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Role of dorsal raphe nucleus 5-HT1A and 5-HT2 receptors in tonic immobility modulation in guinea pigs
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Role of dorsal raphe nucleus 5-HT1A and 5-HT2 receptors in tonic immobility modulation in guinea pigs Mateus Dalbem Ferreira, Leda Menescal-de-Oliveira⁎ Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, 14049-900 Ribeirão Preto, SP, Brazil
A R T I C LE I N FO
AB S T R A C T
Article history:
Tonic immobility (TI) is an innate defensive behavior characterized by a state of physical
Accepted 10 June 2009
inactivity and diminished responsiveness to environmental stimuli. Behavioral adaptations
Available online 16 June 2009
to changes in the external and internal milieu involve complex neuronal network activity and a large number of chemical neurotransmitters. The TI response is thought to be
Keywords:
influenced by serotonin (5-HT) activity in the central nervous system (CNS) of vertebrates,
Dorsal raphe nucleus
but the neuronal groups involved in the mechanisms underlying this behavior are poorly
Serotonin
understood. Owing to its extensive afferents and efferents, the dorsal raphe nucleus (DRN)
Tonic immobility
has been implicated in a great variety of physiological and behavioral functions. In the
Defensive behavior
current study, we investigated the influence of serotonergic 5-HT1A and 5-HT2 receptor activity within the DRN on the modulation of TI behavior in the guinea pig. Microinjection of a 5-HT1A receptor agonist (8-OH-DPAT, 0.01 and 0.1 μg) decreased TI behavior, an effect blocked by pretreatment with WAY-100635 (0.033 μg), a 5-HT1A antagonist. In contrast, activation of 5-HT2 receptors within the DRN (α-methyl-5-HT, 0.5 μg) increased the TI duration, and this effect could be reversed by pretreatment with an ineffective dose (0.01 μg) of ketanserine. Since the 5-HT1A and 5-HT2 agonists decreased and increased, respectively, the duration of TI, different serotonin receptor subtypes may play distinct roles in the modulation of TI in the guinea pig. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
Tonic immobility (TI), also known as reflex immobility, animal hypnosis, catalepsy, or death feigning, can be defined as an inborn defensive response characterized by a temporary state of profound inactivity and a relative lack of responsiveness to external stimuli. The duration of TI episodes is quite variable, with the animal remaining motionless for seconds or hours depending on the species and environmental conditions (Hoagland, 1974). This response is triggered in many vertebrate and invertebrate species by different types of sensory stimuli, primarily prolonged physical contact with predators
(Gilman et al., 1950; Ratner, 1967; Klemm, 1971). Tonic immobility is an inhibitory behavioral response in which the absence of movement appears to be of great adaptive value since struggling may encourage the predator to continue the attack (Thompson et al., 1981). Thus, according to the literature, the initiation of TI during a confrontation increases the chances of surviving a predatory attack by 50% (Sargent and Eberhardt, 1975). The animal's survival depends on its capacity to appear immobile, which encourages the predator to stop the attack (Rodgers and Randal, 1987). Different experimental approaches have been used to determine the regions of the central nervous system (CNS)
⁎ Corresponding author. Fax: +1 55 16 633 0017. E-mail address: [email protected] (L. Menescal-de-Oliveira). 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.06.030
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that control the TI response. In addition, it has been shown that brainstem structures are fundamental to the expression of this behavior (Klemm, 1976; Franchi-Vasconcelos and Hoffman, 1994; Monassi et al., 1999). Studies carried out in our laboratory have demonstrated that the periaqueductal gray matter (PAG) (Monassi et al., 1997; Ramos Coutinho et al., 2008), the amygdala (Leite-Panissi et al., 1999; Leite-Panissi and Menescal-de-Oliveira, 2002), the parabrachial region (Menescal-de-Oliveira and Hoffmann, 1993), the hypothalamus (Oliveira et al., 1997), the nucleus raphe magnus (NRM) (Silva and Menescal-de-Oliveira, 2006 and 2007) and the hippocampus (unpublished data) are involved in TI modulation. The dorsal raphe nucleus (DRN), located in the ventral part of the periaqueductal gray matter, is the most prominent of the brainstem serotonergic nuclei. A very important mechanism of control of 5-HT neurons is self-inhibition through 5-HT1A autoreceptors. Activation of these receptors by 5-HT leads to opening of potassium channels in the cell membrane, hyperpolarization of the cell and a cessation of cell firing (Sprouse and Aghajanian, 1987). In particular, 5-HT1A autoreceptors in the DR may play a pivotal role in
the physiological control of ascending 5-HT pathways, attenuating excessive activation of 5-HT neurons by excitatory afferents from the different forebrain structures. The proportion of 5-HT-containing cells in the DRN is estimated to range from 1/3 to 2/3 (Descarries et al., 1982; Jacobs and Azmitia, 1992; Baumgarten and Grozdanovic, 1997). The remaining non-serotonergic cells contain a variety of other neurotransmitters and neuromodulators (Jacobs and Azmitia, 1992). Some reports have shown that the serotonergic system is an important neuromodulator of central nervous system activity, and TI is a behavior that seems to be highly influenced by cerebral levels of serotonin (5-HT) (Wallnau and Gallup, 1977). Systemic studies have demonstrated that different doses of serotonin could produce an increase or decrease in the IT duration in chickens (Henning, 1980). In the same way, intraventricular administration of 5-HT produced opposing results in chickens and rabbits (Harston et al., 1976; Hatton et al., 1978). These different results have been attributed to differences in the route of drug administration or the species used. Moreover, systemic injections of 5-HT could alter the levels of 5-HT in the entire CNS.
Fig. 1 – Duration of tonic immobility (TI) episodes. Mean ± S.E.M. (A) TI duration under control conditions (CONT), after surgery (SHAM) and after 5-HT1A agonist 8-OH-DPAT [DPAT; Aa (0.005 μg; n = 12), Ab (0.01 μg; n = 10), Ac (0.1 μg; n = 11)]; (B) TI duration after WAY-100635 (WAY; 0.033 μg) followed 10 min later by DPAT (0.01 μg; n = 7). *p < 0.05 compared with CONT and SHAM; (C) TI duration after two different doses of WAY (0.033 μg and 0.067 μg) injection into the DRN on consecutive days (n = 8); (D) Schematic drawings of frontal sections obtained at representative levels of the guinea pig DRN indicating the site of microinjection (filled circles, ●) of DPAT (0.005 μg, DI), DPAT (0.01 μg, DII), DPAT (0.1 μg, DIII), WAY (0.033 μg) followed by DPAT (0.01 μg, DIV) and WAY (0.033 μg and 0.067 μg, DV). Abbreviations: PAG, periaqueductal gray matter; A, brain aqueduct.
