- Email: [email protected]
Postnatal naltrindole treatments induce behavioural modifications in preweanling rats
Neuroscience Letters 283 (2000) 73±76 www.elsevier.com/locate/neulet
Postnatal naltrindole treatments induce behavioural modi®cations in preweanling rats Beatriz FernaÂndez a, Eva M. Romero a, Ian Kitchen b, M. Paz Viveros a,* a b
Departamento de BiologõÂa Animal II, Facultad de BiologõÂa, Universidad Complutense, 28040 Madrid, Spain Pharmacology Group, School of Biological Sciences, University of Surrey, Guildford, Surrey, GU2 5XH, UK Received 6 December 1999; received in revised form 14 February 2000; accepted 17 February 2000
Abstract To investigate the physiological role of the d -opioid receptor during the preweanling period, we have studied the effects of chronic (daily injections from birth to postnatal day 19) and acute treatments with the selective d -antagonist naltrindole (1 mg/kg), on behavioural and nociceptive responses in 20-day old male rats. Behavioural testing was performed using an open ®eld paradigm. Acute naltrindole induced signi®cant decreases in external and total ambulation (horizontal activity) and rearing behaviour (vertical activity), as well as a signi®cant increase in grooming frequency. In animals chronically treated with naltrindole there was an increase in total ambulation one day after the discontinuation of the treatment. In a test of nociception (tail immersion) no signi®cant effect of chronic naltrindole treatment on baseline latencies or of acute naltrindole on latency quotients (post-treatment latency/pre-treatment latency) were found. However, chronic naltrindole administration signi®cantly decreased the latency quotients. The results show that the d -opioid receptor participates in the tonic regulation of motor activity during the preweanling period and might be involved in certain aspects of stress responsiveness. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Naltrindole; d -Opioid receptor; Open ®eld; Nociception; Preweanling rats
Pharmacological studies using selective d -opioid compounds [2,11,17,18,21,23], as well as other studies using d -receptor knockdown [19,20] and knockout [12] animals have shown the involvement of d -opioid receptors in the modulation of locomotor activity and opioidmediated antinociception, in adult rodents. However, there is much less information about the physiological role of d receptors in neonates. When responses to d -agonists have been reported [3,9,13], these show a lack of functional responses in preweanling animals possibly associated with the late development of the d -receptor [14]. However, induction of d -receptor functionally de®cient animals, by chronic treatment with the d -selective antagonist naltrindole postnatally, has allowed us to show functional interactions between d -receptors and other opioid [8] and nonopioid [1] receptors in preweanling rats. To further address the physiological role of d -receptors during the preweanling period, we have studied the effects of chronic and acute
* Corresponding author. Tel.: 134-91-3944993; fax: 134-913944935. E-mail address: [email protected] (M. Paz Viveros)
naltrindole treatments on behavioural and nociceptive responses in 20-day old rats. Experiments were performed on male Wistar albino rats from the animal house of the Universidad Complutense of Madrid, which is served by Harlan Interfauna IbeÂrica S.A. (Barcelona, Spain). The animals were maintained at a constant temperature of 218C and in a reverse 12:12 h dark-light cycle (lights on at 20:00 h), with free access to food (commercial diet for rodents; Panlab, Barcelona, Spain) and water. Male rats were mated with females (one male £ two females) and spermpositive females were then rehoused in individual cages for the duration of pregnancy. On the day of birth (postnatal day 0), litters were sexbalanced and culled to 10 ^ 1 pups per dam. Although only males were used in this study, the whole litters remained with their mothers to avoid an in¯uence of sexual isolation [6]. From the day of birth to day 19 half of the animals within each litter (males and females) received a daily s.c. injection of naltrindole (RBI) (1 mg/kg, 1 ml/kg) and the other half a s.c. injection with the same volume of 0.9% saline solution. The dose of naltrindole used in this study selectively blocks d -receptors without affecting mreceptors [3] and has been previously used in our recent
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 00 91 8- 6
74
B. FernaÂndez et al. / Neuroscience Letters 283 (2000) 73±76
