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Dorsal periaqueductal gray matter inhibits passive coping strategy elicited by forced swimming stress in rats
Neuroscience Letters 335 (2002) 87–90 www.elsevier.com/locate/neulet
Dorsal periaqueductal gray matter inhibits passive coping strategy elicited by forced swimming stress in rats Cilene Lino-de-Oliveira*, Thereza C.M. De Lima, Antonio P. Carobrez Departamento de Farmacologia, CCB, Universidade Federal de Santa Catarina,Rua Ferreira Lima 82, Floriano´polis, SC, 88015-420, Brazil Received 22 July 2002; received in revised form 24 September 2002; accepted 24 September 2002
Abstract Neuroanatomical evidence suggests that dorsal periaqueductal gray matter (dPAG) plays a role in behavioral changes induced by uncontrollable stress. To investigate this hypothesis, male Wistar rats were stressed (forced swimming, 15 min) and 24 h later received intra-dPAG injections of glutamate (20 nmol), lidocaine (4%) or vehicle 5 min before a forced swimming test (FST). The glutamate injection increased the latency to immobility, while lidocaine treatment increased the time spent in immobility during the FST. Both treatments failed to change exploratory parameters as evaluated in the open field test. These data suggest that while dPAG stimulation inhibits passive coping, dPAG inactivation intensifies uncontrollable stress effects. Thus, it is possible that the dPAG participates in the behavioral expression in the FST, inhibiting the passive coping strategies elicited by uncontrollable stress. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Forced swimming test; Uncontrollable stress; Dorsal periaqueductal gray matter; Superior colliculus; Antidepressant; Depression
Psychological stress is proposed to play a role in the development of affective disturbances [13]. Most of the animal models of depression are based on behavioral changes, induced by exposure to uncontrollable stress, that are attenuated by antidepressant treatment [2]. Uncontrollable stressors usually evoke passive emotional coping characterized by conservation-withdrawal strategies [2,7]. Uncontrollable stress such as that of forced swimming [6] or restraint [8,16] has also been shown to induce c-fos expression, a marker of neural activity, in multiple limbic brain areas. In fact, gamma-aminobutyric acid or antidepressants injected in the dorsal hippocampus, frontal cortex, or in the amygdala nuclei prevent the behavioral effects of uncontrollable stress in animal models of depression [5,10,15]. Forced swimming [Lino-de-Oliveira and Guimara˜es, unpublished data] and restraint stress [8] induce c-fos expression in dorsomedial and dorsolateral columns of the periaqueductal gray (dPAG) neurons, which suggest that activation of the neurons located in dPAG could mediate animal responses elicited by these stressors. However, elec-
* Corresponding author. Tel.: 155-48-331-9491; fax: 155-48222-4164. E-mail address: [email protected] (C. Lino-de-Oliveira).
trical or chemical dPAG stimulation produces responses such as fight-flight-freezing [14] or avoidance behaviors [1] that are similar to the natural strategies used by animals when exposed to controllable (or escapable) stress or threat [7]. Thus, the purpose of the present study was to verify whether the dPAG participates in behavioral changes induced by uncontrollable stress. To achieve this objective, the rats were submitted to a chemical stimulation with glutamate [1] or an inactivation of the dPAG with lidocaine [9] before the forced swimming test (FST). The FST [2,4,12] is an animal model of depression which employs forced swimming to induce passive emotional coping characterized by increased time spent in immobility posture during a subsequent swimming test. All procedures were carried out according to international standards of animal welfare (Brazilian Society of Neuroscience and Behavior, Act 1992) and the guidelines of the local Committee for Animal Care in Research (# 081/ CEUA and 23080.001156//2001-50/UFSC). Four-monthold male Wistar rats (300–400 g, n ¼ 87) were housed in pairs, with access to water and food, kept under a 12/12 h light/dark cycle (lights on at 6:00 am) and controlled temperature (23 ^ 1 8C). Animals were anesthetized (ketamine, 80 mg/kg plus xilazine, 75 mg/kg, v/v; 1 ml/kg, i.p.) for the implantation of a stainless steel guide cannula