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In a recent study, Monassi and Menescal-de-Oliveira (2004) showed that the action of 5-HT inside the ventrolateral and dorsal periaqueductal gray matter appears to be biphasic and dose-dependent. In these regions, the microinjection of low doses (0.1 μg) of 5-HT increased TI, whereas high doses (1, 3 and 6 μg) decreased this behavioral response. The authors suggested that the PAG 5-HT1A and 5-HT2 receptors play different roles in the modulation of TI, since the 5-HT1A and 5HT2 agonists, respectively, increased and decreased the duration of TI. To date, research has established 16 serotonergic receptor subtypes that are divided into 7 families based on operational, structural and transductional information (Hoyer et al., 2002). Utilizing radioligand-binding techniques, Peroutka and Snyder (1983) showed that 5-HT binds with high affinity in specific sites in the rat cortex, and these sites were named 5-HT1 and 5-HT2. The receptors 5-HT2 and 5-HT1A are considered to be the primary 5-HT receptors, with their effects related to excitatory and inhibitory responses, respectively (Brandão et al., 1991; Lovick, 1993). Considering that 5-HT1A and 5-HT2 serotonergic receptors when activated may produce different actions, the objective of the present study was to evaluate the effects of the serotonergic 5-HT2 and 5-HT1A agonists of the dorsal raphe nucleus on the duration of TI in guinea pigs.
2.
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Results
2.1. Experiment 1: Effect of microinjection of the 8-OH-DPAT and WAY-100635 Our results showed that the microinjection of two different doses of 8-OH-DPAT (0.01 and 0.1 μg; selective 5-HT1A agonist) into the DRN significantly decreased the duration of TI (Duncan's post-test) when compared with the control and sham treatments (Fig. 1Ab,c). A one-way ANOVA showed that the effect of the two doses of 8-OH-DPAT (0.01 and 0.1 μg) microinjected into the DRN was different between the groups [F(2, 18) = 5.914, p = 0.011, to the first dose; and F(2,20) = 11.822, p = 0.001 to the second dose]. However, the microinjection of 0.005 μg into the same site did not produce a significant effect (p > 0.05; Fig. 1Aa). Prior application of WAY-100635 (0.033 μg; selective 5-HT1A antagonist) into the DRN blocked the 8-OHDPAT-induced decrease in the TI duration in guinea pigs (0.1 μg; Fig. 1B). ANOVA for repeated measures indicated a significant difference between treatments [F(3,18) = 4.277; p = 0.019], and Duncan's post-test showed that treatment with 8-OH-DPAT differed from the other treatments (control, sham and the combination of antagonist/agonist 5-HT1A), which did not differ from one another. In addition, microinjection of
Fig. 2 – Duration of tonic immobility (TI) episodes. Mean ± S.E.M. (A) TI duration under control conditions (CONT), after surgery (SHAM) and after 5-HT2 agonist α-methyl-5-HT [α-Methyl; Aa (0.1 μg; n = 10) and Ab (0.5 μg; n = 9)]; (B) TI duration after ketanserin (KET; 0.01 μg) followed 10 min later by α-Methyl (0.5 μg; n = 10). *p < 0.05 compared with CONT and SHAM; (C) TI duration after two different doses of KET (0.01 μg and 0.03 μg) injection into the DRN on consecutive days (n = 7); (D) Schematic drawings of frontal sections obtained at representative levels of the guinea pig DRN indicating the site of microinjection (filled circles, ●) of α-Methyl (0.01 μg, DI), α-Methyl (0.5 μg, DII), KET (0.01 μg) followed by α-Methyl (0.5 μg, DIII) and KET (0.01 μg and 0.03 μg, DIV). Abbreviations: PAG, periaqueductal gray matter; A, brain aqueduct.
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the two doses of WAY-100635 alone in consecutive days did not alter the mean duration of TI when compared with the control and sham treatments (p = 0.114; Fig. 1C). Fig. 1D shows a schematic representation of the microinjection sites of the 8-OH-DPAT and WAY-100635 in the DRN.
2.2. Experiment 2: Effect of microinjection of alfa-methyl-5-HT and ketanserin As shown in Fig. 2Ab, the microinjection of 0.5 μg of α-methyl5-HT (selective 5-HT2 agonist) into the DRN increased the duration of TI compared with controls [ANOVA; F(2,16) = 6.007, p = 0.011]. Duncan's post-test demonstrated a difference in the mean duration of TI between animals treated with 0.5 μg of the α-methyl-5-HT and the control and sham groups. However, the microinjection of α-methyl-5-HT (0.1 μg) into the DRN did not significantly alter the duration of TI (p > 0.05; Fig. 2Aa). Prior application of ketanserin (0.01 μg; selective 5-HT2 antagonist) into the DRN blocked the α-methyl-5-HT-induced increase in the duration of TI in guinea pigs (0.5 μg; Fig. 2B). ANOVA for repeated measures indicated a significant difference between treatments [F(3,27) = 4.469; p = 0.011], and Duncan's post-test showed that treatment with α-methyl-5-HT differed from the other treatments (control, sham and the combination of antagonist/agonist 5-HT2), which did not differ from one another. Microinjection of ketanserin (0.01 and 0.03 μg in consecutive days) into the DRN did not significantly modulate the TI duration in guinea pigs at any dose (p > 0.05; Fig. 2C). In addition, the microinjection of saline did not alter the mean duration of TI when compared with the control and sham treatments (p > 0.05; Fig. 3A). Fig. 2D shows a schematic representation of the α-methyl-5-HT and ketanserin micro-
Fig. 3 – Duration of tonic immobility (TI) episodes. Mean ± S.E.M. (A) TI duration under control conditions (CONT), after surgery (SHAM) and after saline (SAL; 0.2 μl; n = 12) microinjection into the dorsal raphe nucleus (DRN); (B) Schematic drawings of frontal sections obtained at representative levels of the guinea pig DRN indicating the site of microinjection (filled circles, ●) of saline (SAL).
injection sites in the DRN and Fig. 3B the microinjection of saline.
3.
