regions of the limbic system of rabbits [60] altered the duration of TI, indicating that other structures may participate in TI modulation. Studies carried out in our laboratory [40,41,46] have shown that some central structures known to be involved in the regulation of nociceptive responses, such as the parabrachial region [33,34], lateral hypothalamus [20] and PAG [7,50], also participate in the modulation of the TI response. Some studies have shown that during TI, and therefore already in the absence of restraining maneuvers, there is activation of pain-suppressing mechanisms [15,16,23]. The parallel occurrence of analgesia during TI suggests the existence of neural substrates shared by the two systems that could be activated simultaneously by the same environmental stimuli. A recent study from our laboratory [41] showed that different regions of the PAG and the cholinergic system are involved in the modulation of tonic immobility (TI) in guinea pigs. The cholinergic stimulation of the dorsal PAG decreases the duration of TI episodes, while stimulation of the ventrolateral region increases it. Further, it has been postulated that this effect is mediated by muscarine receptors. Thus, the study of the role of other neural transmitters may be relevant to the understanding of the intrinsic neural circuits of the PAG involved in the modulation of this behavior. On this basis, the objective of the present study was to investigate the possibility that the ventrolateral PAG modulates TI episodes by an interaction of cholinergic, opioidergic and GABAergic mechanisms. For this purpose, we microinjected carbachol, morphine, naloxone, muscimol and bicuculline into the ventrolateral PAG of different groups of guinea pigs and observed the effects of these drugs on TI duration. MATERIALS AND METHODS Animals Adult male guinea pigs (Cavia porcellus, n ⫽ 59), weighing 450 –500 g from the animal care facility of the Faculty of Medicine of Ribeira˜o Preto were used in the present study. The animals were kept under controlled temperature (24 ⫾ 1°C) and illumination
(12-h cycle) in Plexiglas-wall cages (56 ⫻ 17 ⫻ 39 cm, five animals per cage) with free access to water and food. Conditions of animal housing and all experimental procedures obeyed institutional guidelines, and were in accordance with National Institutes of Health guidelines for animal care. Experimental Procedure Each animal was submitted to five control maneuvers of TI induction and the duration of the episodes was recorded. Tonic immobility induction consisted of sudden inversion, which is characterized by gravitational and tactile stimulation, and manual restraint of the animal. The experimenter held the animal around the thorax with her hands, quickly inverted it and pressed it on its back in a V-shaped plywood box covered with a thick foam layer (25 cm long ⫻ 15 cm high) until struggling ceased. The pressure applied by the hands of the experimenter was proportional to the resistance offered by the animal to the restraining maneuver. The restraint and postural inversion maneuvers used in the laboratory are an attempt to simulate the preying condition. When the animal stopped moving, the experimenter’s hand was slowly withdrawn and a chronometer started to time the duration (in seconds) of the response, which ended when the animal resumed the upright position. When TI occurs, it persists in the absence of restraint for periods varying from a few seconds to several minutes. If the animal did not reach immobility within 60 s, the episode was recorded as having zero duration. A guide cannula (14 mm in length and 0.6 mm in outer diameter, prepared from a hypodermic needle) was implanted unilaterally into the ventrolateral PAG under anesthesia (40 mg/kg pentobarbital sodium, intraperitoneal [i.p.]). The animal was attached to the Kopf stereotaxic apparatus, with the mouthpiece 21.4 mm below the interauricular line. According to the Ro¨ssner atlas for guinea pigs [52], the stereotaxic coordinates for the guide placement were 9.4 mm caudal to the bregma, 0.8 mm lateral to the midline, and 2.8 mm above the intraural line. The guide cannula was lowered to a depth of 1 mm