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Role of cholinergic, opioidergic and GABAergic neurotransmission of the dorsal hippocampus in the modulation of nociception in guinea pigs
Life Sciences 83 (2008) 644–650
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Role of cholinergic, opioidergic and GABAergic neurotransmission of the dorsal hippocampus in the modulation of nociception in guinea pigs Lys Angela Favaroni Mendes, Leda Menescal-de-Oliveira ⁎ Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo, Av. Bandeirantes 3900, CEP 14049-900, Ribeirão Preto, SP, Brazil
a r t i c l e
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Article history: Received 4 July 2007 Accepted 4 September 2008 Keywords: Cholinergic system Dorsal hippocampus GABAergic system Nociception Opioidergic system Vocalization
a b s t r a c t Aims: Several physiological, pharmacological and behavioral lines of evidence suggest that the hippocampal formation is involved in nociception. The hippocampus is also believed to play an important role in the affective and motivational components of pain perception. Thus, our aim was to investigate the participation of cholinergic, opioidergic and GABAergic systems of the dorsal hippocampus (DH) in the modulation of nociception in guinea pigs. Main methods: The test used consisted of the application of a peripheral noxious stimulus (electric shock) that provokes the emission of a vocalization response by the animal. Key findings: Our results showed that, in guinea pigs, microinjection of carbachol, morphine and bicuculline into the DH promoted antinociception, while muscimol promoted pronociception. These results were verified by a decrease and an increase, respectively, in the vocalization index in the vocalization test. This antinociceptive effect of carbachol (2.7 nmol) was blocked by previous administration of atropine (0.7 nmol) or naloxone (1.3 nmol) into the same site. In addition, the decrease in the vocalization index induced by the microinjection of morphine (2.2 nmol) into the DH was prevented by pretreatment with naloxone (1.3 nmol) or muscimol (0.5 nmol). At doses of 1.0 nmol, muscimol microinjection caused pronociception, while bicuculline promoted antinociception. Significance: These results indicate the involvement of the cholinergic, opioidergic and GABAergic systems of the DH in the modulation of antinociception in guinea pigs. In addition, the present study suggests that cholinergic transmission may activate the release of endorphins/enkephalin from interneurons of the DH, which would inhibit GABAergic neurons, resulting in antinociception. © 2008 Elsevier Inc. All rights reserved.
Introduction The hippocampus, as a part of the limbic system, participates both in learning and memory processes, as well as playing a role in attention- and arousal-related changes, in addition to emotional states involving stress- and pain-induced alterations of behavior. The involvement of the hippocampal formation in nociception has been suggested in physiological (Sinclair and Lo, 1986; Khanna and Sinclair, 1989), pharmacological (Soulairac et al., 1967; Khanna and Sinclair, 1989) and behavioral (Klamt and Prado, 1991; McKenna and Melzack, 1992) studies. Several investigations have demonstrated a role of the hippocampus in pain processing using hippocampal electroencephalograms (EEG) (Soulairac et al., 1967; Archer and Roth, 1997), extracellular recordings (Zheng and Khanna, 1999), long-term potentiation (Wei et al., 2000), immediate-early gene expression (Pearse et al., 2001), and functional neuroimaging (Derbyshire et al., 1997). Physiological and pharmacological aspects of the hippocampus'
⁎ Corresponding author. Tel.: +55 16 3602 3025; fax: +55 16 3633 0017. E-mail address: [email protected] (L. Menescal-de-Oliveira). 0024-3205/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2008.09.006
role in the processing of nociceptive information have been thoroughly discussed by Khanna (2007). Additionally, Lico et al. (1974) and Henke (1982) also showed the role of the hippocampus in the affective–motivational component of pain. Lico et al. (1974) showed that, in anesthetized guinea pigs, variation in the frequency of the electrical stimulus without altering the electrode's position in limbic structures, including the “dorsal hippocampus, resulted either in analgesic-like effect or algesic-like effect”, thus indicating limbic modulation in the perception of painful messages. Different neurotransmitters involved in nociception are widely present in the DH. Acetylcholine (ACh) is one of the main neurotransmitters released in the DH and plays an important role in hippocampal nociceptive processing (Khanna and Sinclair, 1992; Ceccarelli et al., 1999). Cholinergic neurons in the medial septum and the vertical limb of the diagonal band of Broca are the major source of cholinergic afferents to the hippocampus. ACh and atropine microinjection promoted opposite effects on the excitability of hippocampus neurons previously modified by the peripheral painful stimulus (Khanna and Sinclair, 1992; Yang et al., 2008). Moreover, apparent conflicting effects have been found in response to a noxious stimulation produced by formalin; while Ceccarelli et al. (1999) found
L.A. Favaroni Mendes, L. Menescal-de-Oliveira / Life Sciences 83 (2008) 644–650
an increase in a release of ACh in the hippocampus of mice, Aloisi et al. (1993) reported a prolonged decrease in activity of the AChsynthesizing enzyme, choline acetyltransferase, in the dorsal hippocampus (DH) of rats. Klamt and Prado (1991), using the tail-flick test, demonstrated that microinjection of the muscarinic receptor agonist carbachol into the hippocampus attenuated pain behaviors in rats. Besides ACh, evidence is accumulating that opioid peptides are important modulators of information processing in the hippocampus. When activated, opioid receptors play a key role in central pain modulation mechanisms (Przewlocki and Przewlocka, 2001) and the hippocampal formation is a structure that expresses significant densities of this kind of receptors (McLean et al., 1987; Drake et al., 1996). It has been demonstrated in several encephalic areas, such as the cerebral cortex, diencephalon, mesencephalon, and striatum (Kalyuzhny and Wessendorf, 1997; 1998), that opioids act in a disinhibitory way. The excitatory effects of opioids, including morphine, on hippocampal pyramidal cells are believed to be due to a reduction of a GABA-mediated synaptic inhibitory transmission (i.e., disinhibition) between interneurons and pyramidal cells in the hippocampus. Within this context, Wiesner et al. (1986) and Neumaier et al. (1988) demonstrated by neuronal labeling that, in the dentate gyrus, all the enkephalin immunoreactive buttons evaluated made synaptic contacts with GABAergic structures, whereas Kalyuzhny and Wessendorf (1998) described double-labeling for μ-opioid receptors and GABAergic neurons in the CA1, CA3 and dentate gyrus regions of the rat hippocampus. Based on these characteristics, therefore, hippocampal enkephalin cells are classified as interneurons, which are specialized to innervate other interneurons, probably containing γaminobutyric acid (Blasco-Ibanez et al., 1998). In this way, a complex interaction among these three neurotransmitter systems, cholinergic, opioidergic and GABAergic, seems to be involved in the modulation of the antinociceptive response in guinea pigs (Leite-Panissi et al., 2004; Da Silva and Menescal-de-Oliveira, 2007). Therefore, based on this data, our aim was to investigate the possible role of modulation on nociception of agonists and antagonists cholinergic; opiodergic and GABAergic, as well as their possible interactions when microinjected into the DH of guinea pigs. In this study, nociception was evaluated based on the vocalization response of guinea pigs promoted by a peripheral noxious stimulus (an electric shock applied to the thigh). Guinea pigs are not commonly used in experiments aimed at the study of the neural mechanisms of analgesia. However, in our laboratory we have for some years been using tests that have proved to be appropriate for this animal species (Menescal-de-Oliveira and Lico, 1985; Leite-Panissi et al., 2004; Da Silva and Menescal-de-Oliveira, 2006). Materials and methods Animals Adult male guinea pigs (Cavia porcellus) weighing 400–500 g were obtained from the animal care facility of the School of Medicine of Ribeirão Preto (FMRP/USP). The animals were kept in Plexiglas wall cages (56 cm × 17 cm × 39 cm, five animals per cage) in a room maintained at 24 ± 1 °C, on a 12 h light cycle (lights on at 7:00 AM), with free access to water and food. Experiments were carried out according to the ethical recommendations of the Committee for Research and Ethical Issues of the International Association for the Study of Pain (Zimmermann, 1983) and with the approval (Proc. no. 032/2004) 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.