Discussion
Present experiment results point to the hypothesis that the modification in the activity of 5-HT cells of the DNR by using 5HT1A and 5-HT2 agonists can modulate TI duration in guinea pigs. We found that the microinjection of the 5-HT1A receptor agonist 8-OH-DPAT into the DRN decreased TI duration, whose effect was blocked by the previous injection of the 5HT1A antagonist, the WAY-100635. As to the effect obtained through the 8-OH-DPAT, an indirect effect on the response studied can be supposed, involving serotonergic projection structures and interaction with other mediators. The indirect effect can occur by the regulation of another neurotransmitter's release in the same or other sites. Literature data show that the release of ACh in the hippocampus receives a tonic inhibitory influence from serotonergic projections coming from the dorsal raphe nucleus (Azmitia and Segal, 1978). Moreover, the existence of functional interaction between serotonin and ACh neurotransmission in the central nervous system is extensively documented (Robinson, 1983; Wenk and Engisch, 1986; Quirion and Richard, 1987). In that sense, some authors (Bianchi et al., 1990; Izumi et al., 1994; Wilkinson et al., 1994; Consolo et al., 1996) have shown that 8-OH-DPAT increases the release of acetylcholine in the cortex and hippocampus of guinea pigs and rats and this effect is blocked by selective (WAY 100 635) 5-HT1A receptor antagonists (Bianchi et al., 1990; Wilkinson et al., 1994; Consolo et al., 1996). Considering that, when activated or inhibited, the serotonergic projections of DRN can influence other structures, one possible explanation for our results is that the TI reduction effect produced by the 8-OH-DPAT in the DRN could occur through the activation of the hippocampus' cholinergic system, increasing the release of acetylcholine (ACh) in this site and thus facilitating TI reduction. In line with the proposition that the TI reduction effect through the 8-OHDPAT can occur indirectly via the hippocampus, data obtained at this research laboratory (Baptista 2000, unpublished data) showed that administering carbachol (cholinergic antagonist) in the dorsal hippocampus decreased the duration of TI response in guinea pigs. The involvement of the dorsal hippocampus in TI response has been well documented. It is known that the hippocampus ablation prolongs TI response in rabbits (Woodruff et al., 1975). The hippocampal formation is involved in two important questions related with TI response. First, with fear (Klemm, 1977, 2001), which is considered an important factor for TI expression (Gallup, 1974). Second, the dorsal hippocampus plays an important role in motor activity, in which acetylcholine is important (Dudar et al., 1979; Day et al., 1991). The sensorial stimulation and the motor activity enhance acetylcholine release in the hippocampus (Dudar et al., 1979) and the carbachol microinjection into dentate gyrus leads to a motor hyperactivity (Flicker and Geyer, 1982; Mogenson and Nielsen, 1984a,b). Thompson et al. (1980) raised the hypothesis that the hippocampus plays an important role in the motor component of the TI response. Hence, one may consider the possibility that the TI reduction effect by the 8-
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OH-DPAT in the DRN indirectly results from the increased release of ACh in the hippocampus. This effect is similar to the results of carbachol (cholinergic antagonist) when injected directly into the hippocampus of guinea pigs. Wilkinson et al. (1994) also showed that the systemic administration of 8-OHDPAT produced a clear dose-dependent increase in the release of acetylcholine in guinea pigs' hippocampus. Although the administration was peripheral, the effect of 8-OH-DPAT on ACh release may occur through its activity in autoreceptors of DRN. However, one cannot totally discard the possibility that autoreceptors are involved in other structures. The role of DRN can be strengthened by the report of Auerbach et al. (1989), where administration of 8-OH-DPAT could have an indirect effect on ACh release through a primary action at the somatodendritic autoreceptor of 5-HT-containing neurons within DRN that would inhibit 5-HT release at the nerve terminal. Our results also show that the (WAY 100 635) 5-HT1A receptor antagonists previously microinjected into the DRN antagonized the increasing effect on the duration of TI, indicating the participation of 5-HT1A receptors. When the WAY 100 635 was injected alone into the DRN, however, no modifications in TI response were observed, possibly indicating that the 5-HT1A receptors of guinea pigs' DRN are not tonically active in these experimental conditions. In the same sense, Hughes and Dawson (2004) suggest that, in guinea pigs, 5-HT1A receptors do not appear to be tonically active, as the administration of WAY100635 does not affect extracellular 5HT in either hippocampus or frontal cortex. Roberts et al. (1999) showed that antagonism of 5-HT1A receptors does not affect extracellular levels of 5-HT in either structures in guinea pigs in vivo. In this research, it was also observed that the injection of the 5-HT2 receptor agonist α-metyl-5-HT into the DN produced an increase in the duration of TI. This effect was blocked by pretreatment with ketanserin, a 5-HT2 antagonist. The increasing effect of alpha-methyl-5-TH on TI duration may also have occurred indirectly, that is, involving the decreased release of ACh in the hippocampus. Hence, as already mentioned, according to Azmitia and Segal (1978), ACh release in the hippocampus receives a tonic inhibitory influence of serotonergic projections coming from the dorsal raphe nucleus. In this sense, it was demonstrated by Barnes et al. (1989) that alpha-methyl-5TH produced inhibition of ACh release from slices of rats' entorhinal cortex. The same effect was also observed in cortex slices of guinea pigs through alpha-methyl-5TH administrations in higher concentrations (Bianchi et al., 1990). It is interesting to observe that, in our research laboratory (unpublished data), the TI reduction effect by carbachol was antagonized by an ineffective dose of atropine (muscarinic cholinergic antagonist). However, higher doses of atropine injected in the HC also produced an increase in TI duration. This effect is supposedly due to the occupation of muscarinic receptors and the inhibition of ACh action. As to the TI increased effect by the administration of alpha-methyl5TH in the DRN, one could think of tonic inhibition by serotonin release in the hippocampus, thus producing a decreased ACh release and a consequent TI increase. Hence, one may suppose that the TI increased effect by alpha-methyl5TH in the DRN could also occur indirectly through the decrease of ACh in the hippocampus.
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In summary, our results show that the stimulation of 5HT1A receptors in the DRN by 8-OH-DPAT decreased the duration of TI episodes, while the activation of 5-HT2 receptors by α-methyl-5-HT increased the TI duration. Thus, the activation of different DRN serotonergic receptors can be responsible for the balance in 5-HT release that may modulate the duration of the defensive TI response in guinea pigs.
4.
Experimental procedures
4.1.
Animals
Adult male guinea pigs (Cavia porcellus) weighing 420–470 g were obtained from the animal care facility of the Faculty of Medicine of Ribeirão Preto (FMRP). The animals were kept in Plexiglas wall cages (56 cm × 37 cm × 39 cm, five animals per cage) in a room maintained at 24 ± 1 °C, on a 12-h light cycle, with free access to water and food, during the entire experimental period. The experiments were carried out in compliance with the recommendations of Brazilian Association for Laboratory Animal Science (COBEA) and with the approval (Proc. no. 069/2004) of the Ethical Committee for Animal Experimentation of the School of Medicine of Ribeirão Preto, University of São Paulo. All efforts were made to minimize animal suffering.
4.2.
Procedures
4.2.1.
Induction of tonic immobility
Each naive guinea pig was submitted to five control maneuvers designed to induce TI, and the duration of the episodes was recorded. Experimentally, the induction of TI was attempted by holding the animal around the thorax with the hands, quickly inverting it and pressing it down into a shaped plywood trough (25 cm long × 15 cm high). The pressure applied by the experimenter's hands was proportional to the resistance offered by the animal to the restraining maneuver. When the animal stops moving, the experimenter slowly withdraws his hands and a chronometer is activated to measure the duration (in seconds) of the response, which ends when the animal resumes the upright position. If the animal does not become motionless within 60 s, the episode is recorded as having zero duration. This procedure was repeated at intervals ranging between 1 and 3 min between each episode. TI episodes were held in the afternoon, in a semi-dark room, with background noise (air-conditioning functioning). Before any manipulation, the animal was maintained in the testing room for 20 min in order to habituate to the experimental situation. For group analysis, the mean of five episodes per animal was calculated. It was established that, in a single trial group, the animals serve as their own controls, that is, the mean of TI durations in the control treatment was compared with the mean of the sham and of the experimental treatments.
4.2.2.