above the target region and fixed to the skull by means of auto-polymerizing resin and an additional anchoring screw. All animals were allowed to recover from surgery for 5– 6 days. After recovery from surgery, the intracerebral injections were performed with a thin dental needle (0.3 mm outside diameter [O.D.] and 1 mm longer than the guide cannula) introduced through the guide cannula until its tip was 1 mm below the cannula end. A volume of 0.2 l was injected over a period of 60 s using a 10 l Hamilton microsyringe (US), and the dental needle was left in place for an additional 60 s to avoid reflux. The movement of an air bubble inside the PE 10 polyethylene tubing connecting the microsyringe to the dental needle confirmed drug flow. The microinjections were made on consecutive days and 1 min after each treatment five TI episodes were successively induced and their durations recorded. The animals were divided into six experimental groups. A group of 12 animals (group 1) received carbachol (CCh, 5.4 nmol) on the first day of the experiment and naloxone (NAL, 2.74 nmol) followed 10 min later by CCh (at the same dose) on the next day. The animals of group 2 (n ⫽ 12) were microinjected with morphine sulphate (MS, 4.48 nmol) on the first day and 24 h later, with MS preceded (10 min) by naloxone. Animals in groups 3, 4 and 5 were microinjected with muscimol and 24 h later with bicuculline at different doses. In group 3 (n ⫽ 12), the animals were microinjected with 1 nmol of muscimol and 0.022 nmol of bicuculline on the next day. In group 4 (n ⫽ 8), the animals were microinjected with 0.5 nmol of muscimol and 24 hours later, with 0.2 nmol of bicuculline. In group 5
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FIG. 1. Schematic drawing of frontal sections obtained at representative levels of the guinea pig periaqueductal gray matter (PAG). The open circles (E) represent the sites where carbachol (CCh) and naloxone followed by CCh were microinjected. The filled circles (F) depict the sites of microinjection of morphine and naloxone followed by morphine. The filled triangles () represent the sites where muscimol and bicuculline were microinjected. The open squares (䊐) indicate the sites of microinjection of morphine and muscimol followed by morphine. All microinjections were made on the right side, but bicuculline and muscimol sites as well as muscimol followed by morphine sites are illustrated on the left for clarity. Abbreviations: A, aqueductus Sylvii; IC, inferior colliculus; SC, superior colliculus.
(n ⫽ 7), the animals were microinjected with 0.26 nmol of muscimol and with 1 nmol of bicuculline on the next day. In group 6 (n ⫽ 8), the animals were microinjected with muscimol (0.13 nmol) and 24 h later, with MS (4.8 nmol) and on the next day with muscimol (0.13 nmol) followed 10 min later by MS. All drugs were administered in a volume of 0.2 l.
group and by one-way ANOVA for each treatment assessed. The degree of freedom of the repeated measure (treatment) was corrected by the Huynh-Feld parameter. A post hoc Duncan’s Multiple Range test ( p ⬍ 0.05) was run for multiple comparisons.
Drugs
The localization of the sites in the ventrolateral PAG where drugs were microinjected is shown in Fig. 1. As shown in a previous report [41], the microinjections of carbachol into the ventrolateral PAG of guinea pig increased the duration of TI episodes when compared to control episodes. However, when the microinjection of this drug was preceded by naloxone the potentiating effect of CCh was blocked (Fig. 2A). Repeated measures ANOVA revealed a significant difference between treatments [F(1, 16) ⫽ 16.81, p ⬍ 0.001]. The Duncan post-hoc test showed a significant difference between CCh and control ( p ⬍ 0.05) and CCh preceded by naloxone ( p ⬍ 0.05). In group 2, the microinjection of morphine (MS) into the ventrolateral PAG also increased significantly the duration of TI episodes and this effect was reversed by previous naloxone microinjection (Fig. 2B). These findings were supported by repeated measures ANOVA, which revealed a significant difference between treatments [F(1, 15) ⫽ 12.12, p ⬍ 0.001]. The Duncan test demonstrated