Anxiogenic-like effect induced by TRPV1 receptor activation within the dorsal periaqueductal gray matter in mice

Behavioural Brain Research 250 (2013) 308–315

Contents lists available at SciVerse ScienceDirect

Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr

Research report

Anxiogenic-like effect induced by TRPV1 receptor activation within the dorsal periaqueductal gray matter in mice Diego Cardozo Mascarenhas a,b , Karina Santos Gomes b , Ricardo Luiz Nunes-de-Souza a,b,∗ a b

Joint Graduate Program in Physiological Sciences, UFSCar/UNESP, São Carlos, SP, 13565-905, Brazil School of Pharmaceutical Sciences, Universidade Estadual Paulista–UNESP, 14801-902, Araraquara, SP, Brazil

h i g h l i g h t s • • • •

Activation of TRPV1 within the periaqueductal gray (dPAG) leads to anxiogenesis. Blockade of the dPAG TRPV1 does not change anxiety-like behavior in mice. dPAG TRPV1 blockade reverses the anxiogenesis induced by vanilloid agonist. TRPV1 located within the dPAG play a role in the modulation of defensive emotions.

a r t i c l e

i n f o

Article history: Received 15 March 2013 Received in revised form 3 May 2013 Accepted 14 May 2013 Available online 21 May 2013 Keywords: Capsaicin Capsazepine Periaqueductal gray matter Anxiety Elevated plus-maze Mice

a b s t r a c t Pharmacological manipulation of TRPV1 (Transient Receptor Potential Vanilloid type-1) receptors has been emerging as a novel target in the investigation of anxiety states. Here, we attempt to show the role played by the TRPV1 receptors within the dorsal periaqueductal gray matter (dPAG), a midbrain structure strongly involved in the modulation of anxiety. Anxiety was assessed by recording spatiotemporal [percent open arm entries (%OE) and percent open arm time (%OT)] and ethological [e.g., head dipping (HD), stretched-attend postures (SAP)] measures in mice exposed to the elevated plus-maze (EPM). Mice received an intra-dPAG injection of the TRPV1 agonist capsaicin (0, 0.01, 0.1 or 1.0 nmol/0.2 ␮L; Experiment 1) or antagonist capsazepine (0, 10, 30 or 60 nmol/0.2 ␮L; Experiment 2), or combined injections of capsazepine (30 nmol) and capsaicin (1.0 nmol) (Experiment 3), and were exposed to the EPM to record spatiotemporal and ethological measures. While capsaicin produced an anxiogenic-like effect (it reduced %OE and %OT and frequency of SAP and HD in the open arms), capsazepine did not change any behavior in the EPM. However, when injected before capsaicin (1.0 nmol), intra-dPAG capsazepine (30 nmol–a dose devoid of intrinsic effects) antagonized completely the anxiogenic-like effect of the TRPV1 agonist. These results suggest that the anxiogenic-like effect produced by capsaicin is primarily due to TRPV1 activation within the dPAG in mice, but that dPAG TRPV1 receptors do not exert a tonic control over defensive behavior in mice exposed to the EPM. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The transient receptors potential cation channel, subfamily V (also called vanilloid receptor; TRPV), are nonselective cation channels characterized mainly by calcium influx when activated [1]. Their activation may be due to temperature stimuli, low pH, or exogenous ligands such as capsaicin, the chili pepper pungent ingredient [2,3]. These receptors, especially TRPV member 1 (TRPV1), have commonly been implicated in pain transmission and inflammatory processes in peripheral regions of the nervous system [4,5]. Although several studies have been carried out on the role

∗ Corresponding author. Tel.: +55 16 33016985; fax: +55 16 33016980. E-mail address: [email protected] (R.L. Nunes-de-Souza). 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.05.023

played by vanilloid compounds in sensory neurons, there is mounting evidence pointing their relevance to the central nervous system (CNS) and, more recently, to emotions such as fear and anxiety [3,6,7]. In addition to their role in the CNS, endocannabinoids such as anandamide (AEA) and N-arachidonoyldopamine can also activate the TRPV1 receptors. Consequently, these endocannabinoids have been considered as endovanilloids [3,8]. The TRPV1 receptors are located in several brain regions related to anxiety and pain, including the periaqueductal gray matter (PAG) [9,10], where they can modulate glutamate release [11,12]. Together with the forebrain areas such as the amygdala and dorso-medial hypothalamus, the dorsal portion of the PAG (dPAG: dorso-medial and dorso-lateral columns) belongs to the brain defensive system [13–15]. Electrical or chemical stimulation of the dPAG elicits several defensive responses, such as flight, escape and