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 01 11 9- 9
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C. Lino-de-Oliveira et al. / Neuroscience Letters 335 (2002) 87–90
(o.d. ¼ 0.7 mm, length ¼ 13 mm) that was lowered in the direction of the dPAG to a distance of 2.0 mm from the skull surface, at an angle of 228 with the sagital plane, 27.6 mm from the bregma, 1.9 mm from the midline. In the FST [2,4,12] rats (n ¼ 51) were individually placed, on two occasions 24 h apart (pre-test and test session) into a cylindrical tank (PVC, 20 £ 40 cm) containing 25 cm of water at 25 8C. After pre-test session (15 min), they were taken out of the water and allowed to dry under a lamp (for 30 min, 40 W) before returning to their home cages. Twenty four hours later, rats received an intracerebral injection (needle length ¼ 16.2 mm) of glutamate (GLU, 20 nmol/0.3 ml, pH adjusted to 7, [1]) or lidocaine (LIDO, 4%/0.3 ml, pH 7, [9]) or vehicle (VEH, artificial cerebrospinal fluid, 0.3 ml, pH 7) 5 min before the test session (5 min). The test was videotaped for subsequent evaluation of the latency, frequency of episodes and the time of immobility, climbing, and swimming behaviors [4]. Immobility was defined as the lack of motion of the whole body (only of the small movements necessary to keep the animal’s head above water). Swimming was coded when large forepaw movements displacing the body around the tank were performed. Climbing was recorded when vigorous movements with forepaws in and out of the water were observed. To test the effect of the treatments on locomotion, an additional group of rats (n ¼ 36) was placed in a Plexiglas square arena (45 £ 45 £ 35 cm) divided into nine equal sectors on the floor. The number of sector crossings (with all four paws), episodes of self-grooming and rearing were recorded for 5 min (open-field test, OFT). After completion of the experiments, rats were deeply anesthetized and perfused with 0.9% NaCl followed by a 10% formaldehyde solution.
Fig. 1. Injection sites of the VEH (X, 0.3 ml), GLU (A, 20 nmol/0.3 ml) or LIDO (B, 4%/0.3 ml) into dPAG or SC. Rats received the injection 5 min before a FST or an OFT. The numbers above the representative sections are the approximate distance from bregma in millimeters according to Paxinos and Watson’s rat brain Atlas [11].
Evans Blue dye (0.3 ml) was injected through the guide cannula to mark the site of the injection. The brains were removed, sectioned (100 mm, vibratome), Nissl stained and examined for location of the injection site. All behavioral experiments were carried out 7 days after the surgery (between 1:00 pm and 6:00 pm) and the behavioral scoring done by a single rater who was blind to the histological analysis. A Kruskall–Wallis and Mann–Whitney test were performed using the Statistica w software package (1995, Statsoft, Tulsa, OK) for each variable obtained from the dPAG or superior colliculus (SC; included in analysis as a control for brain region) injected groups.
Fig. 2. Immobility score in 5 min of the FST. The upper panel shows the latency to immobility (in seconds), middle panel presents the frequency of the immobility (episodes in 5 min) and the lower panel depicts the time spent (in seconds) in immobility. Rats received VEH (0.3 ml, white bar), GLU (20 nmol/0.3 ml, black bar) or LIDO (4%/0.3 ml, gray bar) into the dPAG (left figure) or SC (right figure) 5 min before FST. The bars represent means ^ SEM (number of animals is given in brackets). *P , 0:05 from respective VEH group (Mann–Whitney’s test). Abbreviations as in text.