a significant difference between morphine and control ( p ⬍ 0.05) and the treatment with morphine preceded by naloxone ( p ⬍ 0.05), but not between the treatment preceded by naloxone and control. The effects of muscimol and bicuculline into the ventrolateral PAG are shown in Fig. 3. In groups 3, 4, and 5, the results showed that the microinjection of muscimol (at all doses used) into the ventrolateral PAG decreased the duration of TI episodes, while microinjection of bicuculline did not alter (groups 3 and 4) or increased the duration of TI episodes (1 nmol/0.2 l, group 5). A two-way ANOVA showed that there was a significant main effect of treatment [F(2, 46) ⫽ 41.23, p ⬍ 0.001] and a significant
Carbachol (Sigma), naloxone hydrochloride (Sigma), morphine sulfate (Sigma), muscimol (Sigma), and bicuculline methionide (Sigma) were dissolved in physiological saline (0.9% NaCl). Physiological saline also served as vehicle control. The criteria for selecting the above doses have been described in previous reports [31,40,41,46]. Histological Verification Upon completion of the experiments, the animals received an overdose of sodium pentobabital and was perfused intracardially with saline followed by 10% formalin. The brains were removed and fixed in 10% formalin. The routine histological procedures for sectioning were used and stained preparations were observed under the light microscope to determine the location of the stimulated sites according to the Ro¨ssner atlas [52]. Statistical Analysis The results are reported as the mean ⫾ SEM of the durations of the TI episodes for each treatment. Due to variability and skewness in the TI duration data, a natural logarithmic (ln) transformation was performed on all durations scores prior to statistical analysis. Then, for groups 1, 2, and 6 the transformed data were submitted to repeated measure analysis of variance (ANOVA). However, for groups 3, 4, and 5 the data were submitted to two-way factorial analysis of variance followed, when significant treatment ⫻ dose interactions were found, by a repeated measure ANOVA for each
RESULTS
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FIG. 2. Duration of tonic immobility episodes. (A) Mean TI durations before (CONT) and after carbachol (CCh, 5.4 nmol/0.2 l) and CCh preceded by naloxone (CCh, 5.4 nmol/0.2 l ⫹ NAL, 2.74 nmol/0.2 l) microinjection into the ventrolateral periaqueductal gray matter of guinea pigs (n ⫽ 12). (B) Mean TI durations before (CONT) and after morphine (MS, 4.48 nmol/0.2 l) microinjection, and after pretreatment with naloxone plus MS microinjection (NAL, 2.74 nmol/0.2 l ⫹ MS, 4.48 nmol/0.2 l) into the ventrolateral PAG (n ⫽ 12). *p ⬍ 0.05 compared with control (A, B), after microinjection of naloxone followed by CCh (A) and after naloxone followed by morphine (B). The vertical bars indicate the standard error of the mean.
interaction between treatment and dose [F(4, 46) ⫽ 3.41, p ⬍ 0.01]. The repeated measure ANOVA showed a significant difference between treatments in group 3, F(1, 15) ⫽ 11.88, p ⬍ 0.001; in group 4, F(2, 14) ⫽ 18.28, p ⬍ 0.001; and in group
FIG. 3. Duration of tonic immobility episodes. (A) Mean TI durations before (CONT) and after bicuculline (BIC, 0.022 nmol/0.2 l) and muscimol (MUSC, 1 nmol/0.2 l) microinjection into the ventrolateral PAG of guinea pigs (n ⫽ 12). (B) Mean durations of five TI episodes before (CONT) and after bicuculline (0.2 nmol/0.2 l) and after muscimol (0.5 nmol/0.2 l) microinjection (n ⫽ 8). (C) Mean TI durations before (CONT) and after bicuculline (1 nmol/0.2 l) and after muscimol (0.26 nmol/0.2 l) microinjection (n ⫽ 7). *p ⬍ 0.05 compared to control; ⫹p ⬍ 0.05 compared to bicuculline. The vertical bars indicate the SEM of the mean.
MONASSI ET AL.
FIG. 4. Mean TI durations before (CONT) and after muscimol (MUSC, 0.13 nmol/0.2 l) and after morphine (MS, 4.48 nmol/0.2 l) microinjection and after microinjection of muscimol followed by morphine (MUSC, 0.13 nmol/0.2 l ⫹ MS, 4.48 nmol/0.2 l) into the ventrolateral PAG of guinea pigs (n ⫽ 8). *p ⬍ 0.05 compared with control and ⫹p ⬍ 0.05 compared to morphine. The vertical bars indicate the standard error of the mean.