D.C. Mascarenhas et al. / Behavioural Brain Research 250 (2013) 308–315

immobility [6,16–18], suggesting that this midbrain area is closely related to the modulation of anxiety and fear states. As far as we know, only a few studies have investigated the role played by PAG TRPV1 in the modulation of fear and anxiety in rodents, especially in mice. Systemic administration of capsazepine, a TRPV1 antagonist, produced anxiolytic-like effects in rats exposed to the elevated plus maze (EPM) [19], a widely used animal test of anxiety [20–24]. More recently, studies have demonstrated that animals with genetic modifications in their vanilloid receptors, as in TRPV1 knockout mice, showed anxiolyticlike behavior in the EPM and light-dark test [25]. However, Rodgers et al. [26] have demonstrated a lack of effect when the antagonist capsazepine was systemically administrated in mice exposed to the EPM. It has been shown that cannabinoid (CB1) and vanilloid (TRPV1) receptors play opposite roles with respect to anxiety, since CB1 are inhibitory metabotropic receptors and TRPV1 are excitatory ion channels [6,27]. Corroborating this evidence are results showing that systemic injection of a dual blocker of both FAAH (fatty acid amide hydrolase - enzyme responsible for degrading the endocannabinoid anandamide) and TRPV1 channels, N-arachidonoyl-serotonin, revealed a more potent anxiolytic-like effect, mediated by CB1 receptors, than FAAH and TRPV1 blockers alone [27]. Terzian et al. [6] observed that capsazepine microinjection into the dPAG in rats led to anxiolysis, and curiously, the agonist capsaicin mimicked this effect, attenuating anxiolytic-like behavior. Those authors suggested that while stimulation of CB1 receptors within the dPAG seems to inhibit aversive response, TRPV1 activation could facilitate them. If so, CB1 and TRPV1 located within the dPAG could have opposite roles in the modulation of anxiety responses [6]. An intriguing fact regarding the role of cannabinoid compounds is that they usually display bell-shape response curves. AEA and cannabidiol (CBD), respectively an endocannabinoid and a phytocannabinoid, can also bind to TRPV1 receptors [3,7,8]. This helps to explain the bell-shape curves generally found with these compounds regarding their anxiolytic-like effects. Moreover, capsazepine produced antiaversive-like effects in both EPM and Vogel tests when administered within the dPAG of rats [6]. On the basis of previous findings showing that capsaicin is able to quickly desensitize the TRPV1 channel [2,28,29], Terzian et al. [6] suggested that the apparently discrepant results obtained with capsazepine and capsaicin might be due to this ability of capsaicin. Given the paucity of studies regarding the role played by vanilloid receptors in anxiety, the principal aim of this study was to investigate whether and in what way microinjections of both a TRPV1 agonist and an antagonist (capsaicin and capsazepine, respectively) into the dPAG modulate anxiety-like behavior in mice exposed to the EPM. 2. Materials and methods 2.1. Animals Subjects were adult male Swiss adult mice (São Paulo State University/UNESP, SP, Brazil), weighing 28–35 g at testing. They were housed in groups of 10 per cage (41 cm × 34 cm × 16 cm) and maintained under a 12:12-h light/dark cycle (lights on 07:00 h) in a temperature/humidity-controlled environment (23 ± 1 ◦ C/55 ± 5%). Food and water were freely available except during the brief test periods. All mice were naïve at the beginning of the experiments. 2.2. Drugs The drugs used were capsaicin (Tocris Cookson, Ballwin, MO, USA), a TRPV1 agonist, and capsazepine (Tocris Cookson, Ballwin,MO, USA), a TRPV1 antagonist. The compounds were dissolved in undiluted dimethylsulfoxide (DMSO), which alone served as vehicle. The doses of capsaicin (0, 0.01, 0.1 and 1 nmol) and capsazepine (0, 10, 30 and 60 nmol) were based on previous studies [6]. The total volume injected into the dPAG was 0.2 ␮L.

309

2.3. Surgery and microinjection Mice were unilaterally implanted with a 7-mm stainless steel guide cannula (26-gauge; Insight Equipamentos Científicos Ltda, Brazil) under ketamine + xylazine anesthesia (100 mg/kg and 10 mg/kg, i.p.) in a stereotaxic frame (Kopf Instruments). The guide cannula was fixed to the skull with dental acrylic and jeweler’s screws. Stereotaxic coordinates [30] for the dorsal portion of the PAG (dPAG: dorsolateral and dorsomedial columns) were 4.1 mm posterior to the bregma, 1.4 mm lateral to the midline and 2.3 mm ventral to the skull surface. During implantation, the tip of the guide cannula was angled ± 26◦ to the vertical and positioned 1 mm above the target site. A 7-mm dummy cannula (33-gauge stainless steel wire; Fishtex Industry and Commerce of plastics Ltda.), inserted into each guide cannula immediately after surgery, served to reduce the incidence of occlusion. Postoperative analgesia was provided for 2 days by adding acetaminophen (200 mg/mL) to the drinking water at 0.2 mL in 250 mL (final concentration = 0.16 mg/mL) [31]. Five to seven days after recovery from surgery, solutions were injected into the dPAG through 8-mm microinjection units (33-gauge stainless steel cannula; Insight Equipamentos Científicos Ltda), which extended 1.0 mm beyond the tip of the guide cannula. Each microinjection unit was attached to a 5-␮L Hamilton microsyringe via polyethylene tubing (PE-10), and the flow was controlled by an infusion pump (BI 2000, Insight Equipamentos Científicos Ltda) programmed to deliver a volume of 0.2 ␮L over a period of 45 s. The microinjection procedure consisted of gently restraining the animal, removing the dummy cannula, inserting the injection unit, infusing the solution and then keeping the injection unit in situ for a further 60 s. Confirmation of successful infusion was provided visually by the movement of a small air bubble in the PE-10 tubing. 2.4. Apparatus and general procedure The basic EPM design was very similar to that originally described by Lister (1987) [32] and comprised two open arms (30 cm × 5 cm × 0.25 cm) and two closed arms (30 cm × 5 cm × 15 cm) connected via a common central platform (5 cm × 5 cm). The apparatus was constructed from wood (floor) and transparent glass (clear walls) and was raised to a height of 38.5 cm above floor level. All testing were conducted under dim illumination (one 60 W white light providing 55 lux at the central platform of the EPM) during the light phase of the light-dark cycle. Mice were transported to the experimental room 5–6 days after surgery and left undisturbed for at least 60 min prior to testing. Immediately following microinfusion of capsaicin, capsazepine or vehicle, which obeyed a counterbalanced order for treatment condition, into the dPAG, each mouse was individually placed in a holding cage for 10 min and then transported to the EPM. Testing commenced by placing the subject on the central platform of the maze (facing an open arm), the experimenter withdrawing immediately to an adjacent laboratory. Test sessions lasted 5 min and, between subjects, the maze was thoroughly cleaned with 20% ethanol and dry cloths. All sessions were video-recorded by a camera linked to a monitor and DVD recorder in an adjacent laboratory for further analysis. 2.5. Behavioral analysis Behavioral analysis was performed as described elsewhere [23]. Videotapes were scored blind to treatment by a highly trained observer (intrarater reliability ≥ 0.90) using an ethological analysis software package developed by Dr. Morato’s group at Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, USP, Brazil (unpublished). Behavioral parameters comprised spatiotemporal and ethological measures [33]. Conventional spatiotemporal measures were the frequencies of closed arm entries (arm entry = all four paws on an arm), percentage of open arm entries ((open/total) × 100) and percentage of time spent on the open arms of the maze ((time open/300) × 100). Ethological measures are reported as frequency scores for head dipping (HD = exploratory movement of head/shoulders over the side of the maze) and stretched-attend postures (SAP = exploratory posture in which the body is stretched forward then retracted to the original position without any forward locomotion). In view of the importance of the thigmotactic cues for patterns of plusmaze exploration, HD and SAP were further differentiated by recording where on the maze they were displayed. Consistent with earlier reports [33], the closed arms and the central platform were together designated “protected” areas (i.e., offering relative security), while the open arms were designated “unprotected” areas. Data for the HD and SAP measures are thus reported as separate protected (pHD, pSAP) and unprotected (uHD, uSAP) scores [33]. 2.6. Experiments Experiment 1: Effects of intra-dPAG microinjection of capsaicin on the behavior of mice exposed to the EPM On test days, mice were microinjected with vehicle or capsaicin (0.01, 0.1 and 1 nmol/0.2 ␮L) and left in the holding cage for 10 min, after which they were individually placed in the center of the EPM to record the behavioral (spatiotemporal and ethological) measures (see Section 2.5) for a period of 5 min. Experiment 2: Effects of intra-dPAG microinjection of capsazepine on the behavior of mice exposed to the EPM.