C. Lino-de-Oliveira et al. / Neuroscience Letters 335 (2002) 87–90
The injection sites were located in dorsomedial and dorsolateral PAG (here collectively called dPAG) and in the deepest layer of the SC (Fig. 1). In the FST, latency to immobility (Hð2;30Þ ¼ 12:46, P ¼ 0:002) was significantly (P , 0:05) increased after GLU injection into the dPAG and remained unaltered following LIDO injection into this structure (Fig. 2). In contrast, time spent in immobility (Hð2;30Þ ¼ 6:44, P ¼ 0:04) was significantly (P , 0:05) increased after LIDO injection into the dPAG and did not change with GLU injection into the same brain area (Fig. 2). The frequency of immobility episodes remained unchanged after any treatments (Fig. 2). Active behaviors (swimming and climbing) were not changed by treatments. However, frequency of swimming episodes presented a tendency (Hð2;30Þ ¼ 5:66, P ¼ 0:06) to decrease after injection of GLU or LIDO into the dPAG (Table 1). In the OFT, no significant change was observed in the frequency of the behaviors registered following injections into the dPAG or SC (Table 1). The GLU injection into the dPAG increased the latency to immobility in the FST without significant change in the active behaviors, indicating an attenuation of the consequences of the previous stress. Otherwise, the LIDO injection into the dPAG magnified the stress effect, because it increased the time spent in immobility but did not change the active behaviors. Treatments failed to change behavioral responses in the OFT suggesting that effects observed in the FST do not arise from altered exploratory/motor activity. Furthermore, effects of treatments were not due to the spread of agents into the overlying SC because the GLU or LIDO injected into SC did not change any variable in the FST. Thus, results suggest that injection of GLU or LIDO into the dPAG produces opposite effects on the behavioral consequences of swimming stress. In the FST, immobility is thought to reflect either a failure of escape-directed behavior or the development of passive behavior that disengages the animal from active forms of coping with the uncontrollable stressful stimuli [2]. The increase in the latency to immobility in the FST after the GLU injection into the dPAG could reflect the restoration of
89
the escape-directed behavior or inhibition of the passive coping evoked by swimming stress. This proposition is consistent with experiments addressing the role of the dPAG in anxiety models that employ stress as stimulus [17]. The fear-potentiated startle response is inhibited by the association of the kainic acid injection into dPAG and low footshock training, and the decreased response is mimicked by high footshock training [17]. The fear-potentiated startle is inversely proportional to motor activity in response to footshock exposure [17] suggesting that the dPAG stimulation could produce a qualitative shift from passive forms of coping to active strategies when threat levels are increased. The dPAG inactivation with LIDO increased the time spent in immobility in the FST indicating that dPAG inactivation intensifies the passive coping evoked by swimming stress and are in agreement with evidences showing that lesion of the dPAG enhances the fear-potentiated startle [17] or the freezing [3] responses of animals exposed to high levels of the stress or threat (high footshock training or a cat, respectively). Taken together, data suggest that inactivation of dPAG could facilitate the expression of the passive strategies. Moreover, dPAG might act as an inhibitor of immobility behavior in the FST, substituting the passive form of coping evoked by uncontrollable stress with an active mode. Stimulation and lesion studies of the dPAG (chiefly dorsolateral PAG) suggest its involvement in the elicitation of active behaviors when the animal is exposed to a controllable (escapable) stress with psychological features (for review see Ref. [7]). Our data indicate that dPAG could also inhibits passive behavioral responses elicited by uncontrollable stress. The passive emotional coping, as elicited by forced swimming, is attributed to activation of structures such as the ventrolateral column of PAG (vlPAG) [7]. Thus, the present data are consistent with the idea that dPAG stimulation would produce inhibition of the vlPAG while dPAG inactivation would remove it [3]. The data arising from Fos detection have shown that uncontrollable stress such as that of restraint [8] or FST [Lino-de-Oliveira and Guimara˜ es, unpublished data]
Table 1 Frequency of the active behaviors recorded in 5 min of the FST or the OFT a Forced Swimming
dPAG
SC
VEH GLU LIDO VEH GLU LIDO
Open Field (n)
swimming
climbing
(n)
crossings
rearing
grooming
(11) (8) (11) (10) (5) (6)
25.5 ^ 2.5 17 ^ 1 # 19 ^ 2 # 15 ^ 2 10 ^ 6 15 ^ 3
6^1 3.5 ^ 1 5^1 4 ^ 0.5 3 ^ 0.5 4^1
(7) (8) (5) (6) (4) (6)
50 ^ 5.5 46 ^ 5.5 52 ^ 7 57 ^ 11 41 ^ 8 60.6 ^ 7
22 ^ 3 21 ^ 4 16 ^ 2 21 ^ 3 22 ^ 4.8 19 ^ 4
5^1 5.6 ^ 1 6^1 2.1 ^ 0.6 2.7 ^ 1 5^1
a Frequency indicates episodes in 5 min of each test. Rats received VEH (0.3 ml), GLU (20 nmol/0.3 ml) or LIDO (4%/0.3 ml) into the dPAG or SC 5 min before the respective test. The values represent means ^ SEM (n ¼ number of animals given in brackets). #P , 0:01 from respective VEH group (Mann–Whitney’s test). Abbreviations as in text.