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while atropine was obtained from Merck, Sharp and Dohme. The drugs were dissolved in physiological saline (SAL, 0.9%, NaCl), which also served as vehicle control. The volume injected was always 0.2 μl per side and the doses applied were based on previous studies (Baptista, 2000; Leite-Panissi and Menescal-de-Oliveira, 2002; Leite-Panissi et al., 2004). Experimental procedures Stereotaxic surgery The animals were anesthetized by an intramuscular injection of 40 mg/kg ketamine plus 5 mg/kg xylazine and attached to the Kopf stereotaxic apparatus (David-Kopf Instruments, USA), with the mouthpiece 21.4 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 in length and 0.6 mm in outer diameter, prepared from a hypodermic needle) was implanted bilaterally into the DH. According to the Rössner atlas for guinea pigs (Rössner, 1965), the stereotaxic coordinates for the guide placement were 6.4 mm caudal to bregma, 0.2 mm lateral from the midline, and 7.6 mm above the intra-aural 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. Following surgery, the guide cannula was plugged with a stylet to prevent blocking. All animals were allowed to recover from surgery for 5–6 days. Antinociceptive test For the evaluation of antinociception, the animals were submitted to the vocalization test. The vocalization test is carried out by the application of a peripheral noxious stimulus (electric shock) that provokes the emission of a vocalization response by the animal, which is interpreted as a manifestation of pain. For peripheral noxious stimulation, a pair of non-insulated electrodes was implanted into the subcutaneous region of the thigh. The animal was then placed in an acrylic box lined with nylon foam where some movement was possible. After 20 min of animal habituation to the experimental situation, the electrode was connected to an electronic stimulator that released pulses (square waves, 100 Hz frequency, 0.5 ms duration) of varying intensity (0.5–4.0 V) sufficient to induce vocalization. Once the threshold value was established, voltage was maintained at a constant level throughout the experiment. Three electrical shocks (1 s each stimulation) induced brief motor and vocalization responses that did not persist between the stimuli intervals. The peripheral noxious stimulus was then applied at 2, 5, 15, 30, 45 and 60 min after the different treatments. Vocalization was recorded with the aid of an Aiwa DM-64 microphone connected to the pre-amplifier of a polygraph. In the polygraphic recording of vocalization, peak amplitude is proportional to the intensity of animal vocalization. The mean of the peak of each response is a reliable index of the magnitude of vocalization. The peak amplitude of the graphic recordings of vocalization was measured in millimeters and the mean of each response was used for quantitative evaluation. As a control, a baseline test was performed for the determination of the smallest noxious stimulus necessary to produce a vocalization response by the animal. Three consecutive stimuli were applied, and the mean amplitude of vocalization was calculated during control period controls (without saline or drug administration). 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; 2 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 the respective place for an additional 40 s period to avoid reflux.
Drugs Carbachol, morphine sulfate, naloxone hydrochloride, bicuculline methiodide and muscimol were obtained from Sigma Chemical Co.,
Experimental groups The animals (n = 109) were divided into fifteen experimental groups; group 1 (n = 8) received carbachol (CCh, 2.7 nmol); group 2 (n = 7)
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received atropine (ATR, 0.7 nmol); group 3 (n = 7) received atropine (0.7 nmol) followed 10 min later by carbachol (2.7 nmol; ATR+ CCh), and group 4 (n = 7) received naloxone (NAL, 1.3 nmol) followed 10 min later by carbachol (2.7 nmol; NAL + CCh). Animals in groups 5 and 6 were microinjected with different doses of morphine (MOR; group 5: 1.1 nmol, n = 6; group 6: 2.2 nmol, n = 7). Group 7 (n = 7) received naloxone (NAL, 1.3 nmol); group 8 (n = 7) received naloxone (1.3 nmol) followed 10 min later by morphine (2.2 nmol; NAL + MOR), and group 9 (n = 7) received atropine (0.7 nmol) followed 10 min later by morphine (2.2 nmol; ATR + MOR). Animals in groups 10 and 11 were microinjected with different doses of bicuculline (BIC; group 10: 0.2 nmol, n = 7; group 11: 1.0 nmol, n = 8). Animals in groups 12 and 13 were microinjected with different doses of muscimol (MUS; group 12: 0.5 nmol, n = 9; group 13: 1.0 nmol, n = 7); and group 14 (n =9) received muscimol (MUS, 0.5 nmol) followed 10 min later by morphine (2.2 nmol; MUS + MOR). Animals receiving 0.9% saline (SAL, n = 6) were used as control (group 15). Histological verification After the end of the experiments, the animals were anesthetized with Thionembutal, 50 mg/kg, and were perfused intracardially with saline followed by 10% formalin. The brains were removed and fixed in
10% formalin. The material was then submitted to routine histological processing and sections were observed under the microscope to determine the locations of the stimulated sites according to the Rössner atlas (Rössner, 1965). Only the animals whose microinjection reached the target structure were used for data analysis (Fig. 1). Data analysis Amplitude of vocalization results (mm) were transformed into a vocalization index (VI) using the following formula: VI = (mean vocalization − control value)/control value. Data are reported as mean VI ± SEM and were analyzed by Two Way Repeated Measures analysis of variance (Two Way RM ANOVA), with time (repeated factor) and treatment (independent factor) as variables, and One Way ANOVA followed by the Duncan's test. The level of significance was set at p b 0.05. Results Microinjection of carbachol, morphine sulfate and bicuculline into the DH produced antinociception in guinea pigs as determined by the
Fig. 1. Schematic drawing of frontal sections obtained at representative levels of the guinea pig dorsal hippocampus, indicating the site of microinjection: a) SAL (filled circles (●), n = 6), CCh (crosses (✚), n = 8), ATR (filled triangles (▲), n = 7); b) ATR + CCh (crosses (✚), n = 7), NAL + CCh (filled triangles (▲), n = 7), MOR 1.1 (filled squares (■), n = 6), c) NAL (crosses (✚), n = 7), NAL + MOR (filled triangles (▲), n = 7), ATR + MOR (filled lozenges (♦), n = 7); d) MOR 2.2 (filled circles (●), n = 7), BIC 0.2 (crosses (✚), n = 7), BIC 1.0 (filled triangles (▲), n = 8); e) MUS 0.5 (crosses (✚), n = 9); MUS 1.0 (filled circles (●), n = 7), MUS 0.5 + MOR (filled triangles (▲), n = 9). Sections are from rostral to caudal (5.2–7.6 mm). DH: dorsal hippocampus; GM: corpus geniculatum mediale; NPT: nucleus posterior thalami; VH: ventral hippocampus.