5, F(1, 10) ⫽ 13.42, p ⬍ 0.001. Duncan’s post-hoc comparisons between treatments in group 3 showed that the duration of TI episodes after microinjection of 0.022 nmol of bicuculline did not differ from control durations ( p ⬎ 0.05), while the dose of 1 nmol of muscimol significantly decreased the TI durations when compare to control ( p ⬍ 0.05) and to bicuculline ( p ⬍ 0.05) microinjection. In group 4, the Duncan test showed a significant difference between muscimol (0.5 nmol) and control ( p ⬍ 0.05) and bicuculline ( p ⬍ 0.05), but not between bicuculline (0.2 nmol) and control. In group 5, the microinjection of 0.26 nmol of muscimol also decreased the duration of TI episodes ( p ⬍ 0.05, Duncan), while the microinjection of 1 nmol of bicuculline significantly increased the durations of TI episodes when compare to control ( p ⬍ 0.05, Duncan) and to muscimol microinjection ( p ⬍ 0.05, Duncan). One-way ANOVA followed by the Duncan test applied to the data for each treatment showed that while the control durations did not differ between groups 3, 4, and 5, the effect of bicuculline and muscimol were dose-dependent. The two lowest doses (0.022 and 0.2 nmol) of bicuculline did not alter TI duration, but the highest dose (1 nmol) of bicuculline significantly increased TI duration, and its effect was different from that of the other two doses ( p ⬍ 0.05, Duncan test). The three doses of muscimol used decreased the duration of TI, and their effect were not significantly different. Curiously, the dose of 1 nmol of muscimol appeared to increase the variability of TI duration, although its effect was not significantly different from that of the other two doses. The results for group 6 are shown in Fig. 4. As shown for group 2, the microinjection of MS (4.48 nmol) increased the duration of TI episodes, but this effect was blocked by pretreatment with an ineffective dose of muscimol (0.13 nmol). These findings were supported by the repeated measures ANOVA, which showed a significant difference between treatments [F(3, 13) ⫽ 23.41, p ⬍ 0.01]. The Duncan test revealed that while the treatment with muscimol (0.13 nmol) did not differ from the control durations, there were significant difference between MS and control ( p ⬍ 0.05), and MS and muscimol ( p ⬍ 0.05), and also between MS
PERIAQUEDUCTAL GRAY MATTER AND TONIC IMMOBILITY and the treatment with muscimol followed by MS ( p ⬍ 0.05). Treatment with muscimol followed by MS did not differ from the control situation. DISCUSSION The results of the present study indicate that the ventrolateral PAG possibly modulates TI duration via neural circuits involving cholinergic, opioidergic, and GABAergic mechanisms. In a previous study [41], we demonstrated that cholinergic stimulation of the ventrolateral PAG increases the duration of TI episodes and this effect is blocked by atropine microinjected into the same site, indicating that the effect was mediated by muscarinic receptors. In the present study we observed that the effect of increased TI duration induced by carbachol microinjected into the ventrolateral PAG can also be blocked by previous injection of naloxone into the same site. These results suggest that the action of the cholinergic system inside the ventrolateral PAG seems to be opioiddependent. On this basis, we may propose that the cholinergic transmission may activate the liberation of endorphins/enkephalins from interneurons of the ventrolateral PAG, resulting in increased TI. Indeed, Vicent and Reiner [57] demonstrated that the lack of abundance of choline acetyltransferase-immunoreactive cells and nerve terminals in the cat PAG does not appear to match with the presence of a high density of muscarinic receptors [19,25]. Thus, we may assume that the neurons having opioid receptors in the ventrolateral PAG may also contain muscarinic receptors, because in the present study, the effect of carbachol was blocked both by atropine [41] and by naloxone. We also observed that microinjection of morphine into the ventrolateral PAG also increased the duration of TI episodes and this effect was antagonized by naloxone, indicating that this action is probably mediated by -opioid receptors. Morphine injected into the ventrolateral PAG [21,48] produces antinociception by activating structures of the rostral ventromedial medulla that liberate the centrifugal pain-inhibitory system. As proposed by Basbaum and Fields [7] and Depaulis et al. [21], opiate activation of the descending pain-inhibitory system may result from a disinhibition of PAG output neurons. Opioids are generally considered to exert an indirect effect by decreasing the tonic activity of GABA neurons [42], thus causing a disinhibition of the PAG output neurons. The disinhibition of PAG output neurons may be important not only for the antinociceptive effect but also for the action of the PAG when performing some of the diverse functions that are attributed to it. On the basis of these findings, we may raise the hypothesis that the increased TI induced by morphine occurs in the same way, i.e., by disinhibition of PAG output neurons and the consequent activation of the descending pathways involved in TI modulation. The descending projections are mainly directed at the mesencephalic and pontine tegment, terminating in the cuneiform nucleus, parabrachial nucleus, and lateral pontine tegment [28]. In addition, the neurons of the ventrolateral PAG send fibers to the nucleus raphe magnus and raphe pallidus, and the latter in turn sends projections mainly to groups of somatic and autonomic motor neurons of the ventral horn of the spinal cord [29,30]. During TI there is inhibition of the righting reflex, which is essentially regulated by motor neurons distributed along the ventral horn of the spinal cord. Thus, we may propose that these motor neurons involved in the righting reflex receive inhibitory influences from the descending pathways of the nucleus raphe pallidus originating from, or activated by, the ventrolateral PAG. As proposed by Klemm [36], TI may be considered to be a reflex whose expression appears to be related to the activation of a pool of interneurons in the reticular formation of the brain stem that may influence descending neurons which inhibit the motor neurons of the spinal medulla. Another possible explanation for