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increases the activity of the cell bodies located in the dPAG. Paradoxically, Fos expression in dPAG is also increased by chronic antidepressant treatment [8]. This may be explained by the activation of different subsets of neurons, or receptors, or intracellular cascade in dPAG by uncontrollable stress and antidepressant drugs. Taking the above data into account, the stimulation of neural cell bodies in the dPAG could be necessary to the antidepressant-like effect. Indeed, we have here observed that GLU stimulation of the dPAG produced an antidepressant-like action, while the LIDO dPAG inactivation facilitated uncontrollable stress effects in the FST. In summary, it is possible that the dPAG participates in the behavioral expression in the FST inhibiting the passive coping strategies elicited by uncontrollable stress, and therefore this brain structure could be a target for antidepressant drugs. This work was supported by research grants from FAPESP and CNPq, Brazil. A.P. Carobrez and T.C.M. De Lima are recipients of Research fellowships from CNPq, and C. Lino de Oliveira is the recipient of an RD fellowship from CNPq (Proc. 301334/00-5). We are grateful to Dr J. Marino-Neto for the histological facilities. [1] Carobrez, A.P., Teixeira, K.V. and Graeff, F.G., Modulation of defensive behavior by periaqueductal gray NMDA/ glycine-B receptor, Neurosci. Biobehav. Rev., 25 (2001) 697–709. [2] Cryan, J.F., Markou, A. and Lucki, I., Assessing antidepressant activity in rodents: recent developments and future needs, Trends Pharmacol. Sci., 23 (5) (2002) 238–245. [3] DeOca, B.M., DeCola, J.P., Maren, S. and Fanselow, M.S., Distinct regions of the periaqueductal gray involved in the acquisition and expression of defensive responses, J. Neurosci., 1 (18) (1998) 3426–3432. [4] Detke, M.J., Rickels, M. and Lucki, I., Active behaviors in the rat forced swimming test differentially produced by serotonergic and noradrenergic antidepressants, Psychopharmacology, 121 (1995) 66–72.
[5] Duncan, G.E., Breese, G.R., Criswell, H., Stumpf, W.E., Mueller, R.A. and Covey, J.B., Effects of antidepressant drugs injected into the amygdala on behavioral responses of rats in the forced swim test, J. Pharmacol. Exp. Ther., 238 (1986) 758–762. [6] Duncan, G.E., Knapp, D.J., Johnson, K.B. and Breese, G.R., Functional classification of antidepressants based on antagonism of swim stress-induced Fos-like immunoreactivity, J. Pharmacol. Exp. Ther., 277 (1996) 1076–1089. [7] Keay, K.A. and Bandler, R., Parallel circuits mediating distinct emotional coping reactions to different types of stress, Neurosci. Biobehav. Rev., 25 (2001) 669–678. [8] Lino-de-Oliveira, C., Sales, A.J., Del Bel, E.A., Silveira, M.C.L. and Guimara˜ es, F.S., Effects of acute and chronic treatments on restraint stress-induced Fos expression, Brain Res. Bull., 55 (6) (2001) 747–754. [9] Malpelli, J.G., Reversible inactivation of subcortical sites by drug injection, J. Neurosci. Methods, 86 (1999) 119–128. [10] Padovan, C.M. and Guimara˜ es, F.S., Attenuation of behavioral consequences of immobilization stress by intra hippocampal microinjection of zimelidine, Braz. J. Med. Biol. Res., 26 (1993) 509–515. [11] Paxinos, G. and Watson, C., Stereotaxic Atlas of the Rat Brain, , 4th EditionAcademic Press, New York, 1998. [12] Porsolt, L.D., Anton, G., Blavet, N. and Jalfre, M., Behavioral despair in rats: a new model sensitive to antidepressant treatments, Eur. J. Pharmacol., 47 (1978) 379–391. [13] Post, R.M., Transduction of psychosocial stress into the neurobiology of recurrent affective disorder, Am. J. Psychiatry, 149 (1992) 999–1010. [14] Schenberg, L.C., Bittencourt, A.S., Sudre´ , E.C.M. and Vargas, L.C., Modeling panic attacks, Neurosci. Biobehav. Rev., 25 (2001) 647–659. [15] Sherman, A.D. and Petty, F., Neurochemical basis of the action of antidepressants on learned helplessness, Behav. Neurol. Biol., (1980) 119–134. [16] Titze-de-Almeida, R., Shida, H., Guimara˜ es, F.S. and Del Bel, E.A., Stress-induced expression of the c-fos protooncogene in the hippocampal formation, Braz. J. Med. Biol. Res., 27 (1994) 1080–1088. [17] Walker, D.L., Cassela, J.V., Lee, Y., De Lima, T.C.M. and Davis, M., Opposing roles of the amygdala and dorsolateral periaqueductal gray in fear-potentiated startle, Neurosci. Biobehav. Rev., 21 (1997) 743–753.