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decrease of the VI in the vocalization test (Figs. 2 and 3). On the other hand, muscimol microinjection produced pronociception in these same conditions (Figs. 2c and 3). The antinociceptive effect of carbachol (Figs. 2a, 3) was blocked by the previous microinjection of atropine (a muscarinic receptor antagonist) into the same site (Figs. 2a, 3). Furthermore, previous microinjection of naloxone (an opioidergic antagonist) into the same site (Figs. 2a, 3) blocked the antinociceptive action of carbachol. Two-way repeated measures ANOVA applied to the SAL, CCh, ATR, ATR +CCh and NAL +CCh groups showed a significant difference in VI between different
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treatments (F(4, 180) =48.59; p b 0.001), along time (F(6, 180)=2.94; p = 0.009) and for time vs. treatment (F(24, 180) =6.39; p b 0.001). The post hoc Duncan test showed a difference (p b 0.05) between the CCh group and all other groups (SAL, ATR, ATR + CCh and NAL + CCh) (Fig. 2a). Moreover, the same test (p b 0.05) indicated that the SAL, ATR, ATR + CCh and NAL + CCh groups did not differ from one another (Fig. 2a). We then applied one-way ANOVA followed by the Duncan test to each experimental time interval. At 2 min, the CCh group differed significantly from the SAL, ATR and NAL + CCh treatments (F(4, 30) = 3.35, p = 0.022; Fig. 2a). For each subsequent time (5, 15, 30, 45 and 60 min), the carbachol group differed significantly from the other groups (F(4, 30) = 9.05, F(4, 30) = 14.83, F(4, 30) = 37.62, F(4, 30) = 88.64, and F(4, 30) = 42.16, respectively, p b 0.001; Fig. 2a). In addition, microinjection of 2.2 nmol morphine sulfate, but not 1.1 nmol, into the DH promoted antinociception in guinea pigs as determined by the decrease of the VI in the vocalization test (Figs. 2b, 3). Furthermore, the antinociceptive effect of morphine was blocked by previous administration of naloxone (1.3 nmol; Figs. 2b, 3). In contrast, prior microinjection of atropine (0.7 nmol; Figs. 2b and 3) was not able to prevent the antinociceptive effect of morphine. Two-way repeated measures ANOVA applied to the opioidergic groups (SAL, MOR 1.1, MOR 2.2, NAL, NAL+ MOR, ATR+ MOR) revealed a significant difference in the VI between treatments (F(5, 204)= 21.44; p b 0.001), along time (F(6, 204)= 15.25; p b 0.001) and for time vs. treatment (F(30, 204) = 9.89; p b 0.001). The post hoc Duncan test showed a difference (p b 0.05) between MOR 2.2 and ATR+ MOR and the other treatments (SAL, MOR 1.1, NAL, NAL + MOR) (Fig. 2b). The same test indicated that the SAL, MOR 1.1, NAL and NAL + MOR groups did not differ from one another (Fig. 2b). Furthermore, there was no difference between the MOR 2.2 and ATR+ MOR groups (Fig. 2b). One-way ANOVA showed no significant difference between groups for the 2- and 5-min intervals (F(5, 34) = 0.80, p = 0.56, and F(5, 34) = 2.53, p = 0.052, respectively). At 15, 30, 45 and 60 min (Fig. 2b), the Duncan test revealed a significant difference between the MOR 2.2 and ATR+ MOR groups and the other treatments (F(5, 34) = 14.28, F(5, 34) = 34.64, F(5, 34) = 32.40, and F(5, 34)= 20.18, p b 0.001; Fig. 2b). The microinjection of bicuculline (a GABAergic receptor antagonist) in the amount of 1.0 nmol, but not 0.2 nmol, into the DH promoted antinociception in guinea pigs as determined by the decrease of the VI in the vocalization test (Figs. 2c, 3). On the other hand, microinjection of 1.0 nmol muscimol (GABAergic receptor agonist), but not 0.5 nmol, into the DH promoted pronociception in the animals. Furthermore, the antinociceptive effect of morphine was also blocked by previous administration of muscimol (0.5 nmol) (Figs. 2c, 3). Two-way repeated measures ANOVA applied to the GABAergic groups (SAL, BIC 0.2, BIC 1.0, MUS 0.5, MUS 1.0, MUS + MOR) revealed a significant difference in the VI between treatments (F(5, 240) = 32.11; p b 0.001) and time vs. treatment (F(30, 240) = 8.41; p b 0.001). The post hoc Duncan test showed a difference (p b 0.05) Fig. 2. Mean vocalization index (VI) under control conditions (time zero, before microinjection) and after (2, 5, 15, 30, 45 and 60 min) various treatments obtained for different experimental groups of conscious guinea pigs submitted to a peripheral noxious stimulus. a) Vocalization index after microinjection of 0.9% saline ( , 0.2 μl), carbachol (●, 2.7 nmol/0.2 μl), atropine (♦, 0.7 nmol/0.2 μl), atropine (0.7 nmol/0.2 μl) followed by carbachol (○, 2.7 nmol/0.2 μl), and naloxone (1.3 nmol/0.2 μl) followed by carbachol (△, 2.7 nmol/0.2 μl) into the dorsal hippocampus. # CCh compared to SAL, ATR, NAL + CCh, and ⁎ CCh compared to all other groups (Duncan's test, p b 0.05). b) Vocalization index after microinjection of 0.9% saline ( , 0.2 μl), MOR 1.1 (□, 1.1 nmol/ 0.2 μl), MOR 2.2 (■, 2.2 nmol/0.2 μl), naloxone (△, 1.3 nmol/0.2 μl), naloxone (1.3 nmol/ 0.2 μl) followed by morphine (♦, 2.2 nmol/0.2 μl), and atropine (0.7 nmol/0.2 μl) followed by morphine (ο, 2.2 nmol/0.2 μl) into the dorsal hippocampus. ⁎MOR 2.2 and ATR + MOR compared to SAL, MOR 1.1, NAL and NAL + MOR (Duncan's test, p b 0.05). c) Vocalization index after microinjection of 0.9% saline ( , 0.2 μl), BIC 0.2 (□, 0.2 nmol/ 0.2 μl), BIC 1.0 (■, 1.0 nmol/0.2 μl), MUS 0.5 (ο, 0.5 nmol/0.2 μl), MUS 1.0 (●, 1.0 nmol/ 0.2 μl), and muscimol (0.5 nmol/0.2 μl) followed by morphine (♦, 2.2 nmol/0.2 μl), into the dorsal hippocampus. ⁎ MUS 1.0 and BIC 1.0 compared to all other groups (Duncan's test, p b 0.05). The vertical bars represent the S.E.M.
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Fig. 3. Schematic drawing of the vocalization amplitude (mm) after administration of different drugs into the dorsal hippocampus of guinea pigs. a, Animal microinjected with 0.9% saline (0.2 μl); b, animal treated with carbachol (2.7 nmol/0.2 μl); c, animal treated with atropine (0.7 nmol/0.2 μl); d, animal treated with atropine (0.7 nmol/0.2 μl) followed by carbachol (2.7 nmol/0.2 μl); e, animal treated with naloxone (1.3 nmol/0.2 μl) followed by carbachol (2.7 nmol/0.2 μl); f, animal treated with morphine (1.1 nmol/0.2 μl); g, animal treated with morphine (2.2 nmol/0.2 μl); h, animal treated with naloxone (1.3 nmol/0.2 μl); i, animal treated with naloxone (1.3 nmol/0.2 μl) followed by morphine (2.2 nmol/0.2 μl); j, animal treated with atropine (0.7 nmol/0.2 μl) followed by morphine (2.2 nmol/0.2 μl); k, animal treated with bicuculline (0.2 nmol/0.2 μl); l, animal treated with bicuculline (1.0 nmol/0.2 μl); m, animal treated with muscimol (0.5 nmol/0.2 μl); n, animal treated with muscimol (1.0 nmol/0.2 μl); o, animal treated with muscimol (0.5 nmol/0.2 μl) followed by morphine (2.2 nmol/0.2 μl). The horizontal bar indicates the application of the noxious stimulus (3 s) at the different time intervals (min). Zero represents the vocalization amplitude during the control period (without saline or drug administration).