310

D.C. Mascarenhas et al. / Behavioural Brain Research 250 (2013) 308–315

Fig. 1. A: A sketch of microinfusion sites within the dorsal periaqueductal gray (dPAG) of the mouse. Gray area is corresponding to whole area where different microinjections were placed at different slices (distance from bregma in mm) described in the Paxinos and Franklin [30] Atlas. B: Photomicrograph of a midbrain coronal section from a representative subject showing an injection site into the dPAG. The section was 4.1 mm from the bregma.

Similarly to Exp. 1, mice received an intra-dPAG microinjection of vehicle or capsazepine (10, 30 and 60 nmol/0.2 ␮L) and were left to the holding cage for 10 min before being exposed to the EPM to record the same behavioral parameters as in item 2.5. Experiment 3: Effects of combined microinjections of capsazepine (pretreatment; 30 nmol) and capsaicin (treatment; 1 nmol) into the dPAG on the behavior of mice exposed to the EPM. The third experiment can be outlined as follows. Animals were divided into 4 experimental groups, all of which were subjected to 2 microinjections, the first injection (capsazepine or vehicle) being performed 10 min before the second (capsaicin or vehicle). Immediately after the second injection, the animals were placed in a holding cage for 10 min and then exposed to the EPM. The experimental groups were thus: vehicle-vehicle (veh-veh), capsazepine-vehicle (cpz-veh), vehicle-capsaicin (veh-cpsa) and capsazepine-capsaicin (cpz-cpsa). The doses of capsaicin 1.0 nmol and capsazepine 30 nmol were based on the anxiogenic-like effect and lack of effect on anxiety, respectively, observed in Experiments 1 and 2. Moreover, although intradPAG capsazepine 60 nmol was also incapable to change the behavioral measures of mice exposed to the EPM (see results of Exp. 2 below), we avoided using it in the present experiment, since previous findings have shown intrinsic antiaversive effects produced by intra-dPAG capasazepine 60 nmol in rats exposed to the EPM and Vogel tests [6].

2.7. Histology At the end of testing, all animals received an intra-dPAG 0.1 ␮L infusion of 1% Evans blue, by the microinjection procedure described in item 2.3. The animals were then sacrificed in a CO2 chamber, their brains removed and injection sites histologically verified in coronal sections cut with a cryostat microtome (Leica CM 1850) and a microscope (Leica DMLB), by referring to the Paxinos and Franklin atlas [31]. In Experiment 1, mice receiving injections of the active capsaicin doses (0.1 and 1.0 nmol) outside the dPAG were excluded from the original groups and combined in an extra group (OUT) for analysis. These animals included 4 mice for capsaicin 0.1 nmol and 3 mice for capsaicin 1.0 nmol. Animals with injection sites outside the dPAG (Exps. 2 and 3) were excluded from the experimental groups.

2.8. Statistics All results were initially subjected to Levene’s test for homogeneity of variance. Where this test yielded significant heterogeneity, results were transformed to logs, square roots or cube roots and tested again for homogeneity of variance before being subjected to one-way ANOVA (Exps. 1 & 2) or two-way ANOVA (Exp. 3 – factor 1: pretreatment; factor 2: treatment), followed by a post hoc Duncan test. In all cases, p ≤ 0.05 was required for significance.

2.9. Ethics The experimental protocols were conducted in compliance with the ethical principles of the Brazilian College of Animal Experimentation (COBEA) and approved by the local Research Ethics Committee (CEP/FCF/Car, Universidade Estadual Paulista, resolution 14/2011).

