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Defensive-like behaviors and antinociception induced by NMDA injection into the periaqueductal gray of mice depend on nitric oxide synthesis
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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
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Research Report
Defensive-like behaviors and antinociception induced by NMDA injection into the periaqueductal gray of mice depend on nitric oxide synthesis Tarciso Tadeu Miguel a , Ricardo Luiz Nunes-de-Souza a,b,⁎ a
Programa de Pós-Graduação em Ciências Fisiológicas, UFSCar/Convênio UNESP, Rod. Araraquara-Jau, km 01, 14801-902, Araraquara, SP, Brazil b Lab. Farmacologia, Faculdade de Ciências Farmacêuticas–UNESP, Rod. Araraquara-Jau, km 01, 14801-902, Araraquara, SP, Brazil
A R T I C LE I N FO
AB S T R A C T
Article history:
Glutamate NMDA receptor activation within the periaqueductal gray (PAG) leads to
Accepted 21 December 2005
antinociceptive, autonomic and behavioral responses characterized as the fear reaction. Considering that NMDA receptor triggers activation of neuronal nitric oxide synthase (nNOS), enzyme that produces nitric oxide (NO), this study investigated the effects of intra-
Keywords:
PAG infusions of NPLA (Nω-propyl-L-arginine), an nNOS inhibitor, on behavioral and
NMDA receptor
antinociceptive responses induced by local injection of NMDA receptor agonist in mice. The
Defensive reactions
behaviors measured were frequency of jumping and rearing as well as duration (in seconds)
Antinociception
of running and freezing. Nociception was assessed during the second phase of the formalin
Nitric oxide
test (injection of 50 μl of formalin 2.5% into the dorsal surface of the right hind paw). Five to
nNOS
seven days after stereotaxic surgery for intracerebral cannula implantation, mice were
Periaqueductal gray
injected with formalin into the paw, and 10 min later, they received intra-dPAG injection of NPLA (0, 0.2, or 0.4 nmol/0.1 μl). Ten minutes later, they were injected with NMDA (N-methyl-
Mice
D-aspartate:
0 or 0.04 nmol/0.1 μl) into the same midbrain site and were immediately placed
in glass holding cage for recording the defensive behavior and the time spent on licking the injected paw with formalin during a period of 10 min. Microinjections of NMDA significantly decreased nociception response and produced jumping, running, and freezing reactions. Intra-dPAG injections of NPLA (0.4 nmol) completely blocked the NMDA effects without affecting either behavioral or nociceptive responses in intra-dPAG saline-injected animals, except for the rearing frequency that was increased by the nNOS inhibitor. These results strongly suggest the involvement of NO within the PAG in the antinociceptive and defensive reactions induced by local glutamate NMDA receptor activation in this midbrain structure. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
The midbrain periaqueductal gray (PAG) has been identified as a key component of the pain inhibitory system since electrical
or chemical stimulation of this area suppresses nociceptive transmission from the dorsal horn of the spinal cord (for review, see Fields and Basbaum, 1999). In addition, many lines of evidence point to dorsal region of the PAG (dPAG) as the
⁎ Corresponding author. Lab. Farmacologia, Faculdade de Ciências Farmacêuticas–UNESP, Rod. Araraquara-Jau, km 01, 14801-902, Araraquara, SP, Brazil. E-mail address: [email protected] (R.L. Nunes-de-Souza). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.12.095
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principal substrate of aversion in the brain (for review, see Graeff, 2004). For instance, chemical stimulation of dPAG elicits autonomic and behavioral responses similar to defensive reactions to innate aversive stimuli (Bandler and Carrive, 1988; Bittencourt et al., 2004; Blanchard and Blanchard, 1988, 1989). Excitatory amino acids (e.g., glutamate) receptor agonists are probably the main chemical tool used to stimulate PAG and other brain sites (Bandler, 1988; Bandler and Carrive, 1988; Bittencourt et al., 2004; Friebe and Koesling, 2003). The action of glutamate is mediated at metabotropic receptors and three subtypes of ionotropic receptors, AMPA (amino-3-hydroxy-5methyl-4-isoxazole propionic acid), kainate, and NMDA (Nmethyl-D-aspartate) (Heresco-Levy, 2003; Huntley et al., 1994; Seebury, 1993). Glutamate NMDA receptor activation leads to cellular calcium influx which triggers a cascade of intracellular events including activation of nitric oxide synthase (NOS), enzyme that produces nitric oxide (NO) by conversion of Larginine to L-citroline, having NADPH (nicotinamide adenine dinucleotide phosphate) and Ca2+ as co-factors (Garthwaite et al., 1989; Lohse et al., 1998; Mayer et al., 1991). There are at least three known isoforms of NOS: an inducible (iNOS) and two constitutive forms, which are present under physiological conditions in endothelium (but not only in this tissue, eNOS) and in neurons (nNOS) (Guix et al., 2005; Lamas et al., 1992; Mungrue et al., 2003; Prast and Philippu, 2001). Intracellular NO activates guanylate cyclase (GC) with the subsequent production of cyclic guanosine-3′,5′-monophosphate (cGMP) and protein phosphorylation (Friebe and Koesling, 2003; Ignarro,
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1991; Krumenacker et al., 2004). NO also exerts other cellular effects independent of the GC activation (for review, see Guix et al., 2005; Prast and Philippu, 2001). It presents ubiquitous localization that permits its involvement in a wide range of physiological and pathophysiological processes. For instance, several studies have shown that NO is involved in peripheral and central nociceptive processing (for review, see Riedel and Neeck, 2001) and in defensive reactions (Chiavegatto et al., 1998; De Oliveira et al., 2001; Guimarães et al., 2005). Besides producing neurovegetative activation (e.g., tachycardia, hypertension, defecation) and flight reactions (e.g., jumping, running, freezing) (e.g., Bandler and Carrive, 1988; Bittencourt et al., 2004), intra-dPAG infusions of NMDA receptor agonists also provoke high magnitude antinociception (e.g., Berrino et al., 2001; Jacquet, 1988; Siegfried and Nunes-de-Souza, 1989). This study investigated the effects of nNOS inhibition within this midbrain structure is able to antagonize both behavioral and antinociceptive responses induced by local injection of NMDA receptor agonist in mice.
2.