between MUS 1.0 and BIC 1.0; and between treatment with both drugs and the other treatments (SAL, BIC 0.2, MUS 0.5, MUS + MOR) (Fig. 2c). The same test indicated that the SAL, BIC 0.2, MUS 0.5, MUS + MOR groups did not differ from one another (Fig. 2c). When we applied one-way ANOVA followed by the Duncan test to each experimental time interval, the MUS 1.0 group differed significantly from all the other treatments in all the intervals and the BIC 1.0 group differed significantly from all the other treatments in all the intervals except 2 min (p b 0.001). Our results were: 2 min (F(5, 40) = 8.84), 5 min (F(5, 40) = 18.30), 15 min (F(5, 40) = 18.81), 30 min (F(5, 40) = 26.31), 45 min (F(5, 40) = 31.21), 60 min (F(5, 40) = 32.29; Fig. 2c). Saline administration into the DH did not alter the VI after the animals had been submitted to a peripheral noxious stimulus (Fig. 3), and this group was used as control in all experiments (Fig. 2). Discussion The present results demonstrate the participation of the cholinergic, opioidergic and GABAergic circuits of the DH in the modulation of the nociceptive response. Our results showed that carbachol (2.7 nmol), morphine (2.2 nmol) and bicuculline (1.0 nmol) injected into the DH of the different groups of guinea pigs significantly reduced
nociceptive response, as demonstrated by decreased vocalization induced by the peripheral noxious stimulus. On the other hand, muscimol (1.0 nmol) microinjection into DH promoted pronociception demonstrated by increased vocalization induced by the peripheral noxious stimulus. In different nuclei of the central nervous system, intracerebral microinjection of the cholinergic agonist carbachol has been shown to produce antinociceptive effects, which were abolished by local administration of atropine into of rats (Guimarães et al., 2000; Ma et al., 2001; Abe et al., 2003) and guinea pigs (Menescal-de-Oliveira and Lico, 1985; Leite-Panissi et al., 2004; Da Silva and Menescal-deOliveira, 2006). Aloisi et al. (1993) reported that subcutaneous injection of formalin into the rat hind paw induced a persistent nociceptive behavior and a prolonged decrease in DH activity of choline acetyltransferase. In our study, microinjection of carbachol into the DH of guinea pigs produced antinociception, which was demonstrated by a decrease in the vocalization response after a peripheral noxious stimulus (decreased VI). The animal's vocalization in these conditions indicates the participation of the affective– motivational component of the painful sensation, which essentially depends on the supra-spinal activation, including limbic structures like the hippocampus (Lico et al., 1974). The carbachol effect was
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blocked by prior microinjection of atropine, indicating the participation of muscarinic receptors in this antinociceptive response. Although some works have demonstrated that carbachol and morphine microinjected into the hippocampus can produce motor activation (Flicker and Geyer, 1982; Cain and Corcoran, 1985), our experimental conditions did not permit this observation. However, the same dose of carbachol and morphine injected into the DH provoked a reduction in the duration of tonic immobility episodes, which accords with an increase in locomotion, considering that tonic immobility is a response characterized by a reversible state of akinesia (unpublished data). Acetylcholine is mainly released in the hippocampus by the activation of cholinergic cell bodies located in the medial septal area (Amaral and Kurz, 1985). Several opioid neuropeptides, such as enkephalin and β-endorphin, as well as morphine, promote a decrease in hippocampal ACh levels when administered intraseptally (Moroni et al., 1977). These observations suggest that the opioid agonists may act at the level of cholinergic cell bodies in the septal region, modulating the activity of septal cholinergic afferents terminating in the hippocampus (Ammon's horn). On the other hand, when opioids are applied directly to the hippocampus, ACh levels remain unchanged (Botticelli and Wurtman, 1981). Likewise, our results did not show this kind of opioidergic-cholinergic interaction in the hippocampus, since microinjection of atropine prior to morphine was not effective in blocking the antinociceptive effect promoted by the opioid agonist. On the contrary, our results suggest a possible cholinergic–opioidergic interaction, once we showed that the antinociception induced by carbachol or morphine sulfate administered into the DH is blocked by pretreatment with naloxone injected into the same site. These results can indicate the action of the cholinergic system inside the DH seems to be opioiddependent because in this region, pretreatment with naloxone blocks the effect of carbachol. On this basis, we propose that the local carbachol injection may activate opioidergic neurons, which in turn may inhibit GABAergic neurons, disinhibiting DH output neurons that possibly lead to a succession of events that finally reach descending inhibitory pathways promoting antinociception. Although, in our case, the antinociception obtained in DH may be assigned to activation of cholinergic–opioidergic systems, we cannot reject the participation of other hippocampal neurotransmitters in mediating the antinociceptive response. Concerning the effect produced by the microinjection of morphine into the DH, only the 2.2 nmol dose promoted an antinociceptive response, which was reduced or abolished by prior administration of naloxone. Naloxone per se did not produce any alterations in the VI when microinjected into the same site. Although morphine preferentially acts on μ-type opioid receptors, a high density of κ-type opioid receptors has been found in the hippocampus of guinea pigs (Foote and Maurer, 1986). Therefore, in the present study, the effects of morphine microinjected into the hippocampus of guinea pigs may depend on μ- and/or κ-opioid receptors, since only the higher dose (2.2 nmol) was effective in antinociception induction. There is evidence that GABAergic mediation of opioid effects is a widespread phenomenon and occurs throughout most of CNS. Kalyuzhny and Wessendorf (1998) observed a colocalization between hippocampal μ-opioid receptors and GABAergic interneurons in CA1, CA3 and dentate gyrus in rats. The localization of μ-opioid receptors on GABAergic neurons suggest that these receptors, when activated, can directly control the hippocampal GABAergic neurons' activity (Kalyuzhny and Wessendorf, 1997; 1998). Physiological studies have shown that activation of the opioid receptors can lead to the inhibition of interneuron activity (Madison and Nicoll, 1988; Wimpey and Chavkin, 1991; Svoboda and Lupica, 1998), resulting in diminished GABA release and the disinhibition of hippocampal pyramidal neurons (Zieglgänsberger et al., 1979; Cohen et al., 1992; Lupica et al., 1992). Basbaum and Fields (1984) proposed that µ-opioid receptor activation
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produces antinociception from within the PAG by reducing local inhibitory GABAergic influences (disinhibition) on descending projection neurons. Our results support the hypothesis that modulation of nociceptive response in the DH could occur in a manner similar to that proposed by Basbaum and Fields (1984) into PAG. It is therefore likely that the antinociception observed after microinjection of morphine into the DH occurs through the inhibition of tonically active GABAergic interneurons. We also observed that muscimol (0.5 nmol) at a dose that has no effect per se on nociception, when injected before morphine into the same site, blocks the antinociceptive effect of the latter. This supports the suggestion that opioid effects may occur via inhibition of GABA interneurons and also that the modulation of nociception by the DH might occur, in part, via a neural circuit similar to that proposed for the modulation of antinociception and tonic immobility by the ventrolateral PAG and nucleus raphe magnus (Monassi et al., 1999; Da Silva and Menescal-de-Oliveira, 2007). It is known that GABAA receptors have a prominent role in mediating tonic inhibition, particularly in hippocampal pyramidal cells (Glykys and Mody, 2006; Serwanski et al., 2006). This tonic inhibition is responsible for generating ~75% of the total inhibitory charge received by hippocampal neurons (Mody and Pearce, 2004). In the present work, the GABAA receptors' activation into DH by muscimol in an effective dose (1.0 nmol) provoked pronociception evaluated by the increase of vocalization elicited by peripheral noxious stimulation. We suggest that this effect can occur by GABAA receptor activation, possibly situated in DH pyramidal neurons, producing inhibition of these neurons and consequently leading to successive events that could ultimately activate descending facilitatory pathways promoting pronociception. On the other hand, the injection of bicuculline inhibits GABAA receptors, resulting in disinhibition of DH output neurons that possibly lead to a succession of events that finally reach descending inhibitory pathways promoting antinociception. In summary, the present results demonstrate that the activation of the cholinergic or opioidergic system of the DH promotes antinociception in guinea pigs, while GABAergic activation promotes pronociception, as demonstrated by respective decreases and increases of the VI. In addition, antinociception produced by cholinergic stimulation of the DH depends on opioid synapses present at the same site. On the other hand, antinociception observed after microinjection of morphine into the DH occurs through the inhibition of tonically active GABAergic interneurons. Acknowledgments We would like to thank Mrs. M.R. Brentegani and Mr. R.F. de Melo for technical assistance and histological processing. We are also indebted to Rildo A. Volpini, PhD, for suggestions, and to L.F.S da Silva, MSc, for preparation of the figures. This work was supported by CAPES and CNPq. References Abe, K., Kikuta, J., Kato, M., Ishida, K., Shigenaga, T., Taguchi, K., Miyatake, T., 2003. Effects of microinjected carbachol on the antinociceptive response to noxious heat stimuli. Biological & Pharmaceutical Bulletin 26 (2), 162–165. Aloisi, A.M., Albonetti, M.E., Lodi, L., Lupo, C., Carli, G., 1993. Decrease of hippocampal choline acetyltransferase activity induced by formalin pain. Brain Research 629 (1), 167–170. Amaral, D.G., Kurz, J., 1985. An analysis of the origins of the cholinergic and noncholinergic septal projections to the hippocampal formation of the rat. The Journal of Comparative Neurology 240 (1), 37–59. Archer, D.P., Roth, S.H., 1997. Pharmacodynamics of thiopentone: nocifensive reflex threshold changes correlate with hippocampal electroencephalography. British Journal of Anaesthesia 79, 744–749. Baptista, V., 2000. Participação do sistema colinérgico da formação hipocampal dorsal na modulação da imobilidade tônica em cobaias. Unpublished Dissertation, FMRP/ USP. Ribeirão Preto. Basbaum, A.I., Fields, H.L., 1984. Endogenous pain control systems: brainstem spinal pathways and endorphin circuitry. Annual Review of Neuroscience 7, 309–338.