Stereotaxic surgery and microinjection procedure
One day after the control TI episode, the animals were anesthetized by intramuscular injection of 40 mg/kg ketamine plus 5 mg/kg xylazine, and placed in a stereotaxic apparatus
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(David-Kopf Instruments, USA) with the mouthpiece 17 mm below the interauricular line. A midline incision of the scalp was made, exposing the skull, and a small burr hole was made with a dental drill. A guide cannula (14 mm long and 0.6 mm outer diameter, prepared from a hypodermic needle) was implanted into the DRN. According to the atlas of Rössner (1965) for guinea pigs, the stereotaxic coordinates for placement of the guide cannula implanted in the DRN were + 10.8 mm caudal to the bregma, 0.0 mm lateral to the midline and + 3.5 mm below the intra-aural line. The guide cannula was lowered to a depth of 1 mm above the target regions and fixed to the skull with autopolymerizing resin and an additional anchoring screw. After 6–7 days of recovery from surgery, microinjections were performed with a Hamilton microsyringe (10 μl) connected to a PE-10 polyethylene catheter, which in turn was coupled to a Mizzy needle segment (0.3 mm outer diameter; 1.0 mm longer than the guide cannula). In all experimental groups, a volume of 0.2 μl was microinjected over a period of 1 min and the Mizzy needle was left in place for an additional 1 min to avoid reflux.
4.3.
Drugs and experimental groups
The drugs used were: 8-hydroxy-dipropylaminotretalin (8-OHDPAT; RBI, USA), WAY-100635 Maleate (Sigma, St. Louis, USA), α-methyl-5-hydroxytriptamin (α-methyl-5-HT; RBI, USA), and ketanserin (RBI, USA). The solutions were freshly dissolved in 0.9% saline containing 0.1% ascorbic acid, and sodium bicarbonate was added to adjust the pH to 7.4, which also served as vehicle control. All microinjections were performed in a volume of 0.2 μl. Also, in all experimental groups, each animal served as its own control. The doses were based on previous studies (Zanoveli et al., 2003; Monassi and Menescalde-Oliveira, 2004; Soares and Zangrossi, 2004; Pobbe and Zangrossi, 2005). To evaluate the effect of the drugs used, the animals were assigned into 2 different experimental groups. In experiment 1, the effect of 8-OH-DPAT (selective 5-HT1A agonist) and WAY-100635 (selective 5-HT1A antagonist) on the TI duration were tested, and the drugs were microinjected into the DRN. In this experiment, the animals were distributed among five groups. In group 1 (n = 12), group 2 (n = 10) and group 3 (n = 11), the animals were microinjected with 8-OH-DPAT (0.005, 0.01 and 0.1 μg/0.2 μl, respectively); in group 4 (n = 7) the animals were microinjected with WAY-100635 (0.033 μg/0.2 μl) followed 1 min later by 8-OH-DPAT (0.01 μg/0.2 μl); in group 5 (n = 8), the animals were microinjected with WAY-100635 in two different doses (0.033 and 0.067 μg/0.2 μl) on consecutive days. In experiment 2, we tested the effect of α-methyl-5-HT (selective 5-HT2 agonist) and ketanserin (selective 5-HT2 antagonist) on the TI duration, and the drugs were microinjected into the DRN. In this experiment, the animals were distributed in four groups. In group 1 (n = 10) and group 2 (n = 9), the animals were microinjected with α-methyl-5-HT (0.1 and 0.5 μg/0.2 μl, respectively); in group 3 (n = 10) the animals were microinjected with ketanserin (0.01 μg/0.2 μl), followed 1 min later by α-methyl-5-HT (0.5 μg/0.2 μl); in group 4 (n = 7) the animals were microinjected with ketanserin (0.01 and
0.03 μmol/0.2 μl) on consecutive days; in group 4 (n = 12), animals were microinjected with saline (0.2 μl) to the control of vehicle.
4.4.
Histological verification
Following all tests, the injection site was marked by microinjecting 0.2 μl of 2% pontamine sky blue dye. Each animal was deeply anesthetized with sodium pentobarbital and perfused intracardially with saline followed by 10% formalin. The brains were removed and fixed in 10% formalin for 4 days. Routine histological procedures were used for sectioning, and stained preparations were observed under a light microscope to determine the location of the stimulated sites according to the Rössner atlas (1965). Only guinea pigs with microinjection site located inside the target structure (DRN) were included in the data analysis.
4.5.
Statistical analysis
The TI results are reported as mean ± standard error of the mean (SEM) of the five TI episodes. Data were analyzed by analysis of variance (ANOVA) for repeated measures. The degree of freedom of the repeated measure (treatment) was corrected by the Huynh–Feldt ɛ parameter. The Duncan test was used to determine the difference between treatments, with the level of significance set at p < 0.05.
Acknowledgments We would like to thank Mrs. Mariulza Rocha Brentegani and Mr. Rubens Fernando de Melo for technical assistance and histological processing. This work was supported by CNPq (Proc. 134032/2004-7). REFERENCES
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Menescal-de-Oliveira, L., Hoffmann, A., 1993. The parabrachial region as a possible region modulating simultaneously pain and tonic immobility. Behav. Brain Res. 56, 127–132. Monassi, C.R., Menescal-de-Oliveira, L., 2004. Serotonin 5-HT2 and 5-HT1A receptors in the periaqueductal gray matter differentially modulate tonic immobility in guinea pig. Brain Res. 1009, 169–180. Monassi, C.R., Hoffmann, A., Menescal-de-Oliveira, L., 1997. Involvement of the cholinergic system and periaqueductal gray matter in the modulation of tonic immobility in the guinea pig. Physiol. Behav. 62, 53–59. Monassi, C.R., Leite-Panissi, C.R., Menescal-de-Oliveira, L., 1999. Ventrolateral periaqueductal gray matter and the control of tonic immobility. Brain Res. Bull. 50, 201–208. Mogenson, G.J., Nielsen, M., 1984a. A study of the contribution of hippocampal–accumbens–subpallidal projections to locomotor activity. Behav. Neural Biol. 42, 38–51. Mogenson, G.J., Nielsen, M., 1984b. Neuropharmacological evidence to suggest that the nucleus accumbens and subpallidal region contribute to exploratory locomotion. Behav. Neural Biol. 42, 52–60. Oliveira, L., Hoffmann, A., Menescal-de-Oliveira, L., 1997. Participation of the medial and anterior hypothalamus in the modulation of tonic immobility in guinea pigs. Physiol. Behav. 62, 1171–1178. Peroutka, S.J., Snyder, S.H., 1983. Multiple serotonin receptors and their physiological significance. Fed. Proc. 42, 213–217. Pobbe, R.L., Zangrossi Jr, H., 2005. 5-HT(1A) and 5-HT(2A) receptors in the rat dorsal periaqueductal gray mediate the antipanic-like effect induced by the stimulation of serotonergic neurons in the dorsal raphe nucleus. Psychopharmacology 183, 314–321. Quirion, R., Richard, J., 1987. Differential effects of selective lesions of cholinergic and dopaminergic neurons on serotonin-type 1 receptors in rat brain. Synapse 1, 124–130. Ramos Coutinho, M., da Silva, L.F., Menescal-de-Oliveira, L., 2008. Modulation of tonic immobility in guinea pig PAG by homocysteic acid, a glutamate agonist. Physiol. Behav. 94, 468–473. Ratner, C.S., 1967. Comparative aspects of hypnosis. In: Gordon, J.E. (Ed.), Handbook of Clinical and Experimental Hypnosis. Macmillian, New York, pp. 550–587. Roberts, C., Boyd, D.F., Middlemmis, D.N., Routledge, C., 1999. Enhancement of 5-HT1B and 5-HT1D receptor antagonist effects on extracellular 5-HT levels in the guinea pig brain following concurrent 5-HT1A or 5-HT re-uptake site blockade. Neuropharmacology 38, 1409–1419. Robinson, S.E., 1983. Effect of specific serotonergic lesions on cholinergic neurons in the hippocampus, cortex and striatum. Life Sci. 32, 345–353. Rodgers, J.H., Randal, J.I., 1987. Defensive analgesia in rats and mice. Psychol. Rec. 37, 335–347. Rössner, W., 1965. Stereotaktischer Hirnatlas vom Meerschweinchen. Pallas Verlag Munich. Sargent, A.B., Ebehardt, L.E., 1975. Death feigning by ducks in response to predation by red foxes (Vulpes fulva). Am. Midl. Nat. 94, 108–119. Silva, L.F., Menescal-de-Oliveira, L., 2006. Cholinergic modulation of tonic immobility and nociception in the NRM of guinea pig. Physiol. Behav. 87, 821–827. Silva, L.F., Menescal-de-Oliveira, L., 2007. Role of opioidergic and GABAergic neurotransmission of the nucleus raphe magnus in the modulation of tonic immobility in guinea pigs. Brain Res. Bull. 72, 25–31. Soares, V.P., Zangrossi Jr, H., 2004. Involvement of 5-HT1A and 5-HT2 receptors of the dorsal periaqueductal gray in the regulation of the defensive behaviors generated by the elevated T-maze. Brain Res. 64, 181–188.