Results
Histological analysis demonstrated that 70 mice had accurate cannula placements in the PAG (Fig. 1). Final sample sizes ranged from n = 9–15 animals per group. Fig. 2 shows the effects of intra-PAG injection of NMDA on time spent licking the formalin 2.5% injected paw during 10 min (25–35 min after formalin injection, second phase of
Fig. 1 – (A) Schematic representation of microinfusion sites within the midbrain periaqueductal gray (PAG) of the mouse. The number of the points in the figure is less than the total number of mice because of the overlaps. (B) Photomicrograph of midbrain coronal section from a representative subject showing an injection site into the PAG. Section correspond to −4.24 mm from bregma in the atlas of Paxinos and Franklin (2001).
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Fig. 2 – Effects of intra-PAG injection of NMDA (0.04 nmol) on time spent licking the paw injected with formalin 2.5% during 10 min (second phase of nociception test) in mice pretreated with saline or NPLA (0.2–0.4 nmol) into the same midbrain site (n = 10–15) (see text for details). *P b 0.05 vs. Saline + Saline; #P b 0.05 vs. Saline + NMDA. nociception test) in mice pretreated with saline or NPLA into the same midbrain site. Kruskal–Wallis test revealed significant differences among groups (H = 32.96, P b 0.05). Post hoc comparisons showed that animals treated with Saline + NMDA spent less time licking the injected paw when compared with Saline + Saline group (control). Prior injection of NPLA (0.4 nmol) reversed the NMDA effect without producing any significant differences when compared to Saline + Saline group. Fig. 3 shows the effects of intra-PAG injection of NMDA on frequency of jumps in mice pretreated with saline or NPLA into the same midbrain site. Kruskal–Wallis test revealed significant differences among groups (H = 23.74, P b 0.05) and post hoc comparisons indicated that Saline + NMDA group jumped significantly more than the control group (Saline +
Fig. 3 – Effects of intra-PAG injection of NMDA (0.04 nmol) on jumping frequency during 5 min in mice pretreated with saline or NPLA (0.2–0.4 nmol) into the same midbrain site (n = 9–14) (see text for details). *P b 0.05 vs. Saline + Saline; #P b 0.05 vs. Saline + NMDA.
Saline). Prior intra-PAG treatment with NPLA (0.4 nmol) antagonized the NMDA effects on this behavioral response. No significant effects were obtained with NPLA on frequency of jumps when compared to the control group. The effect of intra-PAG NMDA infusion on time spent running in mice submitted to prior injections of NPLA in the same structure is shown in Fig. 4. Non-parametric statistics revealed significant differences among groups (H = 24.14; P b 0.05) and Dunn's test indicated that Saline + NMDA group spent more time running when compared to the control group (Saline + Saline). This NMDA effect was completely blocked when NPLA (0.4 nmol) was previously injected into the PAG. On the other hand, intra-PAG NPLA did not affect running per se, since no significant differences were observed comparing the two groups treated with the nNOS inhibitor [NPLA (0.2 nmol) + Saline and NPLA (0.4 nmol) + Saline] to the control group. Fig. 5 illustrates the effects of intra-PAG infusion of NMDA on time spent in freezing in mice submitted to a prior injection of NPLA into the same structure. One-way ANOVA revealed significant differences among groups (F5,57 = 6.56, P b 0.05). Post hoc Duncan's tests showed that animals treated with Saline + NMDA or NPLA (0.2 nmol) + NMDA spent more time in immobility than control group (Saline + Saline), while NPLA (0.4 nmol) + NMDA group selectively antagonized the NMDA effect. Fig. 6 shows that intra-PAG infusion of NMDA failed to alter rearing frequency. However, intra-PAG infusion of NPLA (0.4 nmol) increased frequency of rearing in both saline or NMDA treated groups (ANOVA, followed by Duncan's test: F5,57 = 8.21, P b 0.05).
3.
Discussion
The present results are in consonance with previous studies demonstrating that NMDA microinjection into the mouse PAG induces antinociception and elicits defensive behavioral reactions characterized by jumping and running followed by
Fig. 4 – Effects of intra-PAG injection of NMDA (0.04 nmol) on running time (sec) during 5 min in mice pretreated with saline or NPLA (0.2–0.4 nmol) into the same midbrain site (n = 9–14) (see text for details). *P b 0.05 vs. Saline + Saline; #P b 0.05 vs. Saline + NMDA.
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Fig. 5 – Effects of intra-PAG injection of NMDA (0.04 nmol) on freezing time (seconds) during 5 min in mice pretreated with saline or NPLA (0.2–0.4 nmol) into the same midbrain site (n = 9–14) (see text for details). *P b 0.05 vs. Saline + Saline; #P b 0.05 vs. Saline + NMDA.