Dorsal periaqueductal gray matter inhibits passive coping strategy elicited by forced swimming stress in rats Cilene Lino-de-Oliveira*, Thereza C.M. De Lima, Antonio P. Carobrez Departamento de Farmacologia, CCB, Universidade Federal de Santa Catarina,Rua Ferreira Lima 82, Floriano´polis, SC, 88015-420, Brazil Received 22 July 2002; received in revised form 24 September 2002; accepted 24 September 2002
Abstract Neuroanatomical evidence suggests that dorsal periaqueductal gray matter (dPAG) plays a role in behavioral changes induced by uncontrollable stress. To investigate this hypothesis, male Wistar rats were stressed (forced swimming, 15 min) and 24 h later received intra-dPAG injections of glutamate (20 nmol), lidocaine (4%) or vehicle 5 min before a forced swimming test (FST). The glutamate injection increased the latency to immobility, while lidocaine treatment increased the time spent in immobility during the FST. Both treatments failed to change exploratory parameters as evaluated in the open field test. These data suggest that while dPAG stimulation inhibits passive coping, dPAG inactivation intensifies uncontrollable stress effects. Thus, it is possible that the dPAG participates in the behavioral expression in the FST, inhibiting the passive coping strategies elicited by uncontrollable stress. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Forced swimming test; Uncontrollable stress; Dorsal periaqueductal gray matter; Superior colliculus; Antidepressant; Depression
Psychological stress is proposed to play a role in the development of affective disturbances [13]. Most of the animal models of depression are based on behavioral changes, induced by exposure to uncontrollable stress, that are attenuated by antidepressant treatment [2]. Uncontrollable stressors usually evoke passive emotional coping characterized by conservation-withdrawal strategies [2,7]. Uncontrollable stress such as that of forced swimming [6] or restraint [8,16] has also been shown to induce c-fos expression, a marker of neural activity, in multiple limbic brain areas. In fact, gamma-aminobutyric acid or antidepressants injected in the dorsal hippocampus, frontal cortex, or in the amygdala nuclei prevent the behavioral effects of uncontrollable stress in animal models of depression [5,10,15]. Forced swimming [Lino-de-Oliveira and Guimara˜es, unpublished data] and restraint stress [8] induce c-fos expression in dorsomedial and dorsolateral columns of the periaqueductal gray (dPAG) neurons, which suggest that activation of the neurons located in dPAG could mediate animal responses elicited by these stressors. However, elec-
* Corresponding author. Tel.: 155-48-331-9491; fax: 155-48222-4164. E-mail address: [email protected] (C. Lino-de-Oliveira).
trical or chemical dPAG stimulation produces responses such as fight-flight-freezing [14] or avoidance behaviors [1] that are similar to the natural strategies used by animals when exposed to controllable (or escapable) stress or threat [7]. Thus, the purpose of the present study was to verify whether the dPAG participates in behavioral changes induced by uncontrollable stress. To achieve this objective, the rats were submitted to a chemical stimulation with glutamate [1] or an inactivation of the dPAG with lidocaine [9] before the forced swimming test (FST). The FST [2,4,12] is an animal model of depression which employs forced swimming to induce passive emotional coping characterized by increased time spent in immobility posture during a subsequent swimming test. All procedures were carried out according to international standards of animal welfare (Brazilian Society of Neuroscience and Behavior, Act 1992) and the guidelines of the local Committee for Animal Care in Research (# 081/ CEUA and 23080.001156//2001-50/UFSC). Four-monthold male Wistar rats (300–400 g, n ¼ 87) were housed in pairs, with access to water and food, kept under a 12/12 h light/dark cycle (lights on at 6:00 am) and controlled temperature (23 ^ 1 8C). Animals were anesthetized (ketamine, 80 mg/kg plus xilazine, 75 mg/kg, v/v; 1 ml/kg, i.p.) for the implantation of a stainless steel guide cannula
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 01 11 9- 9
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C. Lino-de-Oliveira et al. / Neuroscience Letters 335 (2002) 87–90