3. Results 3.1. Histology Fig. 1 shows (A) a sketched series of coronal brain sections based on the Paxinos and Franklin Atlas [30], indicating microinfusion sites within the midbrain dPAG, and (B) a photomicrograph of a midbrain coronal section of a representative subject, showing an injection site within the dPAG of the mouse. Histology confirmed that a total of 89 mice had accurate cannula placements in the dPAG. A total of thirty three animals were used to investigate the effects of intra-dPAG capsaicin microinjection [Experiment 1: vehicle (n = 8); 0.01 nmol cpsa (n = 8); 0.1 nmol cpsa (n = 9); 1.0 nmol cpsa (n = 8)]. Twenty-seven animals were used to assess the effects of capsazepine microinjections into the dPAG [Experiment 2: vehicle (n = 7); 10 nmol cpz (n = 7); 30 nmol cpz (n = 7); 60 nmol cpz (n = 6)]. Finally, twenty nine animals were used in Experiment 3 [vehiclevehicle (n = 9); 30 nmol cpz-vehicle (n = 6); vehicle-1.0 nmol cpsa (n = 7); 30 nmol cpz-1.0 nmol cpsa (n = 7)]. 3.2. Experiment 1: Effects of capsaicin microinjection into the dPAG on the behavior of mice exposed to the EPM Fig. 2 represents a dose-response curve of the effects of intradPAG capsaicin (0, 0.01, 0.1 or 1.0 nmol) on the anxiety indices [% open-arm entries (% OE) and % open arm time (% OT)] and locomotor activity (closed arm entries, CE) of mice exposed to the EPM. One-way ANOVA revealed significant effects for both %OE (F4,35 = 4.81; p < 0.05) and %OT (F4,35 = 6.01; p < 0.05). Post hoc analysis confirmed a decrease in open-arm exploration (%OE and %OT) in the groups treated with capsaicin 0.1 and 1.0 nmol,

D.C. Mascarenhas et al. / Behavioural Brain Research 250 (2013) 308–315

12

Table 1 Lack of effects (mean ± SEM) of capsazepine (10; 30 and 60 nmol/0.2 ␮L) microinfusions into the dPAG on the behavior of mice exposed to the EPM. n = 6–7.

Closed arm entries

10

Behavior

10 Protected SAP Unprotected SAP Protected HD Unprotected HD

6 4

12 2 8.28 3.28

± ± ± ±

2.58 0.65 1.64 1.06

23 2.57 11.57 2.42

30 ± ± ± ±

6.69 1.41 2.81 1.25

13.29 2.71 10.71 1.57

60 ± ± ± ±

2.94 1.39 2.77 0.42

10.67 2 10.67 3.83

± ± ± ±

2.37 0.73 1.49 1.44

SAP: stretched-attend postures; HD: head dipping

2 0

Fig. 3 shows the effects of intra-dPAG injections of capsaicin on the ethological measures of mice exposed to the EPM. One-way ANOVA revealed a significant drug effect for unprotected head dipping and unprotected SAP [(uHD): F4,35 = 3.35; p < 0.05; (uSAP): F4,35 = 1.62; p < 0.05]. Post hoc analysis confirmed a decrease in the frequency of uSAP for all capsaicin doses tested, and in the frequency of uHD for animals that received the higher doses of capsaicin (0.1 and 1.0 nmol) compared to vehicle (p < 0.05). ANOVA did not reveal any other significant behavioral effects of intra-dPAG capsaicin (higher F value: F4,35 = 1.29; p > 0.05). Furthermore, animals treated with capsaicin outside dPAG were no different from vehicle (p > 0.05).

50 45

Entries

40

% Open arm

Capsazepine (nmol/0.2 ␮L)

Vehicle

8

Time

35 30 25

*

20 15 10

*

5 0

311

vehicle

cpsa 0.01

cpsa 0.1

* * cpsa 1

out

Fig. 2. Effects of capsaicin (0.01, 0.1 and 1 nmol/0.2 ␮L) microinjections into the dPAG on the frequency of closed arm entries, percentage of open arm entries and percentage of open arm time in the EPM. n = 7–9. *P < 0.05 compared to the vehicle group. Animals that received capsaicin active doses (0.1 and 1 nmol) outside the dPAG were analyzed together in an OUT group.

compared to vehicle-injected animals (p < 0.05). There was no significant effect on the frequency of entries into the closed arms (CE: F4,35 = 1.67; p > 0.05). Animals receiving capsaicin outside the dPAG were no different from vehicle (p > 0.05).

3.3. Experiment 2: Lack of effect of intra-dPAG microinjection of capsazepine on the behavior of mice exposed to the EPM The second experiment was designed to assess the effects of increasing doses of capsazepine (0, 10, 30 or 60 nmol) on anxiety indices (%OE and %OT) and locomotor activity (CE) of mice subjected to the EPM (Fig. 4). One-way ANOVA showed no significant drug effects on either locomotion (CE: F3,23 = 0.43; p > 0.05) or anxiety indices (F3,23 ≤ 0.85; p > 0.05). Table 1 represents an ethogram of the animals treated with intra-dPAG capsazepine and subjected to the EPM. One-way ANOVA did not reveal any significant drug effects on the behavioral parameters scored (F3,23 ≤ 1.10; p > 0.05).

Fig. 3. Effects of capsaicin (0.01, 0.1 and 1 nmol/0.2 ␮L) microinjections into the dPAG on the behavior of mice exposed to the EPM. n = 7–9. Animals that received capsaicin active doses (0.1 and 1 nmol) outside the dPAG were analyzed together in an OUT group.

D.C. Mascarenhas et al. / Behavioural Brain Research 250 (2013) 308–315

14

16

12

14

Closed arm entries

Closed arm entries

312

10 8 6 4

12 10 8 6 4

2

2

0

0

45

60

Entries

40 35

Entries

50

% Open arm

Time

30

% Open arm

*

25 20 15

Time

40 30 20

*

10

10

*

0

5

vehicle -

cpz 30 -

vehicle -

cpz 30 -

0

vehicle

vehicle

cpsa 1

cpsa 1

vehicle

cpz 10

cpz 30

cpz 60

Fig. 4. Lack of effect of capsazepine (10, 30 and 60 nmol/0.2 ␮L) microinjections into the dPAG on the frequency of closed arm entries, percentage of open arm entries and percentage of open arm time in the EPM. n = 6–7.