a period of freezing (Bandler, 1988; Bandler and Carrive, 1988; Berrino et al., 2001; Bittencourt et al., 2004; Ferreira-Netto et al., 2005; Jacquet, 1988; Siegfried and Nunes-de-Souza, 1989). These behaviors are proposed to reproduce aversive responses since they are similar to reactions induced by natural aversive stimuli such as the exposure to a predator (Blanchard and Blanchard, 1988, 1989). Our study also demonstrated that these responses depend on NO synthesis, since intra-PAG injection of NPLA, an nNOS inhibitor, completely antagonized both NMDA-induced nociception inhibition and defensive-like behaviors. Previous studies have demonstrated that intra-PAG injections of NMDA decrease nociceptive response assessed by different nociception tests (Berrino et al., 2001; Jacquet, 1988; Siegfried and Nunes-de-Souza, 1989). Our results corroborate these findings and indicate a fundamental importance of nitric oxide on NMDA effects, since prior nNOS inhibition with NPLA injections into the same structure was able to reverse the antinociception induced by this excitatory amino acid. Interestingly, NPLA injections did not alter per se the nociceptive response profile. Animals treated with NPLA (both doses) + Saline spent similar time licking the paw injected with formalin when compared to Saline + Saline group, suggesting that the nNOS inhibitor fails to provoke intrinsic effects on nociception. It has been demonstrated that PAG stimulation produced by local injection of glutamate is related to activation of the descending inhibitory system, which inhibits nociceptive neurons in dorsal horn. This system is composed of a descending neuron bundle that involves neurons from PAG and rostroventral medulla nuclei (e.g., nucleus raphe magnus), which project to dorsal horn making inhibitory connections in ascending nociceptive transmission (Fields and Basbaum, 1999; Jones and Gebhart, 1988; Kharkevich and Churukanov, 1999; Millan, 2002). Importantly, our study suggests that the antinociceptive mechanism mediated by glutamate NMDA receptors within the PAG
45
involves, at least in part, synthesis of nitric oxide in this midbrain structure. Interestingly, intra-PAG infusions of NPLA also changed the behavioral reactions provoked by intra-PAG injections of NMDA. The excitatory amino acid provoked a sudden sequence of jumping and running behaviors that were followed by a period of freezing. These results corroborate many previous studies (Bandler, 1988; Bandler and Carrive, 1988; Beckett et al., 1992; De Oliveira et al., 2001; Molchanov and Guimarães, 1999), which have demonstrated that intra-PAG injections of glutamate receptor agonists produce behavioral responses characteristics of rodent's defensive reactions. In the present study, NPLA antagonized these NMDA-induced behavioral effects, suggesting that NO release within the PAG plays a role on defensive behavior. While intra-PAG infusion of NMDA did not alter rearing frequency, NPLA (higher dose) increased this vertical exploratory behavior, an effect that was independent of the treatment combination received (i.e., saline or NMDA). The increase in the frequency of rearing suggests that this nNOS inhibitor, at doses employed here, did not provoke motor disruption, an effect previously reported with systemic injections with other NOS inhibitors (De Oliveira et al., 1997; Del Bel et al., 2002, 2004). In view of these findings, intra-PAG NPLA injections seem to selectively reduce NMDA-induced behavioral responses (e.g., jumping, running, freezing) in mice. Taken together, these results suggest NO as a strong candidate to modulate anxiety states. Support for this hypothesis is found in studies with intra-PAG injections of glutamate receptor agonists and antagonists as well as with NOS inhibitors. For instance, while NMDA receptor agonists produce flight reactions as demonstrated here and in previous studies (Bandler, 1988; Bandler and Carrive, 1988; Del Bel et al., 1998; Ferreira-Netto et al., 2005), intra-PAG injections of NMDA and non-NMDA receptor antagonists provoke anxiolytic-like effects in animals exposed to elevated plus-maze (EPM) (Guimarães et al., 1991; Hall and Behbehani, 1998; Matheus
Fig. 6 – Effects of intra-PAG injection of NMDA (0.04 nmol) on rearing frequency during 5 min in mice pretreated with saline or NPLA (0.2–0.4 nmol) into the same midbrain site (n = 9–14) (see text for details). *P b 0.05 vs. Saline + Saline and Saline + NMDA.
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and Guimarães, 1997; Molchanov and Guimaraes, 2002), a widely used animal model of anxiety (Handley and Mithani, 1984; Lister, 1987; Pelow et al., 1985; Rodgers et al., 1997). Regarding the NO role on anxiety, both systemic injection of LNAME, an unspecific NOS inhibitor, as well as intra-PAG infusions of L-NAME and L-NOARG attenuated anxiety indices in the EPM in rats (De Oliveira et al., 1997, 2001; Faria et al., 1997; Guimarães et al., 1994). More recently, Guimarães et al. (2005), using a prey–predator model, have demonstrated that local infusion into dorsolateral PAG (dlPAG) of AP-7, a glutamate NMDA receptor antagonist, completely prevented the flight reactions induced by intra-dlPAG administration of SIN-1, a NO donor, suggesting a modulatory role for NO within the PAG, probably by interacting with glutamate-mediated neurotransmission. In this context, the present results confirm a strong interaction between NMDA receptor activation and NO synthesis within this midbrain structure on defensive reactions in mice. In conclusion, this study demonstrated that intra-PAG infusions of NPLA selectively antagonized antinociception and defensive behaviors induced by local NMDA infusions in the same midbrain structure, suggesting that NO plays an important role within the PAG on pain inhibition and behavioral responses induced by threatening situations. It has been emphasized that flight reactions provoked by NOS neurons activation within the PAG are probably related to NO modulatory effects on other neurotransmitter release (e.g., glutamate, gamma-aminobutyric acid-GABA and serotonin (Guimarães et al., 2005)). It remains unclear whether NO effects on nociception reported in the present study involve the activation of other neurotransmitter systems.
respectively, 4.1 mm posterior to bregma, 1.3 mm lateral to the midline, and 2.2 mm ventral to the skull surface, with the guide cannula angled 26° to the vertical. A dummy cannula (33-gauge stainless steel wire; Fishtex Industry and Commerce of plastics Ltda.), inserted into each guide-cannula immediately after of surgery, served to reduce the incidence of occlusion. Five to seven days after surgical recovery, solutions were injected into the PAG by microinjection units (33-gauge stainless steel cannula; Insight Equipamentos Científicos Ltda), which extended 1.0 mm beyond the tips of the guide cannula. Each microinjection unit was attached to a 5-μl Hamilton microsyringe via polyethylene tubing (PE-10), and administration was controlled by an infusion pump (BI 2000, Insight Equipamentos Científicos Ltda) programmed to deliver a volume at rate of 0.1 μl (volume injected) over a period of 30 s. The microinjection procedure consisted of gently restraining the animal, removing of dummy cannula, inserting the injection unit, infusing the solution, keeping the injection unit in situ for a further 60 s. Confirmation of successful infusion was obtained by monitoring the movement of a small air bubble in the PE-10 tubing. 4.4.
General procedure
4.
Experimental procedures
On test days, animals were transported to the experimental room and left undisturbed for at least 30 min prior to testing. They were then injected with 50 μl of formalin (2.5%) into the dorsal surface of the right hind paw and individually placed in a glass holding cage (30 × 20 × 25 cm). Fifteen minutes after formalin injection, animals received intra-PAG injection of saline or NPLA (0.2 or 0.4 nmol/0.1 μl) and were placed back in the glass holding cage. Ten minutes later, they received a second intra-PAG injection either with saline or NMDA (0.04 nmol/0.1 μl). Thus, six groups were formed: Saline + Saline, Saline + NMDA, NPLA (0.2 nmol) + Saline, NPLA (0.4 nmol) + Saline, NPLA (0.2 nmol) + NMDA, NPLA (0.4 nmol) + NMDA. Formalin test (Abbott et al., 1995; Dubuisson and Dennis, 1977) was recorded immediately after the second intra-PAG injection procedure placing the animals individually in the glass cage and recording the time spent licking the injected paw for 10 min (25–35 min after formalin injection).