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Role of cholinergic, opioidergic and GABAergic neurotransmission of the dorsal hippocampus in the modulation of nociception in guinea pigs Lys Angela Favaroni Mendes, Leda Menescal-de-Oliveira ⁎ Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo, Av. Bandeirantes 3900, CEP 14049-900, Ribeirão Preto, SP, Brazil
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Article history: Received 4 July 2007 Accepted 4 September 2008 Keywords: Cholinergic system Dorsal hippocampus GABAergic system Nociception Opioidergic system Vocalization
a b s t r a c t Aims: Several physiological, pharmacological and behavioral lines of evidence suggest that the hippocampal formation is involved in nociception. The hippocampus is also believed to play an important role in the affective and motivational components of pain perception. Thus, our aim was to investigate the participation of cholinergic, opioidergic and GABAergic systems of the dorsal hippocampus (DH) in the modulation of nociception in guinea pigs. Main methods: The test used consisted of the application of a peripheral noxious stimulus (electric shock) that provokes the emission of a vocalization response by the animal. Key findings: Our results showed that, in guinea pigs, microinjection of carbachol, morphine and bicuculline into the DH promoted antinociception, while muscimol promoted pronociception. These results were verified by a decrease and an increase, respectively, in the vocalization index in the vocalization test. This antinociceptive effect of carbachol (2.7 nmol) was blocked by previous administration of atropine (0.7 nmol) or naloxone (1.3 nmol) into the same site. In addition, the decrease in the vocalization index induced by the microinjection of morphine (2.2 nmol) into the DH was prevented by pretreatment with naloxone (1.3 nmol) or muscimol (0.5 nmol). At doses of 1.0 nmol, muscimol microinjection caused pronociception, while bicuculline promoted antinociception. Significance: These results indicate the involvement of the cholinergic, opioidergic and GABAergic systems of the DH in the modulation of antinociception in guinea pigs. In addition, the present study suggests that cholinergic transmission may activate the release of endorphins/enkephalin from interneurons of the DH, which would inhibit GABAergic neurons, resulting in antinociception. © 2008 Elsevier Inc. All rights reserved.
Introduction The hippocampus, as a part of the limbic system, participates both in learning and memory processes, as well as playing a role in attention- and arousal-related changes, in addition to emotional states involving stress- and pain-induced alterations of behavior. The involvement of the hippocampal formation in nociception has been suggested in physiological (Sinclair and Lo, 1986; Khanna and Sinclair, 1989), pharmacological (Soulairac et al., 1967; Khanna and Sinclair, 1989) and behavioral (Klamt and Prado, 1991; McKenna and Melzack, 1992) studies. Several investigations have demonstrated a role of the hippocampus in pain processing using hippocampal electroencephalograms (EEG) (Soulairac et al., 1967; Archer and Roth, 1997), extracellular recordings (Zheng and Khanna, 1999), long-term potentiation (Wei et al., 2000), immediate-early gene expression (Pearse et al., 2001), and functional neuroimaging (Derbyshire et al., 1997). Physiological and pharmacological aspects of the hippocampus'
⁎ Corresponding author. Tel.: +55 16 3602 3025; fax: +55 16 3633 0017. E-mail address: [email protected] (L. Menescal-de-Oliveira). 0024-3205/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2008.09.006
role in the processing of nociceptive information have been thoroughly discussed by Khanna (2007). Additionally, Lico et al. (1974) and Henke (1982) also showed the role of the hippocampus in the affective–motivational component of pain. Lico et al. (1974) showed that, in anesthetized guinea pigs, variation in the frequency of the electrical stimulus without altering the electrode's position in limbic structures, including the “dorsal hippocampus, resulted either in analgesic-like effect or algesic-like effect”, thus indicating limbic modulation in the perception of painful messages. Different neurotransmitters involved in nociception are widely present in the DH. Acetylcholine (ACh) is one of the main neurotransmitters released in the DH and plays an important role in hippocampal nociceptive processing (Khanna and Sinclair, 1992; Ceccarelli et al., 1999). Cholinergic neurons in the medial septum and the vertical limb of the diagonal band of Broca are the major source of cholinergic afferents to the hippocampus. ACh and atropine microinjection promoted opposite effects on the excitability of hippocampus neurons previously modified by the peripheral painful stimulus (Khanna and Sinclair, 1992; Yang et al., 2008). Moreover, apparent conflicting effects have been found in response to a noxious stimulation produced by formalin; while Ceccarelli et al. (1999) found
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an increase in a release of ACh in the hippocampus of mice, Aloisi et al. (1993) reported a prolonged decrease in activity of the AChsynthesizing enzyme, choline acetyltransferase, in the dorsal hippocampus (DH) of rats. Klamt and Prado (1991), using the tail-flick test, demonstrated that microinjection of the muscarinic receptor agonist carbachol into the hippocampus attenuated pain behaviors in rats. Besides ACh, evidence is accumulating that opioid peptides are important modulators of information processing in the hippocampus. When activated, opioid receptors play a key role in central pain modulation mechanisms (Przewlocki and Przewlocka, 2001) and the hippocampal formation is a structure that expresses significant densities of this kind of receptors (McLean et al., 1987; Drake et al., 1996). It has been demonstrated in several encephalic areas, such as the cerebral cortex, diencephalon, mesencephalon, and striatum (Kalyuzhny and Wessendorf, 1997; 1998), that opioids act in a disinhibitory way. The excitatory effects of opioids, including morphine, on hippocampal pyramidal cells are believed to be due to a reduction of a GABA-mediated synaptic inhibitory transmission (i.e., disinhibition) between interneurons and pyramidal cells in the hippocampus. Within this context, Wiesner et al. (1986) and Neumaier et al. (1988) demonstrated by neuronal labeling that, in the dentate gyrus, all the enkephalin immunoreactive buttons evaluated made synaptic contacts with GABAergic structures, whereas Kalyuzhny and Wessendorf (1998) described double-labeling for μ-opioid receptors and GABAergic neurons in the CA1, CA3 and dentate gyrus regions of the rat hippocampus. Based on these characteristics, therefore, hippocampal enkephalin cells are classified as interneurons, which are specialized to innervate other interneurons, probably containing γaminobutyric acid (Blasco-Ibanez et al., 1998). In this way, a complex interaction among these three neurotransmitter systems, cholinergic, opioidergic and GABAergic, seems to be involved in the modulation of the antinociceptive response in guinea pigs (Leite-Panissi et al., 2004; Da Silva and Menescal-de-Oliveira, 2007). Therefore, based on this data, our aim was to investigate the possible role of modulation on nociception of agonists and antagonists cholinergic; opiodergic and GABAergic, as well as their possible interactions when microinjected into the DH of guinea pigs. In this study, nociception was evaluated based on the vocalization response of guinea pigs promoted by a peripheral noxious stimulus (an electric shock applied to the thigh). Guinea pigs are not commonly used in experiments aimed at the study of the neural mechanisms of analgesia. However, in our laboratory we have for some years been using tests that have proved to be appropriate for this animal species (Menescal-de-Oliveira and Lico, 1985; Leite-Panissi et al., 2004; Da Silva and Menescal-de-Oliveira, 2006). Materials and methods Animals Adult male guinea pigs (Cavia porcellus) weighing 400–500 g were obtained from the animal care facility of the School of Medicine of Ribeirão Preto (FMRP/USP). The animals were kept in Plexiglas wall cages (56 cm × 17 cm × 39 cm, five animals per cage) in a room maintained at 24 ± 1 °C, on a 12 h light cycle (lights on at 7:00 AM), with free access to water and food. Experiments were carried out according to the ethical recommendations of the Committee for Research and Ethical Issues of the International Association for the Study of Pain (Zimmermann, 1983) and with the approval (Proc. no. 032/2004) 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.