work on d -receptor de®cient animals [1,8]. At 20 days of age, and within each of the above mentioned chronically treated groups, the effect of a single acute injection of naltrindole (1 mg/kg, 0.1 ml/20 g, i. p.), was studied in comparison to a control group receiving a single injection with an equivalent volume of 0.9% saline solution i.p. Each experimental group contained 9±10 individuals from four different litters which were tested on at least two different days to minimize inter-litter and inter-day variability. Nociception was assessed using the tail immersion test with water at 508C [15]. Nociceptive responses (tail immersion latencies) were measured as the time elapsed prior to removal of the tail from the water surface, and a maximum 10-s cut-off was used. Response latencies were measured 15 min before acute administration of saline or naltrindole and 15 min after treatment. To compare the different treatments, antinociception was quanti®ed as previously described [1,8] using the following formula: Latency quotient Post-treatment latency/pre-treatment latency. Behavioural testing was performed by using a square open ®eld (60 £ 60 £ 45 cm) with a ¯oor divided into 36 squares (10 £ 10 cm). The duration of the test was 5 min. The parameters recorded were: external ambulation (number of entries to the external sectors situated in the periphery, by the walls) and internal ambulation (number of entries to the internal sectors situated in the central area), total ambulation (external 1 internal), frequency of rearing and facial grooming, and defecation score (number of boluses). The animals were tested individually in the open ®eld, 5 min after the completion of the tail immersion test. All experimental procedures were carried out between 09:30 and 14:30 h. On the day of testing the animals were equilibrated in a quiet laboratory at least 1 h before experimental procedures were begun. Behavioural tests were carried out under the same illumination conditions as those in the animal facilities (red light). Data from tail immersion (latency quotients) and open ®eld were analyzed by 2-way analysis of variance (ANOVA) (factors: chronic and acute treatment). Student± Newman±Keuls test was used for posthoc comparisons. A Student t-test was employed to analyze basal nociceptive latencies. There was no signi®cant effect of chronic treatment on the baseline responses (animals injected chronically with saline: 1.73 ^ 0.12, n 20, animals treated chronically with naltrindole: 1.88 ^ 0.09, n 19). The acute naltrindole treatment did not modify the latency quotients. However, a signi®cant reduction of the latency quotients was found after chronic naltrindole administration (P 0:01) (Table 1). In the open ®eld test, the acute naltrindole treatment induced signi®cant decreases in external and total ambulation (horizontal activity) (P , 0:001) and rearing behaviour (vertical activity) (P 0:01), as well as a signi®cant increase in grooming frequency (P , 0:05) (Fig. 1). Although the interaction between factors did not reach statistical signi®cance, the data clearly shows that grooming frequency was increased in the control animals
Table 1 Effects of chronic naltrindole (NTI) treatment (from birth to day 19) on latency quotients (tail immersion) in 20 day old rats. Response latencies were measured 15 min before acute administration of saline (SS) or NTI and 15 min after treatment. Latency quotient post-treatment latency/pre-treatment latency a Chronic
Acute
Latency quotients
SS SS NTI NTI
SS NTI SS NTI
1.24 ^ 0.1 1.48 ^ 0.1 1.07 ^ 0.1* 1.13 ^ 0.1*
a Values represent the mean ^ SEM of nine to ten animals. Student±Newman±Keuls: *P , 0:05 vs. the groups injected chronically with SS.
injected chronically with saline. In contrast to the acute treatment, chronic naltrindole administration produced an increase in external ambulation and in total ambulation (P , 0:05) (Fig. 1). No signi®cant effect of the naltrindole treatments on internal ambulation was found (chronic/acute treatment; SS/SS: 61.9 ^ 5.1, SS/NTI: 56.1 ^ 6.5, NTI/SS: 71.9 ^ 6.7, NTI/NTI: 62.9 ^ 9.1). The mean value for defecation score was 0±1 in all experimental groups.
Fig. 1. Effects of neonatal naltrindole treatments on open ®eld activity in 20-day old rats. The animals were injected chronically with saline (SS) or naltrindole (NTI) (1 mg/kg, s.c.) (from birth to day 19), and 1 day after the discontinuation of this treatment were studied for the acute effects of NTI (1 mg/kg, i.p.). Histograms represent the mean ^ SEM of ten animals. EA, external ambulation; TA, total ambulation; (external 1 internal), R, rearing; G, grooming. Student±Newman±Keuls: *P , 0:05 vs. the control groups injected acutely with SS. ¶P , 0:05 signi®cant difference between groups with different chronic treatment (SS vs. NTI).