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Sprouse, J.S., Aghajanian, G.K., 1987. Electrophysiological responses of serotoninergic dorsal raphe neurons to 5-HT1A and 5-HT1B agonists. Synapse 1, 3–9. Thompson, R.F., Berger, T.W., Berry, S.D., 1980. Brain mechanisms of learning. In: Thompson;, R.F., Hicksans, C.H., Shvyrkov, V.B. (Eds.), Neural Mechanisms of Goal-directed Behaviour and Learning. Academic Press, New York and London. Thompson, R.K.R., Foltin, R.W., Boylan, R.J., Sweet, A., Graves, C.A., Lowitz, C.E., 1981. Tonic immobility in Japanese quail can reduce the probability of sustained attack by cats. Anim. Learn. Behav. 9, 145–149. Wallnau, L.B., Gallup, G.G., 1977. A serotonergic, midbrain-raphe model of tonic immobility. Biobehav. Rev. 1, 35–43.
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Role of dorsal raphe nucleus 5-HT1A and 5-HT2 receptors in tonic immobility modulation in guinea pigs Mateus Dalbem Ferreira, Leda Menescal-de-Oliveira⁎ Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, 14049-900 Ribeirão Preto, SP, Brazil
A R T I C LE I N FO
AB S T R A C T
Article history:
Tonic immobility (TI) is an innate defensive behavior characterized by a state of physical
Accepted 10 June 2009
inactivity and diminished responsiveness to environmental stimuli. Behavioral adaptations
Available online 16 June 2009
to changes in the external and internal milieu involve complex neuronal network activity and a large number of chemical neurotransmitters. The TI response is thought to be
Keywords:
influenced by serotonin (5-HT) activity in the central nervous system (CNS) of vertebrates,
Dorsal raphe nucleus
but the neuronal groups involved in the mechanisms underlying this behavior are poorly
Serotonin
understood. Owing to its extensive afferents and efferents, the dorsal raphe nucleus (DRN)
Tonic immobility
has been implicated in a great variety of physiological and behavioral functions. In the
Defensive behavior
current study, we investigated the influence of serotonergic 5-HT1A and 5-HT2 receptor activity within the DRN on the modulation of TI behavior in the guinea pig. Microinjection of a 5-HT1A receptor agonist (8-OH-DPAT, 0.01 and 0.1 μg) decreased TI behavior, an effect blocked by pretreatment with WAY-100635 (0.033 μg), a 5-HT1A antagonist. In contrast, activation of 5-HT2 receptors within the DRN (α-methyl-5-HT, 0.5 μg) increased the TI duration, and this effect could be reversed by pretreatment with an ineffective dose (0.01 μg) of ketanserine. Since the 5-HT1A and 5-HT2 agonists decreased and increased, respectively, the duration of TI, different serotonin receptor subtypes may play distinct roles in the modulation of TI in the guinea pig. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
Tonic immobility (TI), also known as reflex immobility, animal hypnosis, catalepsy, or death feigning, can be defined as an inborn defensive response characterized by a temporary state of profound inactivity and a relative lack of responsiveness to external stimuli. The duration of TI episodes is quite variable, with the animal remaining motionless for seconds or hours depending on the species and environmental conditions (Hoagland, 1974). This response is triggered in many vertebrate and invertebrate species by different types of sensory stimuli, primarily prolonged physical contact with predators
(Gilman et al., 1950; Ratner, 1967; Klemm, 1971). Tonic immobility is an inhibitory behavioral response in which the absence of movement appears to be of great adaptive value since struggling may encourage the predator to continue the attack (Thompson et al., 1981). Thus, according to the literature, the initiation of TI during a confrontation increases the chances of surviving a predatory attack by 50% (Sargent and Eberhardt, 1975). The animal's survival depends on its capacity to appear immobile, which encourages the predator to stop the attack (Rodgers and Randal, 1987). Different experimental approaches have been used to determine the regions of the central nervous system (CNS)
⁎ Corresponding author. Fax: +1 55 16 633 0017. E-mail address: [email protected] (L. Menescal-de-Oliveira). 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.06.030
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that control the TI response. In addition, it has been shown that brainstem structures are fundamental to the expression of this behavior (Klemm, 1976; Franchi-Vasconcelos and Hoffman, 1994; Monassi et al., 1999). Studies carried out in our laboratory have demonstrated that the periaqueductal gray matter (PAG) (Monassi et al., 1997; Ramos Coutinho et al., 2008), the amygdala (Leite-Panissi et al., 1999; Leite-Panissi and Menescal-de-Oliveira, 2002), the parabrachial region (Menescal-de-Oliveira and Hoffmann, 1993), the hypothalamus (Oliveira et al., 1997), the nucleus raphe magnus (NRM) (Silva and Menescal-de-Oliveira, 2006 and 2007) and the hippocampus (unpublished data) are involved in TI modulation. The dorsal raphe nucleus (DRN), located in the ventral part of the periaqueductal gray matter, is the most prominent of the brainstem serotonergic nuclei. A very important mechanism of control of 5-HT neurons is self-inhibition through 5-HT1A autoreceptors. Activation of these receptors by 5-HT leads to opening of potassium channels in the cell membrane, hyperpolarization of the cell and a cessation of cell firing (Sprouse and Aghajanian, 1987). In particular, 5-HT1A autoreceptors in the DR may play a pivotal role in
the physiological control of ascending 5-HT pathways, attenuating excessive activation of 5-HT neurons by excitatory afferents from the different forebrain structures. The proportion of 5-HT-containing cells in the DRN is estimated to range from 1/3 to 2/3 (Descarries et al., 1982; Jacobs and Azmitia, 1992; Baumgarten and Grozdanovic, 1997). The remaining non-serotonergic cells contain a variety of other neurotransmitters and neuromodulators (Jacobs and Azmitia, 1992). Some reports have shown that the serotonergic system is an important neuromodulator of central nervous system activity, and TI is a behavior that seems to be highly influenced by cerebral levels of serotonin (5-HT) (Wallnau and Gallup, 1977). Systemic studies have demonstrated that different doses of serotonin could produce an increase or decrease in the IT duration in chickens (Henning, 1980). In the same way, intraventricular administration of 5-HT produced opposing results in chickens and rabbits (Harston et al., 1976; Hatton et al., 1978). These different results have been attributed to differences in the route of drug administration or the species used. Moreover, systemic injections of 5-HT could alter the levels of 5-HT in the entire CNS.