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these results is that the ventrolateral PAG may exert an inhibitory influence directly on premotor and/or motor neurons of the ventral horn of the spinal cord involved in the righting reflex. In fact, more recently Mouton and Holstege [44] demonstrated the existence of direct projections from ventrolateral PAG to premotor interneurons in laminae VIII and VII, which extend throughout the length of the spinal cord and are involved in the control of neck and back movements. There is evidence that at least part of the neural circuits and the neurotransmitters involved in TI organization also play an important role in the modulation of antinociceptive responses. A number of studies have demonstrated that systemic injection of morphine increased the duration of TI in chickens [49], guinea pigs [45], and rabbits [15]. Also, microinjection of CCh into the basal midbrain of awake toads, at the doses that increase TI, abolishes the motor reflex triggered by a noxious stimulus [27]. Other studies [11,24, 34] have demonstrated that cholinergic agonists have antinociceptive properties in addition to potentiating the antinociceptive effects triggered by opioids [47]. Rodgers and Randal [51] argued that many of the stimuli that induce analgesia in rodents also elicit defense reactions, suggesting that there may be an important relationship between specific defense reactions and specific forms of analgesia. According to Bandler and Carrive [1] the “defence reaction” is often referred to as the pattern of behavioural and cardiovascular change characteristic of an animal’s reaction to threatening or stressful stimuli. More specifically, Rogers and Randal [51] suggested that non-opioid analgesia is activated by flight-eliciting stimuli, whereas opioid analgesia is activated by stimuli that elicit the terminal defense reaction of TI. Similarly, some investigators observed that electrical stimulation or stimulation with excitatory amino acids of the ventrolateral PAG results in opioid-related antinociception, hypotension, and defensive freezing, whereas dorsomedial and dorsolateral PAG stimulation results in non-opioid hypoalgesia, hypertension, and avoidance escape or aggressive responses [4,22,37]. Since Reynolds’ report [50], a large number of behavioral, pharmacological, and electrophysiological studies have established an important role for the PAG in the modulation of pain. It is possible that disinhibition through inhibitory GABA transmission is involved in the activation of PAG output neurons and behavioral antinociception [42]. The microinjection of muscimol into this substrate antagonizes the analgesia produced by simultaneous morphine microinjection and, conversely, microinjection of bicuculline, a GABA antagonist, produces analgesia [42]. These data suggest that the GABAergic network is an important mechanism for the activation of PAG output neurons. The present results show that microinjection of muscimol into the ventrolateral PAG decreased the duration of TI episodes, suggesting that the GABAergic neurons of the ventrolateral PAG have an inhibitory influence on PAG-output neurons that are involved in the modulation of TI in guinea pigs. On the other hand, the increase duration of TI caused by bicuculline microinjection into the ventrolateral PAG suggests disinhibition of PAG output neurons that may possibly be tonically inhibited. Since the ventrolateral PAG has high densities of GABA receptors and GABA neurons [5] disinhibition of neurons by blockage of GABA receptors with bicuculline is a very powerful method for activation of descending pathways from the PAG [54,55]. Within this context there is the possibility that both morphine and bicuculline act by possibly activating the same pathways that may probably act on the depression of the righting reflex. Indeed, Roychowdhury and Fields [53] observed that the antinociceptive action of bicuculline injected into the ventrolateral PAG was partially reversed by naloxone, indicating that the release of endogenous opioids is necessary for this effect. Thus, on the basis of the above data,
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FIG. 5. Schematic diagram of the hypotheses for the interaction within the ventrolateral periaqueductal gray matter (PAG) of cholinergic, opioidergic, and GABAergic systems involved in TI modulation. Local carbachol injection may activate opioidergic neurons, which in turn may inhibit GABAergic neurons, disinhibiting PAG output neurons and promoting the activation of descending pathway that may act by increasing TI. Local morphine injection activating opioidergic neurons and bicuculline injection inhibiting GABAergic neurons also results in disinhibition of PAG output neurons with activation of descending pathways and with the same effect on TI. Muscimol may facilitate the tonic inhibitory action of GABA inhibiting ventrolateral PAG output neurons and decreasing TI. Œ, inhibitory transmission; ‚, represent excitatory transmission. Abbreviations: CCh, cholinergic termination; GABA, GABAergic neuron; OP, opioidergic neuron.