B. FernaÂndez et al. / Neuroscience Letters 283 (2000) 73±76
In accordance with previous studies [1,8,10], chronic naltrindole administration did not modify the baseline nociceptive responses and acute naltrindole did not modify latency quotients. The slight but signi®cant decrease in latency quotients induced by the chronic naltrindole treatment might re¯ect blunting of a modest antinociceptive effect induced by the ®rst tail immersion test plus the acute injection. It is likely that, at this age, this stress induced antinociception is mediated by m-receptor [14]. Thus, the present data might re¯ect a m-d interaction, which accords with previous results showing that an identical administration schedule of the d -antagonist abolished the antinociception induced by the m-selective agonist alfentanil in the tail immersion test, in 20-day old males [8]. The functional role of d -receptors in motor responses of adult rodents has been well documented in pharmacological studies using d -selective agonists and antagonists. The nonpeptide d -agonists BW 373U86 and SNC 80 have been shown to dose-dependently elicit locomotion, rearing and stereotyped behaviours [23] and other d -agonists induce horizontal hyperlocomotion [18] associated with an increase in rearing [2]. These locomotor stimulatory effects were abolished by d -antagonists. Deltorphins also induced locomotor hyperactivity which is suppressed by naltrindole [21] and by pretreatment with d -antisense oligodeoxynucleotide [19]. However, there is no evidence in the literature for a physiological role of d -receptors in the modulation of motor activity in preweanling rats. The few data available show a lack of d -agonist mediated motor responses in these animals [9]. However, the present results indicate that acute naltrindole administration induces a decrease in both, horizontal and vertical activity in the open ®eld, suggesting the existence of a tonic activity of the d -opioid system in the modulation of motor activity in neonatal rats, and accord with data showing that naltrindole attenuated locomotion in adult animals [11]. Decreased horizontal motor activity has been reported for mice lacking the pre-pro enkephalin gene [16] and kelatorphan, an inhibitor of enkephalin catabolism, induced open ®eld hyperactivity in adult rats, with this effect being suppressed by d -selective antagonists [2]. Moreover, the hyperlocomotion induced by RB 101, a mixed inhibitor of enkephalin-degrading enzymes, has been shown to be mediated by d -receptor stimulation [4]. This data suggests that endogenous enkephalins acting at d opioid receptors might be involved in the tonic positive modulation of motor activity in adult rodents. The observed decrement in locomotor activity after acute blockade of d receptors in 20-day old rats indicates that this opioid-receptor might play a similar role during the preweanling period. The increased ambulation (locomotor horizontal activity) shown by the animals chronically treated with naltrindole 1 day after the discontinuation of the treatment, suggests a compensatory change (supersensitivity/up-regulation) in the d -opioid receptors, which might facilitate the stimulatory action of endogenous enkephalins on locomotion [2,4]. The results obtained in locomotor vertical activity are in
75
part different, since rearing behaviour was affected by acute but not chronic naltrindole administration. This data might suggest that there is a population of d -receptors involved in the modulation of horizontal activity and another different population of d -receptors involved in the modulation of vertical activity. These two different populations of d -receptors appear to be differentially sensitive to repeated blockade by the antagonist. Acute naltrindole administration did not affect internal ambulation, a parameter indicative of emotionality [7]. These results accord with previous data in adult mice indicating that the d agonist DPDPE did not affect internal ambulation [18] and that other d -agonists and kelatorphan were unable to modify the emotional state of adult rats measured in an elevated plus maze [2]. However, the acute naltrindole treatment induced an increase in grooming, and this parameter has been considered to be a possible indication of stress reactivity or emotionality as well as a displacement activity which releases the tension caused by a stressful situation [7]. Thus, our data might indicate that the acute blockade of d -receptors by naltrindole induces a certain level of anxiety, which may be related to the lack of anxiolytic action of endogenous enkephalins [16]. The present data therefore suggests that the d -opioid system may be implicated in certain speci®c aspects of stress responsiveness in preweanling rats. There is evidence for the existence of functional interactions between d -opioid and mesolimbic and mesostriatal dopaminergic systems in the modulation of motor activity in adult rodents [2,5,17]. Dopaminergic and d -opioid receptors appear to mediate the modulation of neural activity in the neostriatum in early stages of development (postnatal days 10±15) [22]. Therefore, it is likely that functional interactions between d -opioid and striatal dopaminergic systems are also involved in the modulation of locomotor activity during the neonatal period. To test this hypothesis, we are currently investigating if chronic naltrindole administration modi®es the activity of brain dopaminergic systems in preweanling rats. In conclusion, acute and chronic administration of the selective d -antagonist naltrindole induced several behavioural modi®cations in 20-day old rats, indicating that the d -opioid system participates in the tonic modulation of locomotor activity in preweanling animals. This study was supported by the European Commission BMH4-CT96±0510 (DG 12-SSMA). [1] Alberti, I., FernaÂndez, B., Alguacil, L.F., Aguilar, A., CaamanÄo, M., Romero, E.M. and Viveros, M.P., Preweanling naltrindole administration differentially affects clonidine induced antinociception and plasma adrenaline levels in male and female neonatal rats. Br. J. Pharmacol., 128 (1999) 953±960. [2] Calenco-Choukroun, G., DaugeÂ, V., Gacel, G., FeÂger, J. and Roques, B.P., Opioid d agonists and endogenous enkephalins induce different emotional reactivity than m agonists