(o.d. ¼ 0.7 mm, length ¼ 13 mm) that was lowered in the direction of the dPAG to a distance of 2.0 mm from the skull surface, at an angle of 228 with the sagital plane, 27.6 mm from the bregma, 1.9 mm from the midline. In the FST [2,4,12] rats (n ¼ 51) were individually placed, on two occasions 24 h apart (pre-test and test session) into a cylindrical tank (PVC, 20 £ 40 cm) containing 25 cm of water at 25 8C. After pre-test session (15 min), they were taken out of the water and allowed to dry under a lamp (for 30 min, 40 W) before returning to their home cages. Twenty four hours later, rats received an intracerebral injection (needle length ¼ 16.2 mm) of glutamate (GLU, 20 nmol/0.3 ml, pH adjusted to 7, [1]) or lidocaine (LIDO, 4%/0.3 ml, pH 7, [9]) or vehicle (VEH, artificial cerebrospinal fluid, 0.3 ml, pH 7) 5 min before the test session (5 min). The test was videotaped for subsequent evaluation of the latency, frequency of episodes and the time of immobility, climbing, and swimming behaviors [4]. Immobility was defined as the lack of motion of the whole body (only of the small movements necessary to keep the animal’s head above water). Swimming was coded when large forepaw movements displacing the body around the tank were performed. Climbing was recorded when vigorous movements with forepaws in and out of the water were observed. To test the effect of the treatments on locomotion, an additional group of rats (n ¼ 36) was placed in a Plexiglas square arena (45 £ 45 £ 35 cm) divided into nine equal sectors on the floor. The number of sector crossings (with all four paws), episodes of self-grooming and rearing were recorded for 5 min (open-field test, OFT). After completion of the experiments, rats were deeply anesthetized and perfused with 0.9% NaCl followed by a 10% formaldehyde solution.
Fig. 1. Injection sites of the VEH (X, 0.3 ml), GLU (A, 20 nmol/0.3 ml) or LIDO (B, 4%/0.3 ml) into dPAG or SC. Rats received the injection 5 min before a FST or an OFT. The numbers above the representative sections are the approximate distance from bregma in millimeters according to Paxinos and Watson’s rat brain Atlas [11].
Evans Blue dye (0.3 ml) was injected through the guide cannula to mark the site of the injection. The brains were removed, sectioned (100 mm, vibratome), Nissl stained and examined for location of the injection site. All behavioral experiments were carried out 7 days after the surgery (between 1:00 pm and 6:00 pm) and the behavioral scoring done by a single rater who was blind to the histological analysis. A Kruskall–Wallis and Mann–Whitney test were performed using the Statistica w software package (1995, Statsoft, Tulsa, OK) for each variable obtained from the dPAG or superior colliculus (SC; included in analysis as a control for brain region) injected groups.
Fig. 2. Immobility score in 5 min of the FST. The upper panel shows the latency to immobility (in seconds), middle panel presents the frequency of the immobility (episodes in 5 min) and the lower panel depicts the time spent (in seconds) in immobility. Rats received VEH (0.3 ml, white bar), GLU (20 nmol/0.3 ml, black bar) or LIDO (4%/0.3 ml, gray bar) into the dPAG (left figure) or SC (right figure) 5 min before FST. The bars represent means ^ SEM (number of animals is given in brackets). *P , 0:05 from respective VEH group (Mann–Whitney’s test). Abbreviations as in text.
C. Lino-de-Oliveira et al. / Neuroscience Letters 335 (2002) 87–90
The injection sites were located in dorsomedial and dorsolateral PAG (here collectively called dPAG) and in the deepest layer of the SC (Fig. 1). In the FST, latency to immobility (Hð2;30Þ ¼ 12:46, P ¼ 0:002) was significantly (P , 0:05) increased after GLU injection into the dPAG and remained unaltered following LIDO injection into this structure (Fig. 2). In contrast, time spent in immobility (Hð2;30Þ ¼ 6:44, P ¼ 0:04) was significantly (P , 0:05) increased after LIDO injection into the dPAG and did not change with GLU injection into the same brain area (Fig. 2). The frequency of immobility episodes remained unchanged after any treatments (Fig. 2). Active behaviors (swimming and climbing) were not changed by treatments. However, frequency of swimming episodes presented a tendency (Hð2;30Þ ¼ 5:66, P ¼ 0:06) to decrease after injection of GLU or LIDO into the dPAG (Table 1). In the OFT, no significant