Fig. 1 – Duration of tonic immobility (TI) episodes. Mean ± S.E.M. (A) TI duration under control conditions (CONT), after surgery (SHAM) and after 5-HT1A agonist 8-OH-DPAT [DPAT; Aa (0.005 μg; n = 12), Ab (0.01 μg; n = 10), Ac (0.1 μg; n = 11)]; (B) TI duration after WAY-100635 (WAY; 0.033 μg) followed 10 min later by DPAT (0.01 μg; n = 7). *p < 0.05 compared with CONT and SHAM; (C) TI duration after two different doses of WAY (0.033 μg and 0.067 μg) injection into the DRN on consecutive days (n = 8); (D) Schematic drawings of frontal sections obtained at representative levels of the guinea pig DRN indicating the site of microinjection (filled circles, ●) of DPAT (0.005 μg, DI), DPAT (0.01 μg, DII), DPAT (0.1 μg, DIII), WAY (0.033 μg) followed by DPAT (0.01 μg, DIV) and WAY (0.033 μg and 0.067 μg, DV). Abbreviations: PAG, periaqueductal gray matter; A, brain aqueduct.
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In a recent study, Monassi and Menescal-de-Oliveira (2004) showed that the action of 5-HT inside the ventrolateral and dorsal periaqueductal gray matter appears to be biphasic and dose-dependent. In these regions, the microinjection of low doses (0.1 μg) of 5-HT increased TI, whereas high doses (1, 3 and 6 μg) decreased this behavioral response. The authors suggested that the PAG 5-HT1A and 5-HT2 receptors play different roles in the modulation of TI, since the 5-HT1A and 5HT2 agonists, respectively, increased and decreased the duration of TI. To date, research has established 16 serotonergic receptor subtypes that are divided into 7 families based on operational, structural and transductional information (Hoyer et al., 2002). Utilizing radioligand-binding techniques, Peroutka and Snyder (1983) showed that 5-HT binds with high affinity in specific sites in the rat cortex, and these sites were named 5-HT1 and 5-HT2. The receptors 5-HT2 and 5-HT1A are considered to be the primary 5-HT receptors, with their effects related to excitatory and inhibitory responses, respectively (Brandão et al., 1991; Lovick, 1993). Considering that 5-HT1A and 5-HT2 serotonergic receptors when activated may produce different actions, the objective of the present study was to evaluate the effects of the serotonergic 5-HT2 and 5-HT1A agonists of the dorsal raphe nucleus on the duration of TI in guinea pigs.
2.
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Results
2.1. Experiment 1: Effect of microinjection of the 8-OH-DPAT and WAY-100635 Our results showed that the microinjection of two different doses of 8-OH-DPAT (0.01 and 0.1 μg; selective 5-HT1A agonist) into the DRN significantly decreased the duration of TI (Duncan's post-test) when compared with the control and sham treatments (Fig. 1Ab,c). A one-way ANOVA showed that the effect of the two doses of 8-OH-DPAT (0.01 and 0.1 μg) microinjected into the DRN was different between the groups [F(2, 18) = 5.914, p = 0.011, to the first dose; and F(2,20) = 11.822, p = 0.001 to the second dose]. However, the microinjection of 0.005 μg into the same site did not produce a significant effect (p > 0.05; Fig. 1Aa). Prior application of WAY-100635 (0.033 μg; selective 5-HT1A antagonist) into the DRN blocked the 8-OHDPAT-induced decrease in the TI duration in guinea pigs (0.1 μg; Fig. 1B). ANOVA for repeated measures indicated a significant difference between treatments [F(3,18) = 4.277; p = 0.019], and Duncan's post-test showed that treatment with 8-OH-DPAT differed from the other treatments (control, sham and the combination of antagonist/agonist 5-HT1A), which did not differ from one another. In addition, microinjection of
Fig. 2 – Duration of tonic immobility (TI) episodes. Mean ± S.E.M. (A) TI duration under control conditions (CONT), after surgery (SHAM) and after 5-HT2 agonist α-methyl-5-HT [α-Methyl; Aa (0.1 μg; n = 10) and Ab (0.5 μg; n = 9)]; (B) TI duration after ketanserin (KET; 0.01 μg) followed 10 min later by α-Methyl (0.5 μg; n = 10). *p < 0.05 compared with CONT and SHAM; (C) TI duration after two different doses of KET (0.01 μg and 0.03 μg) injection into the DRN on consecutive days (n = 7); (D) Schematic drawings of frontal sections obtained at representative levels of the guinea pig DRN indicating the site of microinjection (filled circles, ●) of α-Methyl (0.01 μg, DI), α-Methyl (0.5 μg, DII), KET (0.01 μg) followed by α-Methyl (0.5 μg, DIII) and KET (0.01 μg and 0.03 μg, DIV). Abbreviations: PAG, periaqueductal gray matter; A, brain aqueduct.
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the two doses of WAY-100635 alone in consecutive days did not alter the mean duration of TI when compared with the control and sham treatments (p = 0.114; Fig. 1C). Fig. 1D shows a schematic representation of the microinjection sites of the 8-OH-DPAT and WAY-100635 in the DRN.
2.2. Experiment 2: Effect of microinjection of alfa-methyl-5-HT and ketanserin As shown in Fig. 2Ab, the microinjection of 0.5 μg of α-methyl5-HT (selective 5-HT2 agonist) into the DRN increased the duration of TI compared with controls [ANOVA; F(2,16) = 6.007, p = 0.011]. Duncan's post-test demonstrated a difference in the mean duration of TI between animals treated with 0.5 μg of the α-methyl-5-HT and the control and sham groups. However, the microinjection of α-methyl-5-HT (0.1 μg) into the DRN did not significantly alter the duration of TI (p > 0.05; Fig. 2Aa). Prior application of ketanserin (0.01 μg; selective 5-HT2 antagonist) into the DRN blocked the α-methyl-5-HT-induced increase in the duration of TI in guinea pigs (0.5 μg; Fig. 2B). ANOVA for repeated measures indicated a significant difference between treatments [F(3,27) = 4.469; p = 0.011], and Duncan's post-test showed that treatment with α-methyl-5-HT differed from the other treatments (control, sham and the combination of antagonist/agonist 5-HT2), which did not differ from one another. Microinjection of ketanserin (0.01 and 0.03 μg in consecutive days) into the DRN did not significantly modulate the TI duration in guinea pigs at any dose (p > 0.05; Fig. 2C). In addition, the microinjection of saline did not alter the mean duration of TI when compared with the control and sham treatments (p > 0.05; Fig. 3A). Fig. 2D shows a schematic representation of the α-methyl-5-HT and ketanserin micro-
Fig. 3 – Duration of tonic immobility (TI) episodes. Mean ± S.E.M. (A) TI duration under control conditions (CONT), after surgery (SHAM) and after saline (SAL; 0.2 μl; n = 12) microinjection into the dorsal raphe nucleus (DRN); (B) Schematic drawings of frontal sections obtained at representative levels of the guinea pig DRN indicating the site of microinjection (filled circles, ●) of saline (SAL).
injection sites in the DRN and Fig. 3B the microinjection of saline.