76
[3]
[4]
[5]
[6]
[7]
[8]
[9] [10] [11]
[12]
[13]
B. FernaÂndez et al. / Neuroscience Letters 283 (2000) 73±76 after injection in the rat ventral tegmental area. Psychopharmacology, 103 (1991) 493±502. Crook, T.J., Kitchen, I. and Hill, R.G., Effects of the d opioid receptor antagonist naltrindole on antinociceptive responses to selective d agonists in postweanling rats. Br. J. Pharmacol., 107 (1992) 573±576. Dauge, V., Corringer, P.J. and Roques, B.P., CCKA, but not CCKB, agonists suppress the hyperlocomotion induced by endogenous enkephalins, protected from enzymatic degradation by systemic RB 101, Pharmacol. Biochem. Behav., 50 (1995) 133±139. Dauge, V., Rossignol, P. and Roques, B.P., Blockade of dopamine receptors reverses the behavioral effects of endogenous enkephalins in the nucleus caudatus but not in the nucleus accumbens: differential involvement of delta and mu opioid receptors. Psychopharmacology (Berl.), 99 (1989) 168±175. De Cabo, C., Kelly, M., Kitchen, I. and Viveros, P., Effects of neonatal sexual isolation on m receptor development and nociceptive responses in the rat. Neurosci. Res. Commun., 10 (1992) 79±86. De Cabo de la Vega, C., Pujol, A. and Viveros, M.P., Neonatally administered naltrexone affects several behavioural responses in adult rats of both genders. Pharmacol. Biochem. Behav, 50 (1995) 277±286. FernaÂndez, B., Antelo, M.T., Kitchen, I. and Viveros, M.P., Effects of neonatal naltrindole treatment on antinociceptive and behavioral responses to m and k agonists in rats. Pharmacol. Biochem. Behav., 62 (1999) 145±149. Jackson, H.C. and Kitchen, I., Behavioural effects of selective m- k- and d-opioid agonists in neonatal rats. Psychopharmacology, 97 (1989) 404±409. Jackson, H.C., Ripley, T.L. and Nutt, D.J., Exploring d-receptor function using the selective opioid antagonist naltrindole. Neuropharmacology, 28 (1989) 1427±1430. Kelley, A.E., Bless, E.P. and Swanson, C.J., Investigation of the effects of opiate antagonists infused into the nucleus accumbens on feeding and sucrose drinking in rats. J. Pharmacol. Exp. Ther., 278 (1996) 1499±1507. Kitchen, I., Opioid gene knockouts: New answers to old questions? In E. Kalso, H.J. McQuay and Z. WiesenfeldHallin (Eds.), Progress in Pain Research and Management: Opioid sensitivity in chronic non-cancer pain, Vol. 14, IASP Press, Seattle, 1999, pp. 281±303. Kitchen, I., Kelly, M.D.W. and De Cabo, C., k - and d -opioid
[14]
[15] [16]
[17]
[18]
[19]
[20] [21] [22]
[23]
receptor mediated antinociception in preweanling rats. Analgesia, 1 (1995) 512±515. Kitchen, I., Leslie, F.M., Kelly, M., Barnes, R., Crook, T.J., Hill, R.G., Borsodi, A., Toth, G., Melchiorri, P. and Negri, L., Development of delta-opioid receptor subtypes and the regulatory role of weaning: radioligand binding, autoradiography and in situ hybridization studies. J. Pharmacol. Exp. Ther., 275 (1995) 1597±1607. Kitchen, I., McDowell, J., Winder, C. and Wilson, J.M., Low level lead exposure alters morphine antinociception in neonatal rats. Toxicol. Lett., 22 (1984) 119±123. KoÈnig, M., Zimmer, A.M., Steiner, H., Holmes, P.V., Crawley, J.N., Brownstein, M.J. and Zimmer, A., Pain responses, anxiety and aggression in mice de®cient in pre-proenkephalin. Nature, 383 (1996) 535±538. Longoni, R., Spina, L., Mulas, A., Carboni, E., Garau, L., Melchiorri, P. and Di Chiara, G., (d-Ala2) deltorphin II: D1dependent stereotypies and stimulation of dopamine release in the nucleus accumbens. J. Neurosci., 11 (1991) 1565±1576. Mickley, G.A., Mulvihill, M.A. and Postler, M.A., Brain m and d opioid receptors mediate different locomotor hyperactivity responses of the C57BL/6J mouse. Psychopharmacology, 101 (1990) 332±337. Mizoguchi, H., Narita, M., Nagase, H., Suzuki, T., Quock, R.M. and Tseng, L.F., Use of antisense oligodeoxynucleotide to determine delta-opioid receptor involvement in (dAla2) deltorphin II-induced locomotor hyperactivity. Life Sci, 59 (1996) 69±73. Negri, L., Lattanzi, M., Broccardo, M. and Melchiorri, P., Knockdown of rat brain d -opioid receptors by antisense oligodeoxynucleotides. Dolor, 14 (Suppl. 1) (1999) 11. Negri, L., Noviello, L. and Noviello, V., Antinociceptive and behavioral effects of synthetic deltorphin analogs. Eur. J. Pharmacol., 296 (1996) 9±16. Perez-Navarro, E., Alberch, J. and Marsal, J., Postnatal development of functional dopamine, opioid and tachykinin receptors that regulate acetylcholine release from rat neostriatal slices. Effects of 6-hydroxydopamine lesion. Int. J. Dev. Neurosci., 11 (1993) 701±708. Spina, L., Longoni, R., Mulas, A., Chang, K.J. and Di Chiara, G., Dopamine-dependent behavioural stimulation by nonpeptide delta opioids BW373U86 and SNC 80: 1. Locomotion, rearing and stereotypes in intact rats. Behav. Pharmacol., 9 (1998) 1±8.