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while atropine was obtained from Merck, Sharp and Dohme. The drugs were dissolved in physiological saline (SAL, 0.9%, NaCl), which also served as vehicle control. The volume injected was always 0.2 μl per side and the doses applied were based on previous studies (Baptista, 2000; Leite-Panissi and Menescal-de-Oliveira, 2002; Leite-Panissi et al., 2004). Experimental procedures Stereotaxic surgery The animals were anesthetized by an intramuscular injection of 40 mg/kg ketamine plus 5 mg/kg xylazine and attached to the Kopf stereotaxic apparatus (David-Kopf Instruments, USA), with the mouthpiece 21.4 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 in length and 0.6 mm in outer diameter, prepared from a hypodermic needle) was implanted bilaterally into the DH. According to the Rössner atlas for guinea pigs (Rössner, 1965), the stereotaxic coordinates for the guide placement were 6.4 mm caudal to bregma, 0.2 mm lateral from the midline, and 7.6 mm above the intra-aural 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. Following surgery, the guide cannula was plugged with a stylet to prevent blocking. All animals were allowed to recover from surgery for 5–6 days. Antinociceptive test For the evaluation of antinociception, the animals were submitted to the vocalization test. The vocalization test is carried out by the application of a peripheral noxious stimulus (electric shock) that provokes the emission of a vocalization response by the animal, which is interpreted as a manifestation of pain. For peripheral noxious stimulation, a pair of non-insulated electrodes was implanted into the subcutaneous region of the thigh. The animal was then placed in an acrylic box lined with nylon foam where some movement was possible. After 20 min of animal habituation to the experimental situation, the electrode was connected to an electronic stimulator that released pulses (square waves, 100 Hz frequency, 0.5 ms duration) of varying intensity (0.5–4.0 V) sufficient to induce vocalization. Once the threshold value was established, voltage was maintained at a constant level throughout the experiment. Three electrical shocks (1 s each stimulation) induced brief motor and vocalization responses that did not persist between the stimuli intervals. The peripheral noxious stimulus was then applied at 2, 5, 15, 30, 45 and 60 min after the different treatments. Vocalization was recorded with the aid of an Aiwa DM-64 microphone connected to the pre-amplifier of a polygraph. In the polygraphic recording of vocalization, peak amplitude is proportional to the intensity of animal vocalization. The mean of the peak of each response is a reliable index of the magnitude of vocalization. The peak amplitude of the graphic recordings of vocalization was measured in millimeters and the mean of each response was used for quantitative evaluation. As a control, a baseline test was performed for the determination of the smallest noxious stimulus necessary to produce a vocalization response by the animal. Three consecutive stimuli were applied, and the mean amplitude of vocalization was calculated during control period controls (without saline or drug administration). 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; 2 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 the respective place for an additional 40 s period to avoid reflux.
Drugs Carbachol, morphine sulfate, naloxone hydrochloride, bicuculline methiodide and muscimol were obtained from Sigma Chemical Co.,
Experimental groups The animals (n = 109) were divided into fifteen experimental groups; group 1 (n = 8) received carbachol (CCh, 2.7 nmol); group 2 (n = 7)
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received atropine (ATR, 0.7 nmol); group 3 (n = 7) received atropine (0.7 nmol) followed 10 min later by carbachol (2.7 nmol; ATR+ CCh), and group 4 (n = 7) received naloxone (NAL, 1.3 nmol) followed 10 min later by carbachol (2.7 nmol; NAL + CCh). Animals in groups 5 and 6 were microinjected with different doses of morphine (MOR; group 5: 1.1 nmol, n = 6; group 6: 2.2 nmol, n = 7). Group 7 (n = 7) received naloxone (NAL, 1.3 nmol); group 8 (n = 7) received naloxone (1.3 nmol) followed 10 min later by morphine (2.2 nmol; NAL + MOR), and group 9 (n = 7) received atropine (0.7 nmol) followed 10 min later by morphine (2.2 nmol; ATR + MOR). Animals in groups 10 and 11 were microinjected with different doses of bicuculline (BIC; group 10: 0.2 nmol, n = 7; group 11: 1.0 nmol, n = 8). Animals in groups 12 and 13 were microinjected with different doses of muscimol (MUS; group 12: 0.5 nmol, n = 9; group 13: 1.0 nmol, n = 7); and group 14 (n =9) received muscimol (MUS, 0.5 nmol) followed 10 min later by morphine (2.2 nmol; MUS + MOR). Animals receiving 0.9% saline (SAL, n = 6) were used as control (group 15). Histological verification After the end of the experiments, the animals were anesthetized with Thionembutal, 50 mg/kg, and were perfused intracardially with saline followed by 10% formalin. The brains were removed and fixed in
10% formalin. The material was then submitted to routine histological processing and sections were observed under the microscope to determine the locations of the stimulated sites according to the Rössner atlas (Rössner, 1965). Only the animals whose microinjection reached the target structure were used for data analysis (Fig. 1). Data analysis Amplitude of vocalization results (mm) were transformed into a vocalization index (VI) using the following formula: VI = (mean vocalization − control value)/control value. Data are reported as mean VI ± SEM and were analyzed by Two Way Repeated Measures analysis of variance (Two Way RM ANOVA), with time (repeated factor) and treatment (independent factor) as variables, and One Way ANOVA followed by the Duncan's test. The level of significance was set at p b 0.05. Results Microinjection of carbachol, morphine sulfate and bicuculline into the DH produced antinociception in guinea pigs as determined by the
Fig. 1. Schematic drawing of frontal sections obtained at representative levels of the guinea pig dorsal hippocampus, indicating the site of microinjection: a) SAL (filled circles (●), n = 6), CCh (crosses (✚), n = 8), ATR (filled triangles (▲), n = 7); b) ATR + CCh (crosses (✚), n = 7), NAL + CCh (filled triangles (▲), n = 7), MOR 1.1 (filled squares (■), n = 6), c) NAL (crosses (✚), n = 7), NAL + MOR (filled triangles (▲), n = 7), ATR + MOR (filled lozenges (♦), n = 7); d) MOR 2.2 (filled circles (●), n = 7), BIC 0.2 (crosses (✚), n = 7), BIC 1.0 (filled triangles (▲), n = 8); e) MUS 0.5 (crosses (✚), n = 9); MUS 1.0 (filled circles (●), n = 7), MUS 0.5 + MOR (filled triangles (▲), n = 9). Sections are from rostral to caudal (5.2–7.6 mm). DH: dorsal hippocampus; GM: corpus geniculatum mediale; NPT: nucleus posterior thalami; VH: ventral hippocampus.