4.1.
Subjects
4.5.
Subjects were male Swiss adults mice weighing 25–35 g (Paulista State University/UNESP, SP, Brazil), housed in groups of 10 per cage (cage size: 41 × 34 × 16 cm). They were maintained under a normal 12-h light cycle (lights on: 7:00 a.m.) in a temperature (23 ± 1 °C)and humidity (55 ± 5%)-controlled environment. Food and drinking water were freely available except during the brief test periods. All mice were experimentally naïve and used only once. 4.2.
Drugs
The drugs used were Nù-propyl-L-arginine (NPLA–Tocris Cookson Inc., Ballwin, USA), a highly selective and potent inhibitor of nNOS (Ki = 57 nM), which displays 3158-fold and 149-fold selectivity over iNOS and eNOS, respectively (Zhang et al., 1997), and NMDA (Nmethyl-D-aspartic acid–RBI, USA). The doses used were based on previous studies: NMDA 0.04 nmol/0.1 μl (Siegfried and Nunes-deSouza, 1989) and NPLA, 0.2 nmol/0.1 μl–0.4 nmol/0.1 μl (El-Haddad et al., 2002; Kakoki et al., 2001; Zhang et al., 1997). The drugs were dissolved in physiological saline (NaCl 0.9%). 4.3.
Behavioral analysis
Videotapes were scored blind by a highly trained observer using an ethological analysis packing developed by Dr. Morato's group at Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, USP. In addition to recording the time spent licking the formalin-injected paw (see above), the observer scored the frequency of jumps (i.e., upward leaps directed to the border of the glass cage) and rearing (i.e., standing on hind limbs, with both forelimbs off the floor, including unsupported rearing and rearing against the wall), the time (in seconds) spent running (i.e., time of trotting [running keeping the same pattern of walking] and galloping [fast running alternating anterior and posterior limb pairs]), and in freezing (complete absence of movement except breathing, while the animal a characteristic tense posture). The behavioral observation was performed during 5 min following intra-PAG saline or NMDA injection procedure (see above). All experiments were carried out during the light phase of the light–dark cycle. The illumination intensity in the room where the experiments were carried out was 1 × 100 W at the room with illumination (1 × 100 W). All sessions were videorecorded by a camera linked to a monitor and VCR in the adjacent laboratory.
Surgery and microinjection 4.6.
Mice were implanted with 7-mm stainless steel guide cannula (26gauge; Insight Equipamentos Científicos Ltda) under sodium thiopental (100 mg/kg, i.p.) anesthesia. Guide cannula was fixed to the skull using dental acrylic and jeweler's screws. Stereotaxic coordinates (Paxinos and Franklin, 2001) for the PAG were,
Statistics
All results were initially submitted to Levene's test for homogeneity of variance. When appropriate, the data were transformed to square root, cube root or log before being submitted to one-way analyses of variance (ANOVA). Where indicated by significant F
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values, Duncan's multiple comparisons tests were used. Where Levene's test remained significant even after data transformation non-parametric tests (Kruskal–Wallis test followed by Dunn's multiple comparison test) were used. In all cases, a P value ≤ 0.05 was required for significance. 4.7.
Histology
At the end of testing, all animals were deeply anaesthetized with sodium thiopental and received a microinfusion of 1% Evans blue (according to the microinjection procedure described above). Brains were removed and injection sites verified histologically according to the atlas of Paxinos and Franklin (2001). Data from animals with injection sites outside the PAG were excluded from the study. 4.8.
Ethics
The experiments carried out in this study comply with the norms of Brazilian Neuroscience and Behavior Society (SBNeC), based on the US National Institutes of Health Guide for Care and Use of Laboratory Animals.
Acknowledgments This study was supported by FAPESP (Proc.02/03735-0), CNPq, and PADC/FCF-Unesp. T.T.Miguel was a recipient of FAPESP (Proc. 03/11571-7), and R.L. Nunes-de-Souza received a CNPq research fellowship.
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modulation and the mechanisms of antipsychotyc atypicality. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 27, 1113–1123. Huntley, G.W., Vickers, J.C., Morrison, J.H., 1994. Cellular and synaptic localization of NMDA and non-NMDA receptor subunits in neocortex: organizational features related to cortical circuitry, function and disease. Trends Neurosci. 17 (2), 536–543. Ignarro, L.J., 1991. Signal transduction mechanisms involving nitric oxide. Biochem. Pharmacol. 41, 485–490. Jacquet, Y.F., 1988. The NMDA receptor: central role in pain inhibition in rat periaqueductal gray. Eur. J. Pharmacol. 154 (3), 271–276. Jones, S.L., Gebhart, G.F., 1988. Inhibition of spinal nociceptive transmission from the midbrain, pons and medulla in the rat: activation of descending inhibition by morphine, glutamate and electrical stimulation. Brain Res. 20–460 (2), 281–296. Kakoki, M., Zou, A.P., Matson, D.L., 2001. The influence of nitric oxide synthase 1 on blood flow and interstitial nitric oxide in the kidney. Am. J. Physiol.: Regul., Integr. Comp. Physiol. 281 (1), R91–R97. Kharkevich, D.A., Churukanov, V.V., 1999. Pharmacological regulation of descending cortical control of the nociceptive processing. Eur. J. Pharmacol. 375 (1–3), 121–131. Krumenacker, J.S., Hanafy, K.A., Murad, F., 2004. Regulation of nitric oxide and soluble guanylyl cyclase. Brain Res. Bull. 62, 505–515. Lamas, S., Marsden, P.A., Li, G.K., Tempst, P., Michel, T., 1992. Endothelial nitric oxide synthase: molecular cloning and characterization of a distinct constitutive enzyme isoform. Proc. Natl. Acad. Sci. U. S. A. 89, 6348–6352. Lister, R.G., 1987. The use a plus-maze to measure anxiety in the mouse. Psychopharmacology (Berlin) 92, 180–185. Lohse, M.J., Forstermann, U., Schmidt, H.H.H.W., 1998. Pharmacology of NO: cGMP signal transduction. Naunyn-Schmiedeberg's Arch. Pharmacol. 358, 111–112. Matheus, M.G., Guimarães, F.S., 1997. Antagonism of the non-NMDA receptors in the dorsal periaqueductal gray induces anxiolytic effect in the elevated plus maze. Psychopharmacology (Berlin) 132, 14–18.