Postnatal naltrindole treatments induce behavioural modi®cations in preweanling rats Beatriz FernaÂndez a, Eva M. Romero a, Ian Kitchen b, M. Paz Viveros a,* a b
Departamento de BiologõÂa Animal II, Facultad de BiologõÂa, Universidad Complutense, 28040 Madrid, Spain Pharmacology Group, School of Biological Sciences, University of Surrey, Guildford, Surrey, GU2 5XH, UK Received 6 December 1999; received in revised form 14 February 2000; accepted 17 February 2000
Abstract To investigate the physiological role of the d -opioid receptor during the preweanling period, we have studied the effects of chronic (daily injections from birth to postnatal day 19) and acute treatments with the selective d -antagonist naltrindole (1 mg/kg), on behavioural and nociceptive responses in 20-day old male rats. Behavioural testing was performed using an open ®eld paradigm. Acute naltrindole induced signi®cant decreases in external and total ambulation (horizontal activity) and rearing behaviour (vertical activity), as well as a signi®cant increase in grooming frequency. In animals chronically treated with naltrindole there was an increase in total ambulation one day after the discontinuation of the treatment. In a test of nociception (tail immersion) no signi®cant effect of chronic naltrindole treatment on baseline latencies or of acute naltrindole on latency quotients (post-treatment latency/pre-treatment latency) were found. However, chronic naltrindole administration signi®cantly decreased the latency quotients. The results show that the d -opioid receptor participates in the tonic regulation of motor activity during the preweanling period and might be involved in certain aspects of stress responsiveness. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Naltrindole; d -Opioid receptor; Open ®eld; Nociception; Preweanling rats
Pharmacological studies using selective d -opioid compounds [2,11,17,18,21,23], as well as other studies using d -receptor knockdown [19,20] and knockout [12] animals have shown the involvement of d -opioid receptors in the modulation of locomotor activity and opioidmediated antinociception, in adult rodents. However, there is much less information about the physiological role of d receptors in neonates. When responses to d -agonists have been reported [3,9,13], these show a lack of functional responses in preweanling animals possibly associated with the late development of the d -receptor [14]. However, induction of d -receptor functionally de®cient animals, by chronic treatment with the d -selective antagonist naltrindole postnatally, has allowed us to show functional interactions between d -receptors and other opioid [8] and nonopioid [1] receptors in preweanling rats. To further address the physiological role of d -receptors during the preweanling period, we have studied the effects of chronic and acute
* Corresponding author. Tel.: 134-91-3944993; fax: 134-913944935. E-mail address: [email protected] (M. Paz Viveros)
naltrindole treatments on behavioural and nociceptive responses in 20-day old rats. Experiments were performed on male Wistar albino rats from the animal house of the Universidad Complutense of Madrid, which is served by Harlan Interfauna IbeÂrica S.A. (Barcelona, Spain). The animals were maintained at a constant temperature of 218C and in a reverse 12:12 h dark-light cycle (lights on at 20:00 h), with free access to food (commercial diet for rodents; Panlab, Barcelona, Spain) and water. Male rats were mated with females (one male £ two females) and spermpositive females were then rehoused in individual cages for the duration of pregnancy. On the day of birth (postnatal day 0), litters were sexbalanced and culled to 10 ^ 1 pups per dam. Although only males were used in this study, the whole litters remained with their mothers to avoid an in¯uence of sexual isolation [6]. From the day of birth to day 19 half of the animals within each litter (males and females) received a daily s.c. injection of naltrindole (RBI) (1 mg/kg, 1 ml/kg) and the other half a s.c. injection with the same volume of 0.9% saline solution. The dose of naltrindole used in this study selectively blocks d -receptors without affecting mreceptors [3] and has been previously used in our recent
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 00 91 8- 6
74
B. FernaÂndez et al. / Neuroscience Letters 283 (2000) 73±76