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decrease of the VI in the vocalization test (Figs. 2 and 3). On the other hand, muscimol microinjection produced pronociception in these same conditions (Figs. 2c and 3). The antinociceptive effect of carbachol (Figs. 2a, 3) was blocked by the previous microinjection of atropine (a muscarinic receptor antagonist) into the same site (Figs. 2a, 3). Furthermore, previous microinjection of naloxone (an opioidergic antagonist) into the same site (Figs. 2a, 3) blocked the antinociceptive action of carbachol. Two-way repeated measures ANOVA applied to the SAL, CCh, ATR, ATR +CCh and NAL +CCh groups showed a significant difference in VI between different
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treatments (F(4, 180) =48.59; p b 0.001), along time (F(6, 180)=2.94; p = 0.009) and for time vs. treatment (F(24, 180) =6.39; p b 0.001). The post hoc Duncan test showed a difference (p b 0.05) between the CCh group and all other groups (SAL, ATR, ATR + CCh and NAL + CCh) (Fig. 2a). Moreover, the same test (p b 0.05) indicated that the SAL, ATR, ATR + CCh and NAL + CCh groups did not differ from one another (Fig. 2a). We then applied one-way ANOVA followed by the Duncan test to each experimental time interval. At 2 min, the CCh group differed significantly from the SAL, ATR and NAL + CCh treatments (F(4, 30) = 3.35, p = 0.022; Fig. 2a). For each subsequent time (5, 15, 30, 45 and 60 min), the carbachol group differed significantly from the other groups (F(4, 30) = 9.05, F(4, 30) = 14.83, F(4, 30) = 37.62, F(4, 30) = 88.64, and F(4, 30) = 42.16, respectively, p b 0.001; Fig. 2a). In addition, microinjection of 2.2 nmol morphine sulfate, but not 1.1 nmol, into the DH promoted antinociception in guinea pigs as determined by the decrease of the VI in the vocalization test (Figs. 2b, 3). Furthermore, the antinociceptive effect of morphine was blocked by previous administration of naloxone (1.3 nmol; Figs. 2b, 3). In contrast, prior microinjection of atropine (0.7 nmol; Figs. 2b and 3) was not able to prevent the antinociceptive effect of morphine. Two-way repeated measures ANOVA applied to the opioidergic groups (SAL, MOR 1.1, MOR 2.2, NAL, NAL+ MOR, ATR+ MOR) revealed a significant difference in the VI between treatments (F(5, 204)= 21.44; p b 0.001), along time (F(6, 204)= 15.25; p b 0.001) and for time vs. treatment (F(30, 204) = 9.89; p b 0.001). The post hoc Duncan test showed a difference (p b 0.05) between MOR 2.2 and ATR+ MOR and the other treatments (SAL, MOR 1.1, NAL, NAL + MOR) (Fig. 2b). The same test indicated that the SAL, MOR 1.1, NAL and NAL + MOR groups did not differ from one another (Fig. 2b). Furthermore, there was no difference between the MOR 2.2 and ATR+ MOR groups (Fig. 2b). One-way ANOVA showed no significant difference between groups for the 2- and 5-min intervals (F(5, 34) = 0.80, p = 0.56, and F(5, 34) = 2.53, p = 0.052, respectively). At 15, 30, 45 and 60 min (Fig. 2b), the Duncan test revealed a significant difference between the MOR 2.2 and ATR+ MOR groups and the other treatments (F(5, 34) = 14.28, F(5, 34) = 34.64, F(5, 34) = 32.40, and F(5, 34)= 20.18, p b 0.001; Fig. 2b). The microinjection of bicuculline (a GABAergic receptor antagonist) in the amount of 1.0 nmol, but not 0.2 nmol, into the DH promoted antinociception in guinea pigs as determined by the decrease of the VI in the vocalization test (Figs. 2c, 3). On the other hand, microinjection of 1.0 nmol muscimol (GABAergic receptor agonist), but not 0.5 nmol, into the DH promoted pronociception in the animals. Furthermore, the antinociceptive effect of morphine was also blocked by previous administration of muscimol (0.5 nmol) (Figs. 2c, 3). Two-way repeated measures ANOVA applied to the GABAergic groups (SAL, BIC 0.2, BIC 1.0, MUS 0.5, MUS 1.0, MUS + MOR) revealed a significant difference in the VI between treatments (F(5, 240) = 32.11; p b 0.001) and time vs. treatment (F(30, 240) = 8.41; p b 0.001). The post hoc Duncan test showed a difference (p b 0.05) Fig. 2. Mean vocalization index (VI) under control conditions (time zero, before microinjection) and after (2, 5, 15, 30, 45 and 60 min) various treatments obtained for different experimental groups of conscious guinea pigs submitted to a peripheral noxious stimulus. a) Vocalization index after microinjection of 0.9% saline ( , 0.2 μl), carbachol (●, 2.7 nmol/0.2 μl), atropine (♦, 0.7 nmol/0.2 μl), atropine (0.7 nmol/0.2 μl) followed by carbachol (○, 2.7 nmol/0.2 μl), and naloxone (1.3 nmol/0.2 μl) followed by carbachol (△, 2.7 nmol/0.2 μl) into the dorsal hippocampus. # CCh compared to SAL, ATR, NAL + CCh, and ⁎ CCh compared to all other groups (Duncan's test, p b 0.05). b) Vocalization index after microinjection of 0.9% saline ( , 0.2 μl), MOR 1.1 (□, 1.1 nmol/ 0.2 μl), MOR 2.2 (■, 2.2 nmol/0.2 μl), naloxone (△, 1.3 nmol/0.2 μl), naloxone (1.3 nmol/ 0.2 μl) followed by morphine (♦, 2.2 nmol/0.2 μl), and atropine (0.7 nmol/0.2 μl) followed by morphine (ο, 2.2 nmol/0.2 μl) into the dorsal hippocampus. ⁎MOR 2.2 and ATR + MOR compared to SAL, MOR 1.1, NAL and NAL + MOR (Duncan's test, p b 0.05). c) Vocalization index after microinjection of 0.9% saline ( , 0.2 μl), BIC 0.2 (□, 0.2 nmol/ 0.2 μl), BIC 1.0 (■, 1.0 nmol/0.2 μl), MUS 0.5 (ο, 0.5 nmol/0.2 μl), MUS 1.0 (●, 1.0 nmol/ 0.2 μl), and muscimol (0.5 nmol/0.2 μl) followed by morphine (♦, 2.2 nmol/0.2 μl), into the dorsal hippocampus. ⁎ MUS 1.0 and BIC 1.0 compared to all other groups (Duncan's test, p b 0.05). The vertical bars represent the S.E.M.
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Fig. 3. Schematic drawing of the vocalization amplitude (mm) after administration of different drugs into the dorsal hippocampus of guinea pigs. a, Animal microinjected with 0.9% saline (0.2 μl); b, animal treated with carbachol (2.7 nmol/0.2 μl); c, animal treated with atropine (0.7 nmol/0.2 μl); d, animal treated with atropine (0.7 nmol/0.2 μl) followed by carbachol (2.7 nmol/0.2 μl); e, animal treated with naloxone (1.3 nmol/0.2 μl) followed by carbachol (2.7 nmol/0.2 μl); f, animal treated with morphine (1.1 nmol/0.2 μl); g, animal treated with morphine (2.2 nmol/0.2 μl); h, animal treated with naloxone (1.3 nmol/0.2 μl); i, animal treated with naloxone (1.3 nmol/0.2 μl) followed by morphine (2.2 nmol/0.2 μl); j, animal treated with atropine (0.7 nmol/0.2 μl) followed by morphine (2.2 nmol/0.2 μl); k, animal treated with bicuculline (0.2 nmol/0.2 μl); l, animal treated with bicuculline (1.0 nmol/0.2 μl); m, animal treated with muscimol (0.5 nmol/0.2 μl); n, animal treated with muscimol (1.0 nmol/0.2 μl); o, animal treated with muscimol (0.5 nmol/0.2 μl) followed by morphine (2.2 nmol/0.2 μl). The horizontal bar indicates the application of the noxious stimulus (3 s) at the different time intervals (min). Zero represents the vocalization amplitude during the control period (without saline or drug administration).