Mayer, B., John, M., Heinzel, B., Werner, E.R., Wachter, H., Schultz, G., Bohme, E., 1991. Brain nitric oxide synthase is a biopterinand flavin-containing multi-functional oxido-reductase. FEBS Lett. 288, 187–191. Millan, M.J., 2002. Descending control of pain. Prog. Neurobiol. 66 (6), 355–474. Molchanov, M.L., Guimaraes, F.S., 1999. Defense reaction induced by a metabotropic glutamate receptor agonist microinjected into the dorsal periaqueductal gray of rats. Braz. J. Med. Biol. Res. 32 (12), 1533–1537. Molchanov, M.L., Guimaraes, F.S., 2002. Anxiolytic-like effects of AP-7 injected into the dorsolateral or ventrolateral columns of the periaqueductal gray of rats. Psychopharmacology (Berlin) 160 (1), 30–38. Mungrue, I.M., Bredt, D.S., Stewart, D.J., Husain, M., 2003. From molecules to mammals: what's NOS got to do with it? Acta Physiol. Scand. 179, 123–135. Paxinos, G., Franklin, K.B.J., 2001. The Mouse Brain in Stereotaxic Coordinates. Academic Press, California, USA. Pelow, S., Chopin, P., File, S.E., Briley, M., 1985. Validation of open: enclosed arm entries in a elevated plus-maze as a measure of anxiety in the rat. J. Neurosci. Methods 14, 149–167. Prast, H., Philippu, A., 2001. Nitric oxide as a modulator of neuronal function. Prog. Neurobiol. 64, 51–59. Riedel, W., Neeck, G., 2001. Nociception, pain, and antinociception: current concepts. Z. Rheumatol. 60, 404–415. Rodgers, R.J., Cao, B.J., Dalvi, A., Holmes, A., 1997. Animals models of anxiety an ethological perspective. Braz. J. Med. Biol. Res. 30 (3), 289–304. Seebury, P.H., 1993. The tips/tins lecture: the molecular biology of mammalian glutamate receptors channels. Trends Pharmacol. Sci. 14, 297–303. Siegfried, B., Nunes-de-Souza, R.L., 1989. NMDA receptors blockade in the periaqueductal gray prevents stress-induced analgesia in attacked mice. Eur. J. Pharmacol. 168 (8), 239–242. Zhang, H.Q., Fast, W., Marletta, M.A., Martasek, P., Silverman, R.B., 1997. Potent and selective inhibition of neuronal nitric oxide synthase by Nw-Propil-L-arginina. J. Med. Chem. 40, 3869–3870.
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
Defensive-like behaviors and antinociception induced by NMDA injection into the periaqueductal gray of mice depend on nitric oxide synthesis Tarciso Tadeu Miguel a , Ricardo Luiz Nunes-de-Souza a,b,⁎ a
Programa de Pós-Graduação em Ciências Fisiológicas, UFSCar/Convênio UNESP, Rod. Araraquara-Jau, km 01, 14801-902, Araraquara, SP, Brazil b Lab. Farmacologia, Faculdade de Ciências Farmacêuticas–UNESP, Rod. Araraquara-Jau, km 01, 14801-902, Araraquara, SP, Brazil
A R T I C LE I N FO
AB S T R A C T
Article history:
Glutamate NMDA receptor activation within the periaqueductal gray (PAG) leads to
Accepted 21 December 2005
antinociceptive, autonomic and behavioral responses characterized as the fear reaction. Considering that NMDA receptor triggers activation of neuronal nitric oxide synthase (nNOS), enzyme that produces nitric oxide (NO), this study investigated the effects of intra-
Keywords:
PAG infusions of NPLA (Nω-propyl-L-arginine), an nNOS inhibitor, on behavioral and
NMDA receptor
antinociceptive responses induced by local injection of NMDA receptor agonist in mice. The
Defensive reactions
behaviors measured were frequency of jumping and rearing as well as duration (in seconds)
Antinociception
of running and freezing. Nociception was assessed during the second phase of the formalin
Nitric oxide
test (injection of 50 μl of formalin 2.5% into the dorsal surface of the right hind paw). Five to
nNOS
seven days after stereotaxic surgery for intracerebral cannula implantation, mice were
Periaqueductal gray
injected with formalin into the paw, and 10 min later, they received intra-dPAG injection of NPLA (0, 0.2, or 0.4 nmol/0.1 μl). Ten minutes later, they were injected with NMDA (N-methyl-
Mice
D-aspartate:
0 or 0.04 nmol/0.1 μl) into the same midbrain site and were immediately placed
in glass holding cage for recording the defensive behavior and the time spent on licking the injected paw with formalin during a period of 10 min. Microinjections of NMDA significantly decreased nociception response and produced jumping, running, and freezing reactions. Intra-dPAG injections of NPLA (0.4 nmol) completely blocked the NMDA effects without affecting either behavioral or nociceptive responses in intra-dPAG saline-injected animals, except for the rearing frequency that was increased by the nNOS inhibitor. These results strongly suggest the involvement of NO within the PAG in the antinociceptive and defensive reactions induced by local glutamate NMDA receptor activation in this midbrain structure. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
The midbrain periaqueductal gray (PAG) has been identified as a key component of the pain inhibitory system since electrical
or chemical stimulation of this area suppresses nociceptive transmission from the dorsal horn of the spinal cord (for review, see Fields and Basbaum, 1999). In addition, many lines of evidence point to dorsal region of the PAG (dPAG) as the
⁎ Corresponding author. Lab. Farmacologia, Faculdade de Ciências Farmacêuticas–UNESP, Rod. Araraquara-Jau, km 01, 14801-902, Araraquara, SP, Brazil. E-mail address: [email protected] (R.L. Nunes-de-Souza). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.12.095