3.4. Experiment 3. Effects of combined microinjections of capsazepine (pretreatment, 30 nmol) and capsaicin (treatment, 1.0 nmol) into the dPAG on the behavior of mice exposed to the EPM Fig. 5 summarizes the effects of prior intra-dPAG injections of 30 nmol capsazepine or vehicle followed by vehicle or 1.0 nmol capsaicin on anxiety indices (%OE and %OT) and locomotor activity (CE) of mice exposed to the EPM. Two-way ANOVA revealed significant effect for treatment factor (F1,25 = 6.55; p < 0.05) and interaction between factors (F1,21 = 13.42; p < 0.05) for %OE, but not for pretreatment factor (pretreatment: F1,25 = 4.18; p > 0.05). Post hoc Duncan test confirmed that the groups veh-cpsa and cpz-veh replicated the results obtained in Experiments 1 and 2 (anxiogeniclike effect and lack of effect relative to the vehicle-vehicle group, respectively, p < 0.05). Furthermore, the anxiogenic-like effect of capsaicin was completely blocked by prior treatment with the TRPV1 antagonist capsazepine (p < 0.05). Two-way ANOVA followed by post hoc Duncan test for %OT revealed significant effects for pretreatment factor (F1,25 = 8.05, p < 0.05) and interaction between factors (F1,25 = 7.72; p < 0.05), but not for treatment factor (F1,25 = 3.09, p > 0.05). Regarding CE, two-way ANOVA revealed significant effects only for pretreatment versus treatment interaction (F1,25 = 7.04; p < 0.05; pretreatment factor: F1,25 = 0.72, p > 0.05; treatment factor: F1,25 = 0.87, p > 0.05). Duncan post hoc analysis for CE confirmed an increase in closed arm exploration in capsaicin group (veh + capsa) compared to vehicle group (veh + veh) (p < 0.05). Fig. 6 shows the effects of prior intra-dPAG injections of vehicle or 30 nmol capsazepine followed by vehicle or 1.0 nmol capsaicin, on the ethological measures displayed by mice exposed to the EPM. Two-way ANOVA did not reveal any significant effects for protected

Fig. 5. Effects of prior microinjections of capsazepine (vehicle or 30 nmol) and capsaicin (vehicle and 1.0 nmol) into the dPAG on the frequency of closed arm entries, percentage of open arm entries and percentage of open arm time in mice subjected to the EPM. n = 6–9. *P < 0.05 compared to other groups.

and unprotected SAP and pHD (higher F1,25 value = 2.96, p > 0.05). However, the same analysis showed an interaction between factors for uHD (F1,25 = 12.34, p < 0.05) and a nonsignificant effect for pretreatment and treatment factors (F1,25 = 1.72 and F1,25 = 1.90, respectively; p > 0.05). Duncan post hoc test confirmed a decrease in the frequency of uHD for the group veh-cpsa, compared to veh-veh (p < 0.05). Interestingly, pretreatment with capsazepine (cpz-cpa) completely blocked this effect (p < 0.05 compared to veh-cpa). 4. Discussion The main results obtained in the present study indicate that the TRPV1 receptors located within the mouse dPAG play an important role in the modulation of anxiety-like behavior assessed in the EPM. While intra-dPAG capsaicin increased open-arm avoidance, prior capsazepine infusion into the same site completely blocked the anxiogenic-like effects observed with the TRPV1 agonist, without changing any behavior when injected alone. Intra-dPAG capsaicin microinjection produced a dosedependent anxiogenic-like effect in mice exposed to the EPM. This TRPV1 agonist provoked a reduction in open-arm exploration without altering closed-arm entries, a widely used measure of general locomotion [22–24]. Importantly, this proaversive-like effect is site-dependent, since animals which had the capsaicin active dose (0.1 and 1 nmol) infused outside the dPAG explored the open arms of the EPM similarly to the control group. This site-dependent effect was also observed in the ethological analysis. Given the physiological mechanism of action of these ion channels, which are part of a subfamily of receptors TRPV (Transient Receptor Potential cation channel V, or vanilloid receptors), the receptor TRPV1 represents a critical Ca2+ path of entry into excitable cells and is responsible for reducing the threshold for

D.C. Mascarenhas et al. / Behavioural Brain Research 250 (2013) 308–315

Protected head dipping

Protected stretched-aend postures

Frequency

15

10

12

vehicle - vehicle

10

cpz 30 - vehicle

8

vehicle - cpsa 1 cpz 30 - cpsa 1

Frequency

20

5

4

0

Unprotected stretched-aend postures

Unprotected head dipping 12

5

10

4

8

3

2

Frequency

Frequency

6

2

0

6

313

6 4

1

2

0

0

*

Fig. 6. Effects of prior microinjections of capsazepine (vehicle or 30 nmol) and capsaicin (vehicle and 1.0 nmol) into the dPAG on the behavior of mice exposed to the EPM. n = 6–9. *P < 0.05 compared to veh-veh and cpz-cpsa groups.