between MUS 1.0 and BIC 1.0; and between treatment with both drugs and the other treatments (SAL, BIC 0.2, MUS 0.5, MUS + MOR) (Fig. 2c). The same test indicated that the SAL, BIC 0.2, MUS 0.5, MUS + MOR groups did not differ from one another (Fig. 2c). When we applied one-way ANOVA followed by the Duncan test to each experimental time interval, the MUS 1.0 group differed significantly from all the other treatments in all the intervals and the BIC 1.0 group differed significantly from all the other treatments in all the intervals except 2 min (p b 0.001). Our results were: 2 min (F(5, 40) = 8.84), 5 min (F(5, 40) = 18.30), 15 min (F(5, 40) = 18.81), 30 min (F(5, 40) = 26.31), 45 min (F(5, 40) = 31.21), 60 min (F(5, 40) = 32.29; Fig. 2c). Saline administration into the DH did not alter the VI after the animals had been submitted to a peripheral noxious stimulus (Fig. 3), and this group was used as control in all experiments (Fig. 2). Discussion The present results demonstrate the participation of the cholinergic, opioidergic and GABAergic circuits of the DH in the modulation of the nociceptive response. Our results showed that carbachol (2.7 nmol), morphine (2.2 nmol) and bicuculline (1.0 nmol) injected into the DH of the different groups of guinea pigs significantly reduced
nociceptive response, as demonstrated by decreased vocalization induced by the peripheral noxious stimulus. On the other hand, muscimol (1.0 nmol) microinjection into DH promoted pronociception demonstrated by increased vocalization induced by the peripheral noxious stimulus. In different nuclei of the central nervous system, intracerebral microinjection of the cholinergic agonist carbachol has been shown to produce antinociceptive effects, which were abolished by local administration of atropine into of rats (Guimarães et al., 2000; Ma et al., 2001; Abe et al., 2003) and guinea pigs (Menescal-de-Oliveira and Lico, 1985; Leite-Panissi et al., 2004; Da Silva and Menescal-deOliveira, 2006). Aloisi et al. (1993) reported that subcutaneous injection of formalin into the rat hind paw induced a persistent nociceptive behavior and a prolonged decrease in DH activity of choline acetyltransferase. In our study, microinjection of carbachol into the DH of guinea pigs produced antinociception, which was demonstrated by a decrease in the vocalization response after a peripheral noxious stimulus (decreased VI). The animal's vocalization in these conditions indicates the participation of the affective– motivational component of the painful sensation, which essentially depends on the supra-spinal activation, including limbic structures like the hippocampus (Lico et al., 1974). The carbachol effect was
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blocked by prior microinjection of atropine, indicating the participation of muscarinic receptors in this antinociceptive response. Although some works have demonstrated that carbachol and morphine microinjected into the hippocampus can produce motor activation (Flicker and Geyer, 1982; Cain and Corcoran, 1985), our experimental conditions did not permit this observation. However, the same dose of carbachol and morphine injected into the DH provoked a reduction in the duration of tonic immobility episodes, which accords with an increase in locomotion, considering that tonic immobility is a response characterized by a reversible state of akinesia (unpublished data). Acetylcholine is mainly released in the hippocampus by the activation of cholinergic cell bodies located in the medial septal area (Amaral and Kurz, 1985). Several opioid neuropeptides, such as enkephalin and β-endorphin, as well as morphine, promote a decrease in hippocampal ACh levels when administered intraseptally (Moroni et al., 1977). These observations suggest that the opioid agonists may act at the level of cholinergic cell bodies in the septal region, modulating the activity of septal cholinergic afferents terminating in the hippocampus (Ammon's horn). On the other hand, when opioids are applied directly to the hippocampus, ACh levels remain unchanged (Botticelli and Wurtman, 1981). Likewise, our results did not show this kind of opioidergic-cholinergic interaction in the hippocampus, since microinjection of atropine prior to morphine was not effective in blocking the antinociceptive effect promoted by the opioid agonist. On the contrary, our results suggest a possible cholinergic–opioidergic interaction, once we showed that the antinociception induced by carbachol or morphine sulfate administered into the DH is blocked by pretreatment with naloxone injected into the same site. These results can indicate the action of the cholinergic system inside the DH seems to be opioiddependent because in this region, pretreatment with naloxone blocks the effect of carbachol. On this basis, we propose that the local carbachol injection may activate opioidergic neurons, which in turn may inhibit GABAergic neurons, disinhibiting DH output neurons that possibly lead to a succession of events that finally reach descending inhibitory pathways promoting antinociception. Although, in our case, the antinociception obtained in DH may be assigned to activation of cholinergic–opioidergic systems, we cannot reject the participation of other hippocampal neurotransmitters in mediating the antinociceptive response. Concerning the effect produced by the microinjection of morphine into the DH, only the 2.2 nmol dose promoted an antinociceptive response, which was reduced or abolished by prior administration of naloxone. Naloxone per se did not produce any alterations in the VI when microinjected into the same site. Although morphine preferentially acts on μ-type opioid receptors, a high density of κ-type opioid receptors has been found in the hippocampus of guinea pigs (Foote and Maurer, 1986). Therefore, in the present study, the effects of morphine microinjected into the hippocampus of guinea pigs may depend on μ- and/or κ-opioid receptors, since only the higher dose (2.2 nmol) was effective in antinociception induction. There is evidence that GABAergic mediation of opioid effects is a widespread phenomenon and occurs throughout most of CNS. Kalyuzhny and Wessendorf (1998) observed a colocalization between hippocampal μ-opioid receptors and GABAergic interneurons in CA1, CA3 and dentate gyrus in rats. The localization of μ-opioid receptors on GABAergic neurons suggest that these receptors, when activated, can directly control the hippocampal GABAergic neurons' activity (Kalyuzhny and Wessendorf, 1997; 1998). Physiological studies have shown that activation of the opioid receptors can lead to the inhibition of interneuron activity (Madison and Nicoll, 1988; Wimpey and Chavkin, 1991; Svoboda and Lupica, 1998), resulting in diminished GABA release and the disinhibition of hippocampal pyramidal neurons (Zieglgänsberger et al., 1979; Cohen et al., 1992; Lupica et al., 1992). Basbaum and Fields (1984) proposed that µ-opioid receptor activation
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produces antinociception from within the PAG by reducing local inhibitory GABAergic influences (disinhibition) on descending projection neurons. Our results support the hypothesis that modulation of nociceptive response in the DH could occur in a manner similar to that proposed by Basbaum and Fields (1984) into PAG. It is therefore likely that the antinociception observed after microinjection of morphine into the DH occurs through the inhibition of tonically active GABAergic interneurons. We also observed that muscimol (0.5 nmol) at a dose that has no effect per se on nociception, when injected before morphine into the same site, blocks the antinociceptive effect of the latter. This supports the suggestion that opioid effects may occur via inhibition of GABA interneurons and also that the modulation of nociception by the DH might occur, in part, via a neural circuit similar to that proposed for the modulation of antinociception and tonic immobility by the ventrolateral PAG and nucleus raphe magnus (Monassi et al., 1999; Da Silva and Menescal-de-Oliveira, 2007). It is known that GABAA receptors have a prominent role in mediating tonic inhibition, particularly in hippocampal pyramidal cells (Glykys and Mody, 2006; Serwanski et al., 2006). This tonic inhibition is responsible for generating ~75% of the total inhibitory charge received by hippocampal neurons (Mody and Pearce, 2004). In the present work, the GABAA receptors' activation into DH by muscimol in an effective dose (1.0 nmol) provoked pronociception evaluated by the increase of vocalization elicited by peripheral noxious stimulation. We suggest that this effect can occur by GABAA receptor activation, possibly situated in DH pyramidal neurons, producing inhibition of these neurons and consequently leading to successive events that could ultimately activate descending facilitatory pathways promoting pronociception. On the other hand, the injection of bicuculline inhibits GABAA receptors, resulting in disinhibition of DH output neurons that possibly lead to a succession of events that finally reach descending inhibitory pathways promoting antinociception. In summary, the present results demonstrate that the activation of the cholinergic or opioidergic system of the DH promotes antinociception in guinea pigs, while GABAergic activation promotes pronociception, as demonstrated by respective decreases and increases of the VI. In addition, antinociception produced by cholinergic stimulation of the DH depends on opioid synapses present at the same site. On the other hand, antinociception observed after microinjection of morphine into the DH occurs through the inhibition of tonically active GABAergic interneurons. Acknowledgments We would like to thank Mrs. M.R. Brentegani and Mr. R.F. de Melo for technical assistance and histological processing. We are also indebted to Rildo A. Volpini, PhD, for suggestions, and to L.F.S da Silva, MSc, for preparation of the figures. This work was supported by CAPES and CNPq. References Abe, K., Kikuta, J., Kato, M., Ishida, K., Shigenaga, T., Taguchi, K., Miyatake, T., 2003. Effects of microinjected carbachol on the antinociceptive response to noxious heat stimuli. Biological & Pharmaceutical Bulletin 26 (2), 162–165. Aloisi, A.M., Albonetti, M.E., Lodi, L., Lupo, C., Carli, G., 1993. Decrease of hippocampal choline acetyltransferase activity induced by formalin pain. Brain Research 629 (1), 167–170. Amaral, D.G., Kurz, J., 1985. An analysis of the origins of the cholinergic and noncholinergic septal projections to the hippocampal formation of the rat. The Journal of Comparative Neurology 240 (1), 37–59. Archer, D.P., Roth, S.H., 1997. Pharmacodynamics of thiopentone: nocifensive reflex threshold changes correlate with hippocampal electroencephalography. British Journal of Anaesthesia 79, 744–749. Baptista, V., 2000. Participação do sistema colinérgico da formação hipocampal dorsal na modulação da imobilidade tônica em cobaias. Unpublished Dissertation, FMRP/ USP. Ribeirão Preto. Basbaum, A.I., Fields, H.L., 1984. Endogenous pain control systems: brainstem spinal pathways and endorphin circuitry. Annual Review of Neuroscience 7, 309–338.
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