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principal substrate of aversion in the brain (for review, see Graeff, 2004). For instance, chemical stimulation of dPAG elicits autonomic and behavioral responses similar to defensive reactions to innate aversive stimuli (Bandler and Carrive, 1988; Bittencourt et al., 2004; Blanchard and Blanchard, 1988, 1989). Excitatory amino acids (e.g., glutamate) receptor agonists are probably the main chemical tool used to stimulate PAG and other brain sites (Bandler, 1988; Bandler and Carrive, 1988; Bittencourt et al., 2004; Friebe and Koesling, 2003). The action of glutamate is mediated at metabotropic receptors and three subtypes of ionotropic receptors, AMPA (amino-3-hydroxy-5methyl-4-isoxazole propionic acid), kainate, and NMDA (Nmethyl-D-aspartate) (Heresco-Levy, 2003; Huntley et al., 1994; Seebury, 1993). Glutamate NMDA receptor activation leads to cellular calcium influx which triggers a cascade of intracellular events including activation of nitric oxide synthase (NOS), enzyme that produces nitric oxide (NO) by conversion of Larginine to L-citroline, having NADPH (nicotinamide adenine dinucleotide phosphate) and Ca2+ as co-factors (Garthwaite et al., 1989; Lohse et al., 1998; Mayer et al., 1991). There are at least three known isoforms of NOS: an inducible (iNOS) and two constitutive forms, which are present under physiological conditions in endothelium (but not only in this tissue, eNOS) and in neurons (nNOS) (Guix et al., 2005; Lamas et al., 1992; Mungrue et al., 2003; Prast and Philippu, 2001). Intracellular NO activates guanylate cyclase (GC) with the subsequent production of cyclic guanosine-3′,5′-monophosphate (cGMP) and protein phosphorylation (Friebe and Koesling, 2003; Ignarro,
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1991; Krumenacker et al., 2004). NO also exerts other cellular effects independent of the GC activation (for review, see Guix et al., 2005; Prast and Philippu, 2001). It presents ubiquitous localization that permits its involvement in a wide range of physiological and pathophysiological processes. For instance, several studies have shown that NO is involved in peripheral and central nociceptive processing (for review, see Riedel and Neeck, 2001) and in defensive reactions (Chiavegatto et al., 1998; De Oliveira et al., 2001; Guimarães et al., 2005). Besides producing neurovegetative activation (e.g., tachycardia, hypertension, defecation) and flight reactions (e.g., jumping, running, freezing) (e.g., Bandler and Carrive, 1988; Bittencourt et al., 2004), intra-dPAG infusions of NMDA receptor agonists also provoke high magnitude antinociception (e.g., Berrino et al., 2001; Jacquet, 1988; Siegfried and Nunes-de-Souza, 1989). This study investigated the effects of nNOS inhibition within this midbrain structure is able to antagonize both behavioral and antinociceptive responses induced by local injection of NMDA receptor agonist in mice.
2.
Results
Histological analysis demonstrated that 70 mice had accurate cannula placements in the PAG (Fig. 1). Final sample sizes ranged from n = 9–15 animals per group. Fig. 2 shows the effects of intra-PAG injection of NMDA on time spent licking the formalin 2.5% injected paw during 10 min (25–35 min after formalin injection, second phase of
Fig. 1 – (A) Schematic representation of microinfusion sites within the midbrain periaqueductal gray (PAG) of the mouse. The number of the points in the figure is less than the total number of mice because of the overlaps. (B) Photomicrograph of midbrain coronal section from a representative subject showing an injection site into the PAG. Section correspond to −4.24 mm from bregma in the atlas of Paxinos and Franklin (2001).
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Fig. 2 – Effects of intra-PAG injection of NMDA (0.04 nmol) on time spent licking the paw injected with formalin 2.5% during 10 min (second phase of nociception test) in mice pretreated with saline or NPLA (0.2–0.4 nmol) into the same midbrain site (n = 10–15) (see text for details). *P b 0.05 vs. Saline + Saline; #P b 0.05 vs. Saline + NMDA. nociception test) in mice pretreated with saline or NPLA into the same midbrain site. Kruskal–Wallis test revealed significant differences among groups (H = 32.96, P b 0.05). Post hoc comparisons showed that animals treated with Saline + NMDA spent less time licking the injected paw when compared with Saline + Saline group (control). Prior injection of NPLA (0.4 nmol) reversed the NMDA effect without producing any significant differences when compared to Saline + Saline group. Fig. 3 shows the effects of intra-PAG injection of NMDA on frequency of jumps in mice pretreated with saline or NPLA into the same midbrain site. Kruskal–Wallis test revealed significant differences among groups (H = 23.74, P b 0.05) and post hoc comparisons indicated that Saline + NMDA group jumped significantly more than the control group (Saline +
Fig. 3 – Effects of intra-PAG injection of NMDA (0.04 nmol) on jumping frequency during 5 min in mice pretreated with saline or NPLA (0.2–0.4 nmol) into the same midbrain site (n = 9–14) (see text for details). *P b 0.05 vs. Saline + Saline; #P b 0.05 vs. Saline + NMDA.
Saline). Prior intra-PAG treatment with NPLA (0.4 nmol) antagonized the NMDA effects on this behavioral response. No significant effects were obtained with NPLA on frequency of jumps when compared to the control group. The effect of intra-PAG NMDA infusion on time spent running in mice submitted to prior injections of NPLA in the same structure is shown in Fig. 4. Non-parametric statistics revealed significant differences among groups (H = 24.14; P b 0.05) and Dunn's test indicated that Saline + NMDA group spent more time running when compared to the control group (Saline + Saline). This NMDA effect was completely blocked when NPLA (0.4 nmol) was previously injected into the PAG. On the other hand, intra-PAG NPLA did not affect running per se, since no significant differences were observed comparing the two groups treated with the nNOS inhibitor [NPLA (0.2 nmol) + Saline and NPLA (0.4 nmol) + Saline] to the control group. Fig. 5 illustrates the effects of intra-PAG infusion of NMDA on time spent in freezing in mice submitted to a prior injection of NPLA into the same structure. One-way ANOVA revealed significant differences among groups (F5,57 = 6.56, P b 0.05). Post hoc Duncan's tests showed that animals treated with Saline + NMDA or NPLA (0.2 nmol) + NMDA spent more time in immobility than control group (Saline + Saline), while NPLA (0.4 nmol) + NMDA group selectively antagonized the NMDA effect. Fig. 6 shows that intra-PAG infusion of NMDA failed to alter rearing frequency. However, intra-PAG infusion of NPLA (0.4 nmol) increased frequency of rearing in both saline or NMDA treated groups (ANOVA, followed by Duncan's test: F5,57 = 8.21, P b 0.05).
3.