3.
Discussion
Present experiment results point to the hypothesis that the modification in the activity of 5-HT cells of the DNR by using 5HT1A and 5-HT2 agonists can modulate TI duration in guinea pigs. We found that the microinjection of the 5-HT1A receptor agonist 8-OH-DPAT into the DRN decreased TI duration, whose effect was blocked by the previous injection of the 5HT1A antagonist, the WAY-100635. As to the effect obtained through the 8-OH-DPAT, an indirect effect on the response studied can be supposed, involving serotonergic projection structures and interaction with other mediators. The indirect effect can occur by the regulation of another neurotransmitter's release in the same or other sites. Literature data show that the release of ACh in the hippocampus receives a tonic inhibitory influence from serotonergic projections coming from the dorsal raphe nucleus (Azmitia and Segal, 1978). Moreover, the existence of functional interaction between serotonin and ACh neurotransmission in the central nervous system is extensively documented (Robinson, 1983; Wenk and Engisch, 1986; Quirion and Richard, 1987). In that sense, some authors (Bianchi et al., 1990; Izumi et al., 1994; Wilkinson et al., 1994; Consolo et al., 1996) have shown that 8-OH-DPAT increases the release of acetylcholine in the cortex and hippocampus of guinea pigs and rats and this effect is blocked by selective (WAY 100 635) 5-HT1A receptor antagonists (Bianchi et al., 1990; Wilkinson et al., 1994; Consolo et al., 1996). Considering that, when activated or inhibited, the serotonergic projections of DRN can influence other structures, one possible explanation for our results is that the TI reduction effect produced by the 8-OH-DPAT in the DRN could occur through the activation of the hippocampus' cholinergic system, increasing the release of acetylcholine (ACh) in this site and thus facilitating TI reduction. In line with the proposition that the TI reduction effect through the 8-OHDPAT can occur indirectly via the hippocampus, data obtained at this research laboratory (Baptista 2000, unpublished data) showed that administering carbachol (cholinergic antagonist) in the dorsal hippocampus decreased the duration of TI response in guinea pigs. The involvement of the dorsal hippocampus in TI response has been well documented. It is known that the hippocampus ablation prolongs TI response in rabbits (Woodruff et al., 1975). The hippocampal formation is involved in two important questions related with TI response. First, with fear (Klemm, 1977, 2001), which is considered an important factor for TI expression (Gallup, 1974). Second, the dorsal hippocampus plays an important role in motor activity, in which acetylcholine is important (Dudar et al., 1979; Day et al., 1991). The sensorial stimulation and the motor activity enhance acetylcholine release in the hippocampus (Dudar et al., 1979) and the carbachol microinjection into dentate gyrus leads to a motor hyperactivity (Flicker and Geyer, 1982; Mogenson and Nielsen, 1984a,b). Thompson et al. (1980) raised the hypothesis that the hippocampus plays an important role in the motor component of the TI response. Hence, one may consider the possibility that the TI reduction effect by the 8-
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OH-DPAT in the DRN indirectly results from the increased release of ACh in the hippocampus. This effect is similar to the results of carbachol (cholinergic antagonist) when injected directly into the hippocampus of guinea pigs. Wilkinson et al. (1994) also showed that the systemic administration of 8-OHDPAT produced a clear dose-dependent increase in the release of acetylcholine in guinea pigs' hippocampus. Although the administration was peripheral, the effect of 8-OH-DPAT on ACh release may occur through its activity in autoreceptors of DRN. However, one cannot totally discard the possibility that autoreceptors are involved in other structures. The role of DRN can be strengthened by the report of Auerbach et al. (1989), where administration of 8-OH-DPAT could have an indirect effect on ACh release through a primary action at the somatodendritic autoreceptor of 5-HT-containing neurons within DRN that would inhibit 5-HT release at the nerve terminal. Our results also show that the (WAY 100 635) 5-HT1A receptor antagonists previously microinjected into the DRN antagonized the increasing effect on the duration of TI, indicating the participation of 5-HT1A receptors. When the WAY 100 635 was injected alone into the DRN, however, no modifications in TI response were observed, possibly indicating that the 5-HT1A receptors of guinea pigs' DRN are not tonically active in these experimental conditions. In the same sense, Hughes and Dawson (2004) suggest that, in guinea pigs, 5-HT1A receptors do not appear to be tonically active, as the administration of WAY100635 does not affect extracellular 5HT in either hippocampus or frontal cortex. Roberts et al. (1999) showed that antagonism of 5-HT1A receptors does not affect extracellular levels of 5-HT in either structures in guinea pigs in vivo. In this research, it was also observed that the injection of the 5-HT2 receptor agonist α-metyl-5-HT into the DN produced an increase in the duration of TI. This effect was blocked by pretreatment with ketanserin, a 5-HT2 antagonist. The increasing effect of alpha-methyl-5-TH on TI duration may also have occurred indirectly, that is, involving the decreased release of ACh in the hippocampus. Hence, as already mentioned, according to Azmitia and Segal (1978), ACh release in the hippocampus receives a tonic inhibitory influence of serotonergic projections coming from the dorsal raphe nucleus. In this sense, it was demonstrated by Barnes et al. (1989) that alpha-methyl-5TH produced inhibition of ACh release from slices of rats' entorhinal cortex. The same effect was also observed in cortex slices of guinea pigs through alpha-methyl-5TH administrations in higher concentrations (Bianchi et al., 1990). It is interesting to observe that, in our research laboratory (unpublished data), the TI reduction effect by carbachol was antagonized by an ineffective dose of atropine (muscarinic cholinergic antagonist). However, higher doses of atropine injected in the HC also produced an increase in TI duration. This effect is supposedly due to the occupation of muscarinic receptors and the inhibition of ACh action. As to the TI increased effect by the administration of alpha-methyl5TH in the DRN, one could think of tonic inhibition by serotonin release in the hippocampus, thus producing a decreased ACh release and a consequent TI increase. Hence, one may suppose that the TI increased effect by alpha-methyl5TH in the DRN could also occur indirectly through the decrease of ACh in the hippocampus.