nerve depolarization of that fiber. Therefore, at the physiological level, the excitability blockade provoked by antagonists could explain the antiaversive-like behavior reported in some studies [6,34,35]. Corroborating these findings, the present study demonstrated that TRPV1 activation by intra-dPAG capsaicin microinjection produced an anxiogenic-like effect in mice exposed to the EPM. Furthermore, the activation of TRPV1 receptors and Ca2+ influx can promote the release of glutamate [10,11], which is a well-established anxiogenic neurotransmitter [18,24,36,37]. In addition, the present results corroborate previous findings of proaversive-like effects in mice exposed to the social interaction test after intracerebroventricular (ICV) administration of capsaicin [38]. However, in contrast to this hypothesis are the results reported by Terzian et al. [6], who demonstrated that capsaicin microinjection into the dPAG attenuated the defensive behaviors of rats exposed to the EPM. Although not tested in their work, the authors suggested that these discrepant results could be associated with the capacity for fast desensitization of TRPV1 receptors [6]. Activation of TRPV1 receptors by intra-dPAG infusion of capsaicin in mice showed that these receptors are important in the modulation of emotional states such as fear/anxiety, since their activation produced a robust anxiogenic-like effect, characterized by a reduction on conventional (i.e. %OE and %OT) and ethological (frequency of uHD and uSAP) measures. Regarding the ethological measures, intra-dPAG capsaicin reduced the frequency of uHD and uSAP and tended to increase pSAP (p = 0.10). Fernandez Espejo [39] grouped these behavioral parameters according to their relevance to the understanding of anxiety states. Parameters such as open-arm entries, uHD, pHD and pSAP were considered crucial to the analysis of anxiety in the EPM. Furthermore, while open-arm entries and uHD were positively loaded, pHD and pSAP related negatively to them. uSAP is an anxiety-like behavior that reflects a state of avoidance of approach to a threatening situation [39]. In fact, the present study shows that the uHD and uSAP behaviors go along with the reduction of open arm entries, as well as a tendency (p = 0.10) to increase the pSAP (Experiment 1). While TRPV1 receptor activation within the dPAG led to anxiogenesis, its blockade alone did not produce any effect on the

behavior of mice exposed to the EPM. Intra-dPAG microinjection of capsazepine did not change spatiotemporal or ethological measures of anxiety in the EPM. These results corroborate previous findings showing a lack of effect of capsazepine systemically administrated in mice exposed to the EPM [26]. However, some results in the literature report an antiaversive-like effect when capsazepine was injected into the dPAG of rats [6,34,35]. For instance, Terzian et al. [6] showed that intra-dPAG infusions of capsazepine attenuated anxiety in rats exposed to the Vogel or EPM tests. Evidence demonstrating an anxiolytic-like effect following the blockade of TRPV1 with capsazepine injected into the prefrontal cortex of rats has also been reported [40]. Regarding the role played by TRPV1 within the dPAG in the modulation of anxiety, Campos and Guimarães [34] showed that intra-dPAG capsazepine prevented the decrease in open arm exploration caused by the highest dose of WIN 55,212-2, a cannabinoid agonist. Briefly, cannabinoid agonists, such as the endocannabinoid anandamide (AEA), promote anxiolytic-like effects in a bell-shaped response curve, low doses being anxiolytic while this effect is abolished at higher doses [41]. Those authors suggested that these compounds, in high doses, could also activate the TRPV1 receptors when injected into the dPAG, counteracting their own anxiolytic effects. Therefore, endocannabinoids are also called endovanilloids [34,40]. Moreover, capsazepine even in combination with high doses of cannabinoid agonists, resulted in anxiolytic-like effects in the EPM, via blockade of cannabinoid agonists binding to the TRPV1 receptors [34]. Although the administration of capsazepine in rats can provoke an anxiolytic-like effect, suggesting a tonic modulation of endovanilloid neurotransmission in anxiety [6,35], in the present study, intra-dPAG microinjection of similar doses of this TRPV1 antagonist failed to change the defensive behavior of mice exposed to the EPM. Although corroborating previous work [26] showing a lack of effect of capsazepine systemically administrated in mice exposed to the EPM, the reason for these discrepant results remains unclear. Our results suggest that the TRPV1 receptors located within the midbrain dPAG do not exert a tonic control over the anxiety elicited by the EPM in mice. Moreover, the discrepancies between our results and those reported by

314

D.C. Mascarenhas et al. / Behavioural Brain Research 250 (2013) 308–315

others may be related to differences in the apparatus or experimental conditions, the animal species and even the possibility that the modulation of TRPV1 signaling in mice differs from that in rats. In line with this idea, Nunes-de-Souza et al. [36] demonstrated that the anxiolytic-like effects following inhibition of the neuronal nitric oxide synthase (nNOS), an enzyme that produces nitric oxide, depend on the animal test employed. For instance, intra-dPAG injection of NPLA (N-propyl-l-arginine), an inhibitor of nNOS, attenuated the defensive behavior seen in mice exposed to a predator, but failed to alter anxiety indices in mice exposed to the EPM. Nunes-de-Souza et al. [36] suggested that the prey-predator situation might be more aversive to mice than the EPM and thus would recruit nitric oxide release within the dPAG. Therefore, it is possible that, in the present study, the role played by the TRPV1 receptors may also depend on the magnitude and nature of the aversive stimulus, since defensive responses induced by the exposure to the EPM were not sensitive to the TRPV1 receptor blockade. In other words, the aversive experience in the EPM appears not to be enough to induce endovanilloid release within the mouse dPAG. Perhaps the use of capsazepine in a more aversive test, such as a paradigm involving prey(mouse)/predator(rat) (e.g. the rat exposure test [37]), might result in an antiaversive-like effect. While attractive, this hypothesis would need to be tested empirically in further studies. In order to assess whether the anxiogenic-like effects produced with intra-dPAG capsaicin are selective at TRPV1 receptors, capsazepine [30 nmol, an intrinsically inactive dose (Exp. 2)] was injected prior to capsaicin [1.0 nmol, a dose that produced anxiogenic-like effect (Exp. 1)] into the same site of this midbrain structure. In line with experiments 1 and 2, the TRPV1 agonist and antagonist, respectively, intensified and failed to change spatiotemporal (%OE and %OT) measures in mice exposed to the EPM. Moreover, as observed in Exp. 1, capsaicin (veh + cpsa 1.0) reduced uHD, an effect also blocked by prior intra-dPAG injection of capsazepine (cpz 30 + cpsa 1.0; Exp. 3). Curiously, the decrease of the uSAP frequency observed with intra-dPAG injection of capsaicin (Exp. 1) was not replicated in Exp. 3, suggesting that this parameter is somehow less sensitive to TRPV1 activation or blockade. Besides the spatiotemporal measures, ethological measures are also important to assess anxiety-like behavior, since these complementary forms of behavior provide us with other dimensions that appear to be related to vertical activity, exploration, risk assessment and decision making. It is important to highlight that the adoption of an ethological approach to the scoring of behavior in the EPM clearly confirms and extends the purpose of this behavioral test of anxiety [33]. Importantly, prior injection of an intrinsically inactive dose of capsazepine selectively and completely blocked the anxiogeniclike effects produced by intra-dPAG capsaicin. Present results corroborate previous findings showing that the anxiogenic-like effect of capsaicin, administered ICV in mice exposed to the social interaction test, was completely blocked by a capsazepine dose inactive per se [38]. Taken together, our results suggest that the anxiogenic effects of capsaicin are primarily due to vanilloid receptor activation in this limbic midbrain structure in mice. Although capsaicin-treated animals (veh + cpsa 1 nmol) showed higher frequency of closed-arm entries compared to control group, it must be emphasized that this effect was accompanied by a remarkable reduction of the %OE and %OT, suggesting that the activation of the dPAG TRPV1 increased the avoidance to the aversive areas (i.e. the open arms) of the EPM. These results are consistent in demonstrating the importance of the vanilloid compounds within the PAG in the modulation of anxiety states in mice subjected to the EPM paradigm. Further studies employing other animal models of fear/anxiety in mice are needed to investigate further the role of