Discussion
The present results are in consonance with previous studies demonstrating that NMDA microinjection into the mouse PAG induces antinociception and elicits defensive behavioral reactions characterized by jumping and running followed by
Fig. 4 – Effects of intra-PAG injection of NMDA (0.04 nmol) on running time (sec) during 5 min in mice pretreated with saline or NPLA (0.2–0.4 nmol) into the same midbrain site (n = 9–14) (see text for details). *P b 0.05 vs. Saline + Saline; #P b 0.05 vs. Saline + NMDA.
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Fig. 5 – Effects of intra-PAG injection of NMDA (0.04 nmol) on freezing time (seconds) during 5 min in mice pretreated with saline or NPLA (0.2–0.4 nmol) into the same midbrain site (n = 9–14) (see text for details). *P b 0.05 vs. Saline + Saline; #P b 0.05 vs. Saline + NMDA.
a period of freezing (Bandler, 1988; Bandler and Carrive, 1988; Berrino et al., 2001; Bittencourt et al., 2004; Ferreira-Netto et al., 2005; Jacquet, 1988; Siegfried and Nunes-de-Souza, 1989). These behaviors are proposed to reproduce aversive responses since they are similar to reactions induced by natural aversive stimuli such as the exposure to a predator (Blanchard and Blanchard, 1988, 1989). Our study also demonstrated that these responses depend on NO synthesis, since intra-PAG injection of NPLA, an nNOS inhibitor, completely antagonized both NMDA-induced nociception inhibition and defensive-like behaviors. Previous studies have demonstrated that intra-PAG injections of NMDA decrease nociceptive response assessed by different nociception tests (Berrino et al., 2001; Jacquet, 1988; Siegfried and Nunes-de-Souza, 1989). Our results corroborate these findings and indicate a fundamental importance of nitric oxide on NMDA effects, since prior nNOS inhibition with NPLA injections into the same structure was able to reverse the antinociception induced by this excitatory amino acid. Interestingly, NPLA injections did not alter per se the nociceptive response profile. Animals treated with NPLA (both doses) + Saline spent similar time licking the paw injected with formalin when compared to Saline + Saline group, suggesting that the nNOS inhibitor fails to provoke intrinsic effects on nociception. It has been demonstrated that PAG stimulation produced by local injection of glutamate is related to activation of the descending inhibitory system, which inhibits nociceptive neurons in dorsal horn. This system is composed of a descending neuron bundle that involves neurons from PAG and rostroventral medulla nuclei (e.g., nucleus raphe magnus), which project to dorsal horn making inhibitory connections in ascending nociceptive transmission (Fields and Basbaum, 1999; Jones and Gebhart, 1988; Kharkevich and Churukanov, 1999; Millan, 2002). Importantly, our study suggests that the antinociceptive mechanism mediated by glutamate NMDA receptors within the PAG
45
involves, at least in part, synthesis of nitric oxide in this midbrain structure. Interestingly, intra-PAG infusions of NPLA also changed the behavioral reactions provoked by intra-PAG injections of NMDA. The excitatory amino acid provoked a sudden sequence of jumping and running behaviors that were followed by a period of freezing. These results corroborate many previous studies (Bandler, 1988; Bandler and Carrive, 1988; Beckett et al., 1992; De Oliveira et al., 2001; Molchanov and Guimarães, 1999), which have demonstrated that intra-PAG injections of glutamate receptor agonists produce behavioral responses characteristics of rodent's defensive reactions. In the present study, NPLA antagonized these NMDA-induced behavioral effects, suggesting that NO release within the PAG plays a role on defensive behavior. While intra-PAG infusion of NMDA did not alter rearing frequency, NPLA (higher dose) increased this vertical exploratory behavior, an effect that was independent of the treatment combination received (i.e., saline or NMDA). The increase in the frequency of rearing suggests that this nNOS inhibitor, at doses employed here, did not provoke motor disruption, an effect previously reported with systemic injections with other NOS inhibitors (De Oliveira et al., 1997; Del Bel et al., 2002, 2004). In view of these findings, intra-PAG NPLA injections seem to selectively reduce NMDA-induced behavioral responses (e.g., jumping, running, freezing) in mice. Taken together, these results suggest NO as a strong candidate to modulate anxiety states. Support for this hypothesis is found in studies with intra-PAG injections of glutamate receptor agonists and antagonists as well as with NOS inhibitors. For instance, while NMDA receptor agonists produce flight reactions as demonstrated here and in previous studies (Bandler, 1988; Bandler and Carrive, 1988; Del Bel et al., 1998; Ferreira-Netto et al., 2005), intra-PAG injections of NMDA and non-NMDA receptor antagonists provoke anxiolytic-like effects in animals exposed to elevated plus-maze (EPM) (Guimarães et al., 1991; Hall and Behbehani, 1998; Matheus
Fig. 6 – Effects of intra-PAG injection of NMDA (0.04 nmol) on rearing frequency during 5 min in mice pretreated with saline or NPLA (0.2–0.4 nmol) into the same midbrain site (n = 9–14) (see text for details). *P b 0.05 vs. Saline + Saline and Saline + NMDA.
46
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and Guimarães, 1997; Molchanov and Guimaraes, 2002), a widely used animal model of anxiety (Handley and Mithani, 1984; Lister, 1987; Pelow et al., 1985; Rodgers et al., 1997). Regarding the NO role on anxiety, both systemic injection of LNAME, an unspecific NOS inhibitor, as well as intra-PAG infusions of L-NAME and L-NOARG attenuated anxiety indices in the EPM in rats (De Oliveira et al., 1997, 2001; Faria et al., 1997; Guimarães et al., 1994). More recently, Guimarães et al. (2005), using a prey–predator model, have demonstrated that local infusion into dorsolateral PAG (dlPAG) of AP-7, a glutamate NMDA receptor antagonist, completely prevented the flight reactions induced by intra-dlPAG administration of SIN-1, a NO donor, suggesting a modulatory role for NO within the PAG, probably by interacting with glutamate-mediated neurotransmission. In this context, the present results confirm a strong interaction between NMDA receptor activation and NO synthesis within this midbrain structure on defensive reactions in mice. In conclusion, this study demonstrated that intra-PAG infusions of NPLA selectively antagonized antinociception and defensive behaviors induced by local NMDA infusions in the same midbrain structure, suggesting that NO plays an important role within the PAG on pain inhibition and behavioral responses induced by threatening situations. It has been emphasized that flight reactions provoked by NOS neurons activation within the PAG are probably related to NO modulatory effects on other neurotransmitter release (e.g., glutamate, gamma-aminobutyric acid-GABA and serotonin (Guimarães et al., 2005)). It remains unclear whether NO effects on nociception reported in the present study involve the activation of other neurotransmitter systems.