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In summary, our results show that the stimulation of 5HT1A receptors in the DRN by 8-OH-DPAT decreased the duration of TI episodes, while the activation of 5-HT2 receptors by α-methyl-5-HT increased the TI duration. Thus, the activation of different DRN serotonergic receptors can be responsible for the balance in 5-HT release that may modulate the duration of the defensive TI response in guinea pigs.
4.
Experimental procedures
4.1.
Animals
Adult male guinea pigs (Cavia porcellus) weighing 420–470 g were obtained from the animal care facility of the Faculty of Medicine of Ribeirão Preto (FMRP). The animals were kept in Plexiglas wall cages (56 cm × 37 cm × 39 cm, five animals per cage) in a room maintained at 24 ± 1 °C, on a 12-h light cycle, with free access to water and food, during the entire experimental period. The experiments were carried out in compliance with the recommendations of Brazilian Association for Laboratory Animal Science (COBEA) and with the approval (Proc. no. 069/2004) of the Ethical Committee for Animal Experimentation of the School of Medicine of Ribeirão Preto, University of São Paulo. All efforts were made to minimize animal suffering.
4.2.
Procedures
4.2.1.
Induction of tonic immobility
Each naive guinea pig was submitted to five control maneuvers designed to induce TI, and the duration of the episodes was recorded. Experimentally, the induction of TI was attempted by holding the animal around the thorax with the hands, quickly inverting it and pressing it down into a shaped plywood trough (25 cm long × 15 cm high). The pressure applied by the experimenter's hands was proportional to the resistance offered by the animal to the restraining maneuver. When the animal stops moving, the experimenter slowly withdraws his hands and a chronometer is activated to measure the duration (in seconds) of the response, which ends when the animal resumes the upright position. If the animal does not become motionless within 60 s, the episode is recorded as having zero duration. This procedure was repeated at intervals ranging between 1 and 3 min between each episode. TI episodes were held in the afternoon, in a semi-dark room, with background noise (air-conditioning functioning). Before any manipulation, the animal was maintained in the testing room for 20 min in order to habituate to the experimental situation. For group analysis, the mean of five episodes per animal was calculated. It was established that, in a single trial group, the animals serve as their own controls, that is, the mean of TI durations in the control treatment was compared with the mean of the sham and of the experimental treatments.
4.2.2.
Stereotaxic surgery and microinjection procedure
One day after the control TI episode, the animals were anesthetized by intramuscular injection of 40 mg/kg ketamine plus 5 mg/kg xylazine, and placed in a stereotaxic apparatus
74
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(David-Kopf Instruments, USA) with the mouthpiece 17 mm below the interauricular line. A midline incision of the scalp was made, exposing the skull, and a small burr hole was made with a dental drill. A guide cannula (14 mm long and 0.6 mm outer diameter, prepared from a hypodermic needle) was implanted into the DRN. According to the atlas of Rössner (1965) for guinea pigs, the stereotaxic coordinates for placement of the guide cannula implanted in the DRN were + 10.8 mm caudal to the bregma, 0.0 mm lateral to the midline and + 3.5 mm below the intra-aural line. The guide cannula was lowered to a depth of 1 mm above the target regions and fixed to the skull with autopolymerizing resin and an additional anchoring screw. After 6–7 days of recovery from surgery, microinjections were performed with a Hamilton microsyringe (10 μl) connected to a PE-10 polyethylene catheter, which in turn was coupled to a Mizzy needle segment (0.3 mm outer diameter; 1.0 mm longer than the guide cannula). In all experimental groups, a volume of 0.2 μl was microinjected over a period of 1 min and the Mizzy needle was left in place for an additional 1 min to avoid reflux.
4.3.
Drugs and experimental groups
The drugs used were: 8-hydroxy-dipropylaminotretalin (8-OHDPAT; RBI, USA), WAY-100635 Maleate (Sigma, St. Louis, USA), α-methyl-5-hydroxytriptamin (α-methyl-5-HT; RBI, USA), and ketanserin (RBI, USA). The solutions were freshly dissolved in 0.9% saline containing 0.1% ascorbic acid, and sodium bicarbonate was added to adjust the pH to 7.4, which also served as vehicle control. All microinjections were performed in a volume of 0.2 μl. Also, in all experimental groups, each animal served as its own control. The doses were based on previous studies (Zanoveli et al., 2003; Monassi and Menescalde-Oliveira, 2004; Soares and Zangrossi, 2004; Pobbe and Zangrossi, 2005). To evaluate the effect of the drugs used, the animals were assigned into 2 different experimental groups. In experiment 1, the effect of 8-OH-DPAT (selective 5-HT1A agonist) and WAY-100635 (selective 5-HT1A antagonist) on the TI duration were tested, and the drugs were microinjected into the DRN. In this experiment, the animals were distributed among five groups. In group 1 (n = 12), group 2 (n = 10) and group 3 (n = 11), the animals were microinjected with 8-OH-DPAT (0.005, 0.01 and 0.1 μg/0.2 μl, respectively); in group 4 (n = 7) the animals were microinjected with WAY-100635 (0.033 μg/0.2 μl) followed 1 min later by 8-OH-DPAT (0.01 μg/0.2 μl); in group 5 (n = 8), the animals were microinjected with WAY-100635 in two different doses (0.033 and 0.067 μg/0.2 μl) on consecutive days. In experiment 2, we tested the effect of α-methyl-5-HT (selective 5-HT2 agonist) and ketanserin (selective 5-HT2 antagonist) on the TI duration, and the drugs were microinjected into the DRN. In this experiment, the animals were distributed in four groups. In group 1 (n = 10) and group 2 (n = 9), the animals were microinjected with α-methyl-5-HT (0.1 and 0.5 μg/0.2 μl, respectively); in group 3 (n = 10) the animals were microinjected with ketanserin (0.01 μg/0.2 μl), followed 1 min later by α-methyl-5-HT (0.5 μg/0.2 μl); in group 4 (n = 7) the animals were microinjected with ketanserin (0.01 and
0.03 μmol/0.2 μl) on consecutive days; in group 4 (n = 12), animals were microinjected with saline (0.2 μl) to the control of vehicle.
4.4.
Histological verification
Following all tests, the injection site was marked by microinjecting 0.2 μl of 2% pontamine sky blue dye. Each animal was deeply anesthetized with sodium pentobarbital and perfused intracardially with saline followed by 10% formalin. The brains were removed and fixed in 10% formalin for 4 days. Routine histological procedures were used for sectioning, and stained preparations were observed under a light microscope to determine the location of the stimulated sites according to the Rössner atlas (1965). Only guinea pigs with microinjection site located inside the target structure (DRN) were included in the data analysis.
4.5.
Statistical analysis
The TI results are reported as mean ± standard error of the mean (SEM) of the five TI episodes. Data were analyzed by analysis of variance (ANOVA) for repeated measures. The degree of freedom of the repeated measure (treatment) was corrected by the Huynh–Feldt ɛ parameter. The Duncan test was used to determine the difference between treatments, with the level of significance set at p < 0.05.
Acknowledgments We would like to thank Mrs. Mariulza Rocha Brentegani and Mr. Rubens Fernando de Melo for technical assistance and histological processing. This work was supported by CNPq (Proc. 134032/2004-7). REFERENCES
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