work on d -receptor de®cient animals [1,8]. At 20 days of age, and within each of the above mentioned chronically treated groups, the effect of a single acute injection of naltrindole (1 mg/kg, 0.1 ml/20 g, i. p.), was studied in comparison to a control group receiving a single injection with an equivalent volume of 0.9% saline solution i.p. Each experimental group contained 9±10 individuals from four different litters which were tested on at least two different days to minimize inter-litter and inter-day variability. Nociception was assessed using the tail immersion test with water at 508C [15]. Nociceptive responses (tail immersion latencies) were measured as the time elapsed prior to removal of the tail from the water surface, and a maximum 10-s cut-off was used. Response latencies were measured 15 min before acute administration of saline or naltrindole and 15 min after treatment. To compare the different treatments, antinociception was quanti®ed as previously described [1,8] using the following formula: Latency quotient Post-treatment latency/pre-treatment latency. Behavioural testing was performed by using a square open ®eld (60 £ 60 £ 45 cm) with a ¯oor divided into 36 squares (10 £ 10 cm). The duration of the test was 5 min. The parameters recorded were: external ambulation (number of entries to the external sectors situated in the periphery, by the walls) and internal ambulation (number of entries to the internal sectors situated in the central area), total ambulation (external 1 internal), frequency of rearing and facial grooming, and defecation score (number of boluses). The animals were tested individually in the open ®eld, 5 min after the completion of the tail immersion test. All experimental procedures were carried out between 09:30 and 14:30 h. On the day of testing the animals were equilibrated in a quiet laboratory at least 1 h before experimental procedures were begun. Behavioural tests were carried out under the same illumination conditions as those in the animal facilities (red light). Data from tail immersion (latency quotients) and open ®eld were analyzed by 2-way analysis of variance (ANOVA) (factors: chronic and acute treatment). Student± Newman±Keuls test was used for posthoc comparisons. A Student t-test was employed to analyze basal nociceptive latencies. There was no signi®cant effect of chronic treatment on the baseline responses (animals injected chronically with saline: 1.73 ^ 0.12, n 20, animals treated chronically with naltrindole: 1.88 ^ 0.09, n 19). The acute naltrindole treatment did not modify the latency quotients. However, a signi®cant reduction of the latency quotients was found after chronic naltrindole administration (P 0:01) (Table 1). In the open ®eld test, the acute naltrindole treatment induced signi®cant decreases in external and total ambulation (horizontal activity) (P , 0:001) and rearing behaviour (vertical activity) (P 0:01), as well as a signi®cant increase in grooming frequency (P , 0:05) (Fig. 1). Although the interaction between factors did not reach statistical signi®cance, the data clearly shows that grooming frequency was increased in the control animals
Table 1 Effects of chronic naltrindole (NTI) treatment (from birth to day 19) on latency quotients (tail immersion) in 20 day old rats. Response latencies were measured 15 min before acute administration of saline (SS) or NTI and 15 min after treatment. Latency quotient post-treatment latency/pre-treatment latency a Chronic
Acute
Latency quotients
SS SS NTI NTI
SS NTI SS NTI
1.24 ^ 0.1 1.48 ^ 0.1 1.07 ^ 0.1* 1.13 ^ 0.1*
a Values represent the mean ^ SEM of nine to ten animals. Student±Newman±Keuls: *P , 0:05 vs. the groups injected chronically with SS.
injected chronically with saline. In contrast to the acute treatment, chronic naltrindole administration produced an increase in external ambulation and in total ambulation (P , 0:05) (Fig. 1). No signi®cant effect of the naltrindole treatments on internal ambulation was found (chronic/acute treatment; SS/SS: 61.9 ^ 5.1, SS/NTI: 56.1 ^ 6.5, NTI/SS: 71.9 ^ 6.7, NTI/NTI: 62.9 ^ 9.1). The mean value for defecation score was 0±1 in all experimental groups.
Fig. 1. Effects of neonatal naltrindole treatments on open ®eld activity in 20-day old rats. The animals were injected chronically with saline (SS) or naltrindole (NTI) (1 mg/kg, s.c.) (from birth to day 19), and 1 day after the discontinuation of this treatment were studied for the acute effects of NTI (1 mg/kg, i.p.). Histograms represent the mean ^ SEM of ten animals. EA, external ambulation; TA, total ambulation; (external 1 internal), R, rearing; G, grooming. Student±Newman±Keuls: *P , 0:05 vs. the control groups injected acutely with SS. ¶P , 0:05 signi®cant difference between groups with different chronic treatment (SS vs. NTI).