endovanilloid modulation of the defensive behavior of this murine species. Acknowledgments We thank Elisabete Zocal Paro Lepera and Rosana Finoti Pupim Silva for their excellent technical assistance. This study was supported by CNPq and PADC/FCF-UNESP. D.C. Mascarenhas and K.S. Gomes are recipients of FAPESP research fellowship (Proc. 2010/13442-3 and 2010/01290-4, respectively). R.L. Nunes-deSouza received a CNPq research fellowship (Proc. 305597/2012-4). References [1] Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 1997;389:816–24. [2] Szallasi A, Blumberg PM. Vanilloid (Capsaicin) receptors and mechanisms. Pharmacological Reviews 1999;51:159–212. [3] Van der Stelt M, Di Marzo V. Endovanilloids: putative endogenous ligands of transient receptor potential vanilloid 1 channels. European Journal of Biochemistry 2004;271:1827–34. [4] Gewehr C, da Silva MA, dos Santos GT, Rossato MF, de Oliveira SM, Drewes CC, et al. Contribution of peripheral vanilloid receptor to the nociception induced by injection of spermine in mice. Pharmacology Biochemistry and Behavior 2011;99:775–81. [5] Akopian AN, Ruparel NB, Jeske NA, Patwardhan A, Hargreaves KM. Role of ionotropic cannabinoid receptors in peripheral antinociception and antihyperalgesia. Trends in Pharmacological Sciences 2009;30:79–84. [6] Terzian AL, Aguiar DC, Guimarães FS, Moreira FA. Modulation of anxiety-like behaviour by transient Receptor Potential Vanilloid Type 1 (TRPV1) channels located in the dorsolateral periaqueductal gray. European Neuropsychopharmacology 2009;19:188–95. [7] Moreira FA, Aguiar DC, Campos AC, Lisboa SF, Terzian AL, Resstel LB, et al. Antiaversive Effects of Cannabinoids: Is the Periaqueductal Gray Involved? Neural Plasticity 2009;2009:625469. [8] Marinelli S, Di Marzo V, Florenzano F, Fezza F, Viscomi MT, van der Stelt M, et al. Narachidonoyl-dopamine tunes synaptic transmission onto dopaminergic neurons by activating both cannabinoid and vanilloid receptors. Neuropsychopharmacology 2007;32:298–308. [9] Mezey E, Tóth ZE, Cortright DN, Arzubi MK, Krause JE, Elde R, et al. Distribution of mRNA for vanilloid receptor subtype 1 (VR1), and VR1-like immunoreactivity, in the central nervous system of the rat and human. Proceedings of the National Academy of Sciences of the United States of America 2000;97:3655–60. [10] McGaraughty S, Chu KL, Bitner RS, Martino B, El Kouhen R, Han P, et al. Capsaicin infused into the PAG affects rat tail flick responses to noxious heat and alters neuronal firing in the RVM. Journal of Neurophysiology 2003;90:2702–10. [11] Palazzo E, de Novellis V, Marabese I, Cuomo D, Rossi F, Berrino L, et al. Interaction between vanilloid and glutamate receptors in the central modulation of nociception. European Journal of Pharmacology 2002;439:69–75. [12] Xing J, Li J. TRPV1 receptor mediates glutamatergic synaptic input to dorsolateral periaqueductal gray (dl-PAG) neurons. Journal of Neurophysiology 2007;97:503–11. [13] Graeff FG. Brain defense systems and anxiety. In: Burrows GD, Roth M, Noyers Jr, editors. Handbook of anxiety. Amsterdam: Elsevier Science Publishers; 1990. p. 307–54. [14] Bandler R, Depaulis A, Vergnes M. Identification of midbrain neurons mediating defensive behaviors in the rat by microinjections of excitatory amino acids. Behavioural Brain Research 1985;15:107–19. [15] Bandler R, Shipley MT. Columnar organization in midbrain periaqueductal gray: modules of emotional expression? Trends in Neurosciences 1994;17:379–89. [16] Graeff FG, Del-Ben CM. Neurobiology of panic disorder: From animal models to brain neuroimaging. Neuroscience and Biobehavioral Reviews 2008;32:1326–35. [17] Valenstein ES. Independence of approach and escape reactions to electrical stimulation of the brain. Journal of Comparative and Physiological Psychology 1965;60:20–30. [18] Miguel TT, Nunes-de-Souza RL. Defensive-like behaviors and antinociception induced by NMDA injection into the periaqueductal gray of mice depend on nitric oxide synthesis. Brain Research 2006;1076:42–8. [19] Kasckow JW, Mulchahey JJ, Geracioti Jr TD. Effects of the vanilloid agonist olvanil and antagonist capsazepine on rat behaviours. Progress in NeuroPsychopharmacology and Biological Psychiatry 2004;28:291–5. [20] Lee C, Rodgers RJ. Antinociceptive effects of plus-maze exposure: influence of opiates receptor manipulations. Psychopharmacology 1990;102:507–13. [21] Pellow S, Chopin P, File SE, Briley M. Validation of open: closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. Journal of Neuroscience Methods 1985;14:149–67. [22] Taukulis HK, Goggin CE. Diazepam-stress interactions in the rat: effects on autoanalgesia and a plus-maze model of anxiety. Behavioral and Neural Biology 1990;53:205–16.