MONASSI ET AL. effect on TI may occur directly or indirectly on the motor neurons of the ventral horn of the spinal cord. In summary, the present experiments have demonstrated that the involvement of the ventrolateral PAG in the modulation of TI in guinea pigs possibly occurs via an interaction between cholinergic, opioidergic and GABAergic systems. The cholinergic and opioidergic stimulation of the ventrolateral PAG increases the duration of TI, and both effects can be reversed by pretreatment with naloxone. Thus, these results suggest an interaction between opioid and cholinergic systems into the ventrolateral PAG, causing an increase in TI duration. Furthermore, the effect of muscimol (at effective doses) also showed that the GABAergic neurons of the ventrolateral PAG have an inhibitory influence on PAG-output neurons involved in the modulation of TI, and this effect seems to occur tonically and is mediated by GABAA receptors. We also observed that muscimol at a dose that has no effect per se on TI, when injected before morphine at the same site blocks the facilitatory effect of morphine. This supports the suggestion that the opioid effects may occur via inhibition of GABA interneurons and also that the modulation of TI by the ventrolateral PAG might, in part, occur via a neural circuit similar to that proposed for the antinociceptive system. ACKNOWLEDGEMENTS
another hypothesis for explaining the effect of bicuculline may be via the liberation of endogenous opioids, although in the present study we did not use naloxone to reverse the effect of bicuculline. Many reports have suggested an interaction between opioid and GABAergic circuits inside the PAG in the modulation of nociception [7,21,42,58,59]. Since opioids exert an inhibitory effect on neural excitability and transmitter release, it has been proposed that opioids in the PAG act by releasing descending output neurons by GABAergic inhibition, which may in turn lead to a disinhibition of descending antinociceptive circuits. Activation of this system inhibits nociceptive neurons in the dorsal horn of the spinal cord. In the present study we observed that in the ventrolateral PAG pretreatment with muscimol at a dose that has no effect per se, blocks the action of morphine on the duration of TI. This result suggests that microinjection of muscimol may reinforce the tonic inhibitory influence of GABAergic neurons on ventrolateral PAGoutput neurons, perhaps at the same time preventing the effect of morphine. According to this hypothesis, it is possible that during TI, the opioids in the PAG act by inhibiting the GABA interneurons. These findings represent indirect experimental evidence that the circuits containing opioidergic and GABAergic neurons inside the ventrolateral PAG involved in TI modulation may be similar, in part, to the circuits proposed by Basbaum and Fields [7] for the modulation of antinociception. Figure 5 schematically shows the neural circuits inside the ventrolateral PAG that may possibly be involved in TI modulation. Although in a speculative manner, the present results may permit us to postulate that the ventrolateral PAG may function as an interface in the systems that modulate TI and analgesia. This region contains neurons involved in the organization of different stages of defensive behavior including TI, and neurons related to pain modulation. These neuronal groups may be mobilized simultaneously by the same environmental stimuli in a situation of confrontation, resulting in analgesia that may permit the execution of defensive behaviors. It is possible to propose descending pathways originating from the PAG and modulating both analgesia and TI. There is reason to believe that descending pathways originating from the PAG exert a net inhibitory effect on nociceptive transmission at the level of the dorsal horn, whereas the inhibitory
We would like to thank Dr. Pravin K. Mishira from DBTS/University of Illinois College of Medicine at Peoria, for constructive comments on the manuscript and Mrs Mariulza Rocha Brentegani and Mr Rubens Fernando de Melo for technical assistance and histological processing. This work was supported by FAPESP (Proc. 97/08787-5).
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