B. FernaÂndez et al. / Neuroscience Letters 283 (2000) 73±76
In accordance with previous studies [1,8,10], chronic naltrindole administration did not modify the baseline nociceptive responses and acute naltrindole did not modify latency quotients. The slight but signi®cant decrease in latency quotients induced by the chronic naltrindole treatment might re¯ect blunting of a modest antinociceptive effect induced by the ®rst tail immersion test plus the acute injection. It is likely that, at this age, this stress induced antinociception is mediated by m-receptor [14]. Thus, the present data might re¯ect a m-d interaction, which accords with previous results showing that an identical administration schedule of the d -antagonist abolished the antinociception induced by the m-selective agonist alfentanil in the tail immersion test, in 20-day old males [8]. The functional role of d -receptors in motor responses of adult rodents has been well documented in pharmacological studies using d -selective agonists and antagonists. The nonpeptide d -agonists BW 373U86 and SNC 80 have been shown to dose-dependently elicit locomotion, rearing and stereotyped behaviours [23] and other d -agonists induce horizontal hyperlocomotion [18] associated with an increase in rearing [2]. These locomotor stimulatory effects were abolished by d -antagonists. Deltorphins also induced locomotor hyperactivity which is suppressed by naltrindole [21] and by pretreatment with d -antisense oligodeoxynucleotide [19]. However, there is no evidence in the literature for a physiological role of d -receptors in the modulation of motor activity in preweanling rats. The few data available show a lack of d -agonist mediated motor responses in these animals [9]. However, the present results indicate that acute naltrindole administration induces a decrease in both, horizontal and vertical activity in the open ®eld, suggesting the existence of a tonic activity of the d -opioid system in the modulation of motor activity in neonatal rats, and accord with data showing that naltrindole attenuated locomotion in adult animals [11]. Decreased horizontal motor activity has been reported for mice lacking the pre-pro enkephalin gene [16] and kelatorphan, an inhibitor of enkephalin catabolism, induced open ®eld hyperactivity in adult rats, with this effect being suppressed by d -selective antagonists [2]. Moreover, the hyperlocomotion induced by RB 101, a mixed inhibitor of enkephalin-degrading enzymes, has been shown to be mediated by d -receptor stimulation [4]. This data suggests that endogenous enkephalins acting at d opioid receptors might be involved in the tonic positive modulation of motor activity in adult rodents. The observed decrement in locomotor activity after acute blockade of d receptors in 20-day old rats indicates that this opioid-receptor might play a similar role during the preweanling period. The increased ambulation (locomotor horizontal activity) shown by the animals chronically treated with naltrindole 1 day after the discontinuation of the treatment, suggests a compensatory change (supersensitivity/up-regulation) in the d -opioid receptors, which might facilitate the stimulatory action of endogenous enkephalins on locomotion [2,4]. The results obtained in locomotor vertical activity are in
75
part different, since rearing behaviour was affected by acute but not chronic naltrindole administration. This data might suggest that there is a population of d -receptors involved in the modulation of horizontal activity and another different population of d -receptors involved in the modulation of vertical activity. These two different populations of d -receptors appear to be differentially sensitive to repeated blockade by the antagonist. Acute naltrindole administration did not affect internal ambulation, a parameter indicative of emotionality [7]. These results accord with previous data in adult mice indicating that the d agonist DPDPE did not affect internal ambulation [18] and that other d -agonists and kelatorphan were unable to modify the emotional state of adult rats measured in an elevated plus maze [2]. However, the acute naltrindole treatment induced an increase in grooming, and this parameter has been considered to be a possible indication of stress reactivity or emotionality as well as a displacement activity which releases the tension caused by a stressful situation [7]. Thus, our data might indicate that the acute blockade of d -receptors by naltrindole induces a certain level of anxiety, which may be related to the lack of anxiolytic action of endogenous enkephalins [16]. The present data therefore suggests that the d -opioid system may be implicated in certain speci®c aspects of stress responsiveness in preweanling rats. There is evidence for the existence of functional interactions between d -opioid and mesolimbic and mesostriatal dopaminergic systems in the modulation of motor activity in adult rodents [2,5,17]. Dopaminergic and d -opioid receptors appear to mediate the modulation of neural activity in the neostriatum in early stages of development (postnatal days 10±15) [22]. Therefore, it is likely that functional interactions between d -opioid and striatal dopaminergic systems are also involved in the modulation of locomotor activity during the neonatal period. To test this hypothesis, we are currently investigating if chronic naltrindole administration modi®es the activity of brain dopaminergic systems in preweanling rats. In conclusion, acute and chronic administration of the selective d -antagonist naltrindole induced several behavioural modi®cations in 20-day old rats, indicating that the d -opioid system participates in the tonic modulation of locomotor activity in preweanling animals. This study was supported by the European Commission BMH4-CT96±0510 (DG 12-SSMA). [1] Alberti, I., FernaÂndez, B., Alguacil, L.F., Aguilar, A., CaamanÄo, M., Romero, E.M. and Viveros, M.P., Preweanling naltrindole administration differentially affects clonidine induced antinociception and plasma adrenaline levels in male and female neonatal rats. Br. J. Pharmacol., 128 (1999) 953±960. [2] Calenco-Choukroun, G., DaugeÂ, V., Gacel, G., FeÂger, J. and Roques, B.P., Opioid d agonists and endogenous enkephalins induce different emotional reactivity than m agonists