D.C. Mascarenhas et al. / Behavioural Brain Research 250 (2013) 308–315 [23] Gomes KS, Nunes-De-Souza RL. Implication of the 5-HT2A and 5-HT2C (but not 5HT1A) receptors located within the periaqueductal gray in the elevated plusmaze test-retest paradigm in mice. Progress in Neuro-Psychopharmacology and Biological Psychiatry 2009;33:1261–9. [24] Miguel TT, Nunes-de-Souza RL. Anxiogenic-like effects induced by NMDA receptor activation are prevented by inhibition of neuronal nitric oxide synthase in the periaqueductal gray in mice. Brain Research 2008;1240:39–46. [25] Marsch R, Foeller E, Rammes G, Bunck M, Kössl M, Holsboer F, et al. Reduced anxiety, conditioned fear, and hippocampal long-term potentiation in transient receptor potential vanilloid type 1 receptordeficient mice. Journal of Neuroscience 2007;27:832–9. [26] Rodgers RJ, Bickerdike PA, Dingley HK. Lack of effect of capsazepine, a TRPV1 receptor antagonist, on benzodiazepine-sensitive emotional behaviour in the mouse elevated plus-maze. Journal of Psychopharmacology 2010;24(3):TG15. A76. [27] Micale V, Cristino L, Tamburella A, Petrosino S, Leggio GM, Drago F, et al. Anxiolytic effects in mice of a dual blocker of fatty acid amide hydrolase and transient receptor potential vanilloid type-1 channels. Neuropsychopharmacology 2009;34:593–606. [28] LaMotte RH, Shain CN, Simone DA, Tsai EF. Neurogenic hyperalgesia: psychophysical studies of underlying mechanisms. Journal of Neurophysiology 1991;66:190–211. [29] Szallasi A, Di Marzo V. New perspectives on enigmatic vanilloid receptors. Trends in Neurosciences 2000;23:491–7. [30] Paxinos G, Frankling KBJ. The mouse brain in stereotaxic coordinates. 2nd ed. San Diego: Academic Press; 2001. [31] Messier C, Emond S, Ethier K. New techniques in stereotaxic surgery and anesthesia in mice. Pharmacology Biochemistry and Behavior 1999;63:313–8. [32] Lister RG. The use of a plus-maze to measure anxiety in the mouse. Psychopharmacology 1987;92:180–5.

315

[33] Rodgers RJ, Johnson NJ. Factor analysis of spatiotemporal and ethological measures in the murine elevated plus-maze test of anxiety. Pharmacology Biochemistry and Behavior 1995;52:297–303. [34] Campos AC, Guimarães FS. Evidence for a potential role for TRPV1 receptors in the dorsolateral periaqueductal gray in the attenuation of the anxiolytic effects of cannabinoids. Progress in Neuro-Psychopharmacology and Biological Psychiatry 2009;33:1517–21. [35] Casarotto PC, Terzian AL, Aguiar DC, Zangrossi H, Guimarães FS, Wotjak CT, et al. Opposing Roles for Cannabinoid Receptor Type-1 (CB1) and Transient Receptor Potential Vanilloid Type-1 Channel (TRPV1) on the Modulation of Panic-Like Responses in Rats. Neuropsychopharmacology 2012;37:478–86. [36] Nunes-de-Souza RL, Miguel TT, Gomes KS, Fugimoto JS, Mendes-Gomes J, Amaral VCS, Carvalho-Netto EF. Role of nitric oxide in the periaqueductal gray in defensive behavior in mice: influence of prior local N-methyl-D-aspartate receptor activation and aversive condition. Psychology and Neuroscience 2010;3:59–66. [37] Carvalho-Netto EF, Gomes KS, Amaral VC, Nunes-de-Souza RL. Role of glutamate NMDA receptors and nitric oxide located within the periaqueductal gray on defensive behaviors in mice confronted by predator. Psychopharmacology 2009;204:617–25. [38] Manna SS, Umathe SN. Transient receptor potential vanilloid 1 channels modulate the anxiolytic effect of diazepam. Brain Research 2011;1425:75–82. [39] Fernández Espejo E. Structure of the mouse behaviour on the elevated plusmaze test of anxiety. Behavioural Brain Research 1997;86:105–12. [40] Aguiar DC, Terzian AL, Guimarães FS, Moreira FA. Anxiolytic-like effects induced by blockade of transient receptor potential vanilloid type 1 (TRPV1) channels in the medial prefrontal cortex of rats. Psychopharmacology 2009;205:217–25. [41] Moreira FA, Aguiar DC, Guimarães FS. Anxiolytic-like effect of cannabinoids injected into the rat dorsolateral periaqueductal gray. Neuropharmacology 2007;52:958–65.