change was observed in the frequency of the behaviors registered following injections into the dPAG or SC (Table 1). The GLU injection into the dPAG increased the latency to immobility in the FST without significant change in the active behaviors, indicating an attenuation of the consequences of the previous stress. Otherwise, the LIDO injection into the dPAG magnified the stress effect, because it increased the time spent in immobility but did not change the active behaviors. Treatments failed to change behavioral responses in the OFT suggesting that effects observed in the FST do not arise from altered exploratory/motor activity. Furthermore, effects of treatments were not due to the spread of agents into the overlying SC because the GLU or LIDO injected into SC did not change any variable in the FST. Thus, results suggest that injection of GLU or LIDO into the dPAG produces opposite effects on the behavioral consequences of swimming stress. In the FST, immobility is thought to reflect either a failure of escape-directed behavior or the development of passive behavior that disengages the animal from active forms of coping with the uncontrollable stressful stimuli [2]. The increase in the latency to immobility in the FST after the GLU injection into the dPAG could reflect the restoration of
89
the escape-directed behavior or inhibition of the passive coping evoked by swimming stress. This proposition is consistent with experiments addressing the role of the dPAG in anxiety models that employ stress as stimulus [17]. The fear-potentiated startle response is inhibited by the association of the kainic acid injection into dPAG and low footshock training, and the decreased response is mimicked by high footshock training [17]. The fear-potentiated startle is inversely proportional to motor activity in response to footshock exposure [17] suggesting that the dPAG stimulation could produce a qualitative shift from passive forms of coping to active strategies when threat levels are increased. The dPAG inactivation with LIDO increased the time spent in immobility in the FST indicating that dPAG inactivation intensifies the passive coping evoked by swimming stress and are in agreement with evidences showing that lesion of the dPAG enhances the fear-potentiated startle [17] or the freezing [3] responses of animals exposed to high levels of the stress or threat (high footshock training or a cat, respectively). Taken together, data suggest that inactivation of dPAG could facilitate the expression of the passive strategies. Moreover, dPAG might act as an inhibitor of immobility behavior in the FST, substituting the passive form of coping evoked by uncontrollable stress with an active mode. Stimulation and lesion studies of the dPAG (chiefly dorsolateral PAG) suggest its involvement in the elicitation of active behaviors when the animal is exposed to a controllable (escapable) stress with psychological features (for review see Ref. [7]). Our data indicate that dPAG could also inhibits passive behavioral responses elicited by uncontrollable stress. The passive emotional coping, as elicited by forced swimming, is attributed to activation of structures such as the ventrolateral column of PAG (vlPAG) [7]. Thus, the present data are consistent with the idea that dPAG stimulation would produce inhibition of the vlPAG while dPAG inactivation would remove it [3]. The data arising from Fos detection have shown that uncontrollable stress such as that of restraint [8] or FST [Lino-de-Oliveira and Guimara˜ es, unpublished data]
Table 1 Frequency of the active behaviors recorded in 5 min of the FST or the OFT a Forced Swimming
dPAG
SC
VEH GLU LIDO VEH GLU LIDO
Open Field (n)
swimming
climbing
(n)
crossings
rearing
grooming
(11) (8) (11) (10) (5) (6)
25.5 ^ 2.5 17 ^ 1 # 19 ^ 2 # 15 ^ 2 10 ^ 6 15 ^ 3
6^1 3.5 ^ 1 5^1 4 ^ 0.5 3 ^ 0.5 4^1
(7) (8) (5) (6) (4) (6)
50 ^ 5.5 46 ^ 5.5 52 ^ 7 57 ^ 11 41 ^ 8 60.6 ^ 7
22 ^ 3 21 ^ 4 16 ^ 2 21 ^ 3 22 ^ 4.8 19 ^ 4
5^1 5.6 ^ 1 6^1 2.1 ^ 0.6 2.7 ^ 1 5^1
a Frequency indicates episodes in 5 min of each test. Rats received VEH (0.3 ml), GLU (20 nmol/0.3 ml) or LIDO (4%/0.3 ml) into the dPAG or SC 5 min before the respective test. The values represent means ^ SEM (n ¼ number of animals given in brackets). #P , 0:01 from respective VEH group (Mann–Whitney’s test). Abbreviations as in text.