respectively, 4.1 mm posterior to bregma, 1.3 mm lateral to the midline, and 2.2 mm ventral to the skull surface, with the guide cannula angled 26° to the vertical. A dummy cannula (33-gauge stainless steel wire; Fishtex Industry and Commerce of plastics Ltda.), inserted into each guide-cannula immediately after of surgery, served to reduce the incidence of occlusion. Five to seven days after surgical recovery, solutions were injected into the PAG by microinjection units (33-gauge stainless steel cannula; Insight Equipamentos Científicos Ltda), which extended 1.0 mm beyond the tips of the guide cannula. Each microinjection unit was attached to a 5-μl Hamilton microsyringe via polyethylene tubing (PE-10), and administration was controlled by an infusion pump (BI 2000, Insight Equipamentos Científicos Ltda) programmed to deliver a volume at rate of 0.1 μl (volume injected) over a period of 30 s. The microinjection procedure consisted of gently restraining the animal, removing of dummy cannula, inserting the injection unit, infusing the solution, keeping the injection unit in situ for a further 60 s. Confirmation of successful infusion was obtained by monitoring the movement of a small air bubble in the PE-10 tubing. 4.4.
General procedure
4.
Experimental procedures
On test days, animals were transported to the experimental room and left undisturbed for at least 30 min prior to testing. They were then injected with 50 μl of formalin (2.5%) into the dorsal surface of the right hind paw and individually placed in a glass holding cage (30 × 20 × 25 cm). Fifteen minutes after formalin injection, animals received intra-PAG injection of saline or NPLA (0.2 or 0.4 nmol/0.1 μl) and were placed back in the glass holding cage. Ten minutes later, they received a second intra-PAG injection either with saline or NMDA (0.04 nmol/0.1 μl). Thus, six groups were formed: Saline + Saline, Saline + NMDA, NPLA (0.2 nmol) + Saline, NPLA (0.4 nmol) + Saline, NPLA (0.2 nmol) + NMDA, NPLA (0.4 nmol) + NMDA. Formalin test (Abbott et al., 1995; Dubuisson and Dennis, 1977) was recorded immediately after the second intra-PAG injection procedure placing the animals individually in the glass cage and recording the time spent licking the injected paw for 10 min (25–35 min after formalin injection).
4.1.
Subjects
4.5.
Subjects were male Swiss adults mice weighing 25–35 g (Paulista State University/UNESP, SP, Brazil), housed in groups of 10 per cage (cage size: 41 × 34 × 16 cm). They were maintained under a normal 12-h light cycle (lights on: 7:00 a.m.) in a temperature (23 ± 1 °C)and humidity (55 ± 5%)-controlled environment. Food and drinking water were freely available except during the brief test periods. All mice were experimentally naïve and used only once. 4.2.
Drugs
The drugs used were Nù-propyl-L-arginine (NPLA–Tocris Cookson Inc., Ballwin, USA), a highly selective and potent inhibitor of nNOS (Ki = 57 nM), which displays 3158-fold and 149-fold selectivity over iNOS and eNOS, respectively (Zhang et al., 1997), and NMDA (Nmethyl-D-aspartic acid–RBI, USA). The doses used were based on previous studies: NMDA 0.04 nmol/0.1 μl (Siegfried and Nunes-deSouza, 1989) and NPLA, 0.2 nmol/0.1 μl–0.4 nmol/0.1 μl (El-Haddad et al., 2002; Kakoki et al., 2001; Zhang et al., 1997). The drugs were dissolved in physiological saline (NaCl 0.9%). 4.3.
Behavioral analysis
Videotapes were scored blind by a highly trained observer using an ethological analysis packing developed by Dr. Morato's group at Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, USP. In addition to recording the time spent licking the formalin-injected paw (see above), the observer scored the frequency of jumps (i.e., upward leaps directed to the border of the glass cage) and rearing (i.e., standing on hind limbs, with both forelimbs off the floor, including unsupported rearing and rearing against the wall), the time (in seconds) spent running (i.e., time of trotting [running keeping the same pattern of walking] and galloping [fast running alternating anterior and posterior limb pairs]), and in freezing (complete absence of movement except breathing, while the animal a characteristic tense posture). The behavioral observation was performed during 5 min following intra-PAG saline or NMDA injection procedure (see above). All experiments were carried out during the light phase of the light–dark cycle. The illumination intensity in the room where the experiments were carried out was 1 × 100 W at the room with illumination (1 × 100 W). All sessions were videorecorded by a camera linked to a monitor and VCR in the adjacent laboratory.
Surgery and microinjection 4.6.
Mice were implanted with 7-mm stainless steel guide cannula (26gauge; Insight Equipamentos Científicos Ltda) under sodium thiopental (100 mg/kg, i.p.) anesthesia. Guide cannula was fixed to the skull using dental acrylic and jeweler's screws. Stereotaxic coordinates (Paxinos and Franklin, 2001) for the PAG were,
Statistics
All results were initially submitted to Levene's test for homogeneity of variance. When appropriate, the data were transformed to square root, cube root or log before being submitted to one-way analyses of variance (ANOVA). Where indicated by significant F
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values, Duncan's multiple comparisons tests were used. Where Levene's test remained significant even after data transformation non-parametric tests (Kruskal–Wallis test followed by Dunn's multiple comparison test) were used. In all cases, a P value ≤ 0.05 was required for significance. 4.7.
Histology
At the end of testing, all animals were deeply anaesthetized with sodium thiopental and received a microinfusion of 1% Evans blue (according to the microinjection procedure described above). Brains were removed and injection sites verified histologically according to the atlas of Paxinos and Franklin (2001). Data from animals with injection sites outside the PAG were excluded from the study. 4.8.
Ethics
The experiments carried out in this study comply with the norms of Brazilian Neuroscience and Behavior Society (SBNeC), based on the US National Institutes of Health Guide for Care and Use of Laboratory Animals.
Acknowledgments This study was supported by FAPESP (Proc.02/03735-0), CNPq, and PADC/FCF-Unesp. T.T.Miguel was a recipient of FAPESP (Proc. 03/11571-7), and R.L. Nunes-de-Souza received a CNPq research fellowship.
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