76
[3]
[4]
[5]
[6]
[7]
[8]
[9] [10] [11]
[12]
[13]
B. FernaÂndez et al. / Neuroscience Letters 283 (2000) 73±76 after injection in the rat ventral tegmental area. Psychopharmacology, 103 (1991) 493±502. Crook, T.J., Kitchen, I. and Hill, R.G., Effects of the d opioid receptor antagonist naltrindole on antinociceptive responses to selective d agonists in postweanling rats. Br. J. Pharmacol., 107 (1992) 573±576. Dauge, V., Corringer, P.J. and Roques, B.P., CCKA, but not CCKB, agonists suppress the hyperlocomotion induced by endogenous enkephalins, protected from enzymatic degradation by systemic RB 101, Pharmacol. Biochem. Behav., 50 (1995) 133±139. Dauge, V., Rossignol, P. and Roques, B.P., Blockade of dopamine receptors reverses the behavioral effects of endogenous enkephalins in the nucleus caudatus but not in the nucleus accumbens: differential involvement of delta and mu opioid receptors. Psychopharmacology (Berl.), 99 (1989) 168±175. De Cabo, C., Kelly, M., Kitchen, I. and Viveros, P., Effects of neonatal sexual isolation on m receptor development and nociceptive responses in the rat. Neurosci. Res. Commun., 10 (1992) 79±86. De Cabo de la Vega, C., Pujol, A. and Viveros, M.P., Neonatally administered naltrexone affects several behavioural responses in adult rats of both genders. Pharmacol. Biochem. Behav, 50 (1995) 277±286. FernaÂndez, B., Antelo, M.T., Kitchen, I. and Viveros, M.P., Effects of neonatal naltrindole treatment on antinociceptive and behavioral responses to m and k agonists in rats. Pharmacol. Biochem. Behav., 62 (1999) 145±149. Jackson, H.C. and Kitchen, I., Behavioural effects of selective m- k- and d-opioid agonists in neonatal rats. Psychopharmacology, 97 (1989) 404±409. Jackson, H.C., Ripley, T.L. and Nutt, D.J., Exploring d-receptor function using the selective opioid antagonist naltrindole. Neuropharmacology, 28 (1989) 1427±1430. Kelley, A.E., Bless, E.P. and Swanson, C.J., Investigation of the effects of opiate antagonists infused into the nucleus accumbens on feeding and sucrose drinking in rats. J. Pharmacol. Exp. Ther., 278 (1996) 1499±1507. Kitchen, I., Opioid gene knockouts: New answers to old questions? In E. Kalso, H.J. McQuay and Z. WiesenfeldHallin (Eds.), Progress in Pain Research and Management: Opioid sensitivity in chronic non-cancer pain, Vol. 14, IASP Press, Seattle, 1999, pp. 281±303. Kitchen, I., Kelly, M.D.W. and De Cabo, C., k - and d -opioid
[14]
[15] [16]
[17]
[18]
[19]
[20] [21] [22]
[23]
receptor mediated antinociception in preweanling rats. Analgesia, 1 (1995) 512±515. Kitchen, I., Leslie, F.M., Kelly, M., Barnes, R., Crook, T.J., Hill, R.G., Borsodi, A., Toth, G., Melchiorri, P. and Negri, L., Development of delta-opioid receptor subtypes and the regulatory role of weaning: radioligand binding, autoradiography and in situ hybridization studies. J. Pharmacol. Exp. Ther., 275 (1995) 1597±1607. Kitchen, I., McDowell, J., Winder, C. and Wilson, J.M., Low level lead exposure alters morphine antinociception in neonatal rats. Toxicol. Lett., 22 (1984) 119±123. KoÈnig, M., Zimmer, A.M., Steiner, H., Holmes, P.V., Crawley, J.N., Brownstein, M.J. and Zimmer, A., Pain responses, anxiety and aggression in mice de®cient in pre-proenkephalin. Nature, 383 (1996) 535±538. Longoni, R., Spina, L., Mulas, A., Carboni, E., Garau, L., Melchiorri, P. and Di Chiara, G., (d-Ala2) deltorphin II: D1dependent stereotypies and stimulation of dopamine release in the nucleus accumbens. J. Neurosci., 11 (1991) 1565±1576. Mickley, G.A., Mulvihill, M.A. and Postler, M.A., Brain m and d opioid receptors mediate different locomotor hyperactivity responses of the C57BL/6J mouse. Psychopharmacology, 101 (1990) 332±337. Mizoguchi, H., Narita, M., Nagase, H., Suzuki, T., Quock, R.M. and Tseng, L.F., Use of antisense oligodeoxynucleotide to determine delta-opioid receptor involvement in (dAla2) deltorphin II-induced locomotor hyperactivity. Life Sci, 59 (1996) 69±73. Negri, L., Lattanzi, M., Broccardo, M. and Melchiorri, P., Knockdown of rat brain d -opioid receptors by antisense oligodeoxynucleotides. Dolor, 14 (Suppl. 1) (1999) 11. Negri, L., Noviello, L. and Noviello, V., Antinociceptive and behavioral effects of synthetic deltorphin analogs. Eur. J. Pharmacol., 296 (1996) 9±16. Perez-Navarro, E., Alberch, J. and Marsal, J., Postnatal development of functional dopamine, opioid and tachykinin receptors that regulate acetylcholine release from rat neostriatal slices. Effects of 6-hydroxydopamine lesion. Int. J. Dev. Neurosci., 11 (1993) 701±708. Spina, L., Longoni, R., Mulas, A., Chang, K.J. and Di Chiara, G., Dopamine-dependent behavioural stimulation by nonpeptide delta opioids BW373U86 and SNC 80: 1. Locomotion, rearing and stereotypes in intact rats. Behav. Pharmacol., 9 (1998) 1±8.