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increases the activity of the cell bodies located in the dPAG. Paradoxically, Fos expression in dPAG is also increased by chronic antidepressant treatment [8]. This may be explained by the activation of different subsets of neurons, or receptors, or intracellular cascade in dPAG by uncontrollable stress and antidepressant drugs. Taking the above data into account, the stimulation of neural cell bodies in the dPAG could be necessary to the antidepressant-like effect. Indeed, we have here observed that GLU stimulation of the dPAG produced an antidepressant-like action, while the LIDO dPAG inactivation facilitated uncontrollable stress effects in the FST. In summary, it is possible that the dPAG participates in the behavioral expression in the FST inhibiting the passive coping strategies elicited by uncontrollable stress, and therefore this brain structure could be a target for antidepressant drugs. This work was supported by research grants from FAPESP and CNPq, Brazil. A.P. Carobrez and T.C.M. De Lima are recipients of Research fellowships from CNPq, and C. Lino de Oliveira is the recipient of an RD fellowship from CNPq (Proc. 301334/00-5). We are grateful to Dr J. Marino-Neto for the histological facilities. [1] Carobrez, A.P., Teixeira, K.V. and Graeff, F.G., Modulation of defensive behavior by periaqueductal gray NMDA/ glycine-B receptor, Neurosci. Biobehav. Rev., 25 (2001) 697–709. [2] Cryan, J.F., Markou, A. and Lucki, I., Assessing antidepressant activity in rodents: recent developments and future needs, Trends Pharmacol. Sci., 23 (5) (2002) 238–245. [3] DeOca, B.M., DeCola, J.P., Maren, S. and Fanselow, M.S., Distinct regions of the periaqueductal gray involved in the acquisition and expression of defensive responses, J. Neurosci., 1 (18) (1998) 3426–3432. [4] Detke, M.J., Rickels, M. and Lucki, I., Active behaviors in the rat forced swimming test differentially produced by serotonergic and noradrenergic antidepressants, Psychopharmacology, 121 (1995) 66–72.
[5] Duncan, G.E., Breese, G.R., Criswell, H., Stumpf, W.E., Mueller, R.A. and Covey, J.B., Effects of antidepressant drugs injected into the amygdala on behavioral responses of rats in the forced swim test, J. Pharmacol. Exp. Ther., 238 (1986) 758–762. [6] Duncan, G.E., Knapp, D.J., Johnson, K.B. and Breese, G.R., Functional classification of antidepressants based on antagonism of swim stress-induced Fos-like immunoreactivity, J. Pharmacol. Exp. Ther., 277 (1996) 1076–1089. [7] Keay, K.A. and Bandler, R., Parallel circuits mediating distinct emotional coping reactions to different types of stress, Neurosci. Biobehav. Rev., 25 (2001) 669–678. [8] Lino-de-Oliveira, C., Sales, A.J., Del Bel, E.A., Silveira, M.C.L. and Guimara˜ es, F.S., Effects of acute and chronic treatments on restraint stress-induced Fos expression, Brain Res. Bull., 55 (6) (2001) 747–754. [9] Malpelli, J.G., Reversible inactivation of subcortical sites by drug injection, J. Neurosci. Methods, 86 (1999) 119–128. [10] Padovan, C.M. and Guimara˜ es, F.S., Attenuation of behavioral consequences of immobilization stress by intra hippocampal microinjection of zimelidine, Braz. J. Med. Biol. Res., 26 (1993) 509–515. [11] Paxinos, G. and Watson, C., Stereotaxic Atlas of the Rat Brain, , 4th EditionAcademic Press, New York, 1998. [12] Porsolt, L.D., Anton, G., Blavet, N. and Jalfre, M., Behavioral despair in rats: a new model sensitive to antidepressant treatments, Eur. J. Pharmacol., 47 (1978) 379–391. [13] Post, R.M., Transduction of psychosocial stress into the neurobiology of recurrent affective disorder, Am. J. Psychiatry, 149 (1992) 999–1010. [14] Schenberg, L.C., Bittencourt, A.S., Sudre´ , E.C.M. and Vargas, L.C., Modeling panic attacks, Neurosci. Biobehav. Rev., 25 (2001) 647–659. [15] Sherman, A.D. and Petty, F., Neurochemical basis of the action of antidepressants on learned helplessness, Behav. Neurol. Biol., (1980) 119–134. [16] Titze-de-Almeida, R., Shida, H., Guimara˜ es, F.S. and Del Bel, E.A., Stress-induced expression of the c-fos protooncogene in the hippocampal formation, Braz. J. Med. Biol. Res., 27 (1994) 1080–1088. [17] Walker, D.L., Cassela, J.V., Lee, Y., De Lima, T.C.M. and Davis, M., Opposing roles of the amygdala and dorsolateral periaqueductal gray in fear-potentiated startle, Neurosci. Biobehav. Rev., 21 (1997) 743–753.