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Cannabinoid exposure alters learning of zebra finch vocal patterns
Developmental Brain Research 142 (2003) 215–217 www.elsevier.com / locate / devbrainres
Short communication
Cannabinoid exposure alters learning of zebra finch vocal patterns Ken Soderstrom a , *, Frank Johnson b a
Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA b Department of Psychology and Program in Neuroscience, Florida State University, Tallahassee, FL 32306, USA Accepted 18 February 2003
Abstract Using a well-established songbird model of juvenile vocal development, we have found that daily cannabinoid exposure at modest dosages alters sensory-motor vocal learning. Adult exposure did not change song that had already been learned. Our results demonstrate the potential for cannabinoid exposure to produce distinct effects during post-natal CNS development. 2003 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Learning and memory: pharmacology Keywords: Cannabinoid; Vocal learning; Post-natal CNS development
Similar to human language acquisition [2], vocal learning in song birds requires coordinated perception and motor production of memorized vocal patterns. Because these types of cognitive processes are influenced by cannabinoid receptor activation [3,14], cannabinoid signaling may be relevant to vocal development, a hypothesis supported by dense CB1 cannabinoid receptor expression in brain regions important for zebra finch song learning and production [11] and CB1-dependent reductions in singing behavior [12]. Here we show that daily cannabinoid exposure at modest dosages for 50 days alters learning of song by juvenile birds, while the same treatment in adult birds is without effect on the pattern of already-learned song. Subjects were male zebra finches raised in our breeding aviaries. Seven pairs of sibling or cross-fostered (2062 days of age) fledgling zebra finches were raised together by the same male tutor (so that the same song pattern was memorized) until 50 days of age. Animals were then housed singly in visual isolation and individuals from each pair were randomly assigned to receive either injections of the cannabinoid agonist WIN55212-2 (1 mg / kg, a transiently effective [2 h] dosage determined in prior experi*Corresponding author. Tel.: 11-252-816-2742; fax: 11-252-8163203. E-mail address: [email protected] (K. Soderstrom).
ments [12]) or vehicle (1:1:18, DMSO /Alkamuls [Rhodia, Cranbury, NJ, USA] / PBS). Note that the affinity of WIN55212-2 for zebra finch brain membranes (Ki 563.3 nM [12]) is similar to that reported for mammalian species [7]. From 50 to 100 days of age, animals received daily treatments by i.m. injection into pectoralis of 50 ml 30 min prior to the beginning of 14-h light cycles as described previously [12]. Songs were recorded at adulthood (110 days of age) and 10 bouts from each animal were randomly selected for analysis by two independent observers blind to treatment. Data were pooled prior to analysis as described previously [6]. A second experiment examined effects of the same 50-day treatment regimen in groups of adult male zebra finches (n54). Treated birds learned significantly fewer notes than their vehicle-injected siblings (Fig. 1a). While treated birds developed mature, crystallized song patterns, their songs were significantly less well stereotyped than controls when scored according to a standard method [9] (Fig. 1b). Lower stereotypy was often associated with repetitive note production (compare Fig. 2a and b). Average note duration did not vary as a function of treatment (Table 1). Treatment differences were also not associated with changes in the volume of two song-control brain regions involved with the learning and production of song (the higher vocal center, HVC, and the robust nucleus of the archistriatum, RA, see Table 1). Treatment of adult birds had no effect
0165-3806 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0165-3806(03)00061-0
K. Soderstrom, F. Johnson / Developmental Brain Research 142 (2003) 215–217
216
Fig. 1. Effects of cannabinoid exposure during sensory-motor learning on song patterns produced at adulthood. Individuals from sibling pairs raised by the same adult tutor were randomly assigned to treatment (1 mg / kg WIN55212-2) or vehicle control groups (n57 pairs). Treatments were given from 50 to 100 days of age as described in the text. Songs were recorded at adulthood (110 days of age) and 10 bouts from each animal were randomly selected for analysis by two independent observers blind to treatment. Shown are means6S.E.M. (a) Cannabinoid-treated birds learned significantly fewer note types (mean55.660.5 vs. 7.860.3, *P50.025, paired t-test, two-tailed). (b) Songs of cannabinoid-treated birds were less well-stereotyped (mean scores5 0.5660.06 vs. 0.8160.02, **P50.004, paired t-test, two-tailed). (c) Mean note duration did not vary as a function of treatment (mean duration [ms]5177621 vs. 176613, p50.97, paired t-test, two-tailed).
Fig. 2. Representative adult song patterns produced by a sibling pair. (a) Vehicle-treated animal B379 and (b) WIN55212-2-treated animal B379. Shown are sonograms produced at 110 days of age. Introductory notes are indicated by ‘i’. Song notes are indicated alphabetically according to order of production by the control animal. A unique note type (with similarities to notes ‘h’ and ‘f’) produced by the WIN55212-2-treated animal is indicated by ‘*’. Evident are fewer note types and decreased stereotypy in the song pattern produced by the WIN55212-2-treated sibling.
Table 1 Effect of WIN55212-2 on song quality and volume of related brain regions Treatment group
Number of animals
HVC volume (mm 3 )
RA volume (mm 3 )
Stereotype score
Note duration (ms)
Notes produced
Juvenile VEH-Tx Juvenile WIN-Tx Adult VEH-Tx Adult WIN-Tx (Pre) Adult WIN-Tx (Post)
7 7 4 4 4
0.4360.04 0.3960.02 0.2660.06
0.2460.02 0.2460.01 0.2160.03
0.8160.02 0.5660.06**
176612 177621
7.860.3 5.660.5*
0.2660.02
0.1960.02
0.7560.07 0.7760.08
178621 179621
5.2560.75 5.2560.75
Shown are means6S.E.M., *P,0.05, **P,0.01, paired t-test, two-tailed. VEH-Tx550 daily vehicle injections, WIN-Tx550 daily WIN55212-2 injections. Juveniles were treated from 50 to 100 days of age. Adults were .90 days of age before initiation of treatment. Songs were recorded 10 days after treatments ended. Two animals with four-note songs contributed to low ‘Notes produced’ in Adult WIN-Tx group.
K. Soderstrom, F. Johnson / Developmental Brain Research 142 (2003) 215–217
on song region morphology or on the structure of alreadylearned song patterns, demonstrating that the effects of cannabinoid treatment were specific to the learning and development of song in juveniles (Table 1). Altered vocal learning in juveniles may be the result of cannabinoid effects on neural development, mechanisms of learning, or both. For example, our findings are consistent with developmental changes in CNS gene expression in CB1 deficient mice [13], and underscore the importance of appreciating neurochemical differences between developing and mature animals (cf. Ref. [10]). Cannabinoid-effected reductions in the number of notes learned and repetitive note production may be independent effects on processes related to memory and timing, both of which are wellknown to be influenced by cannabinoid agonists [1]. In the case of note timing, a combination of distinct high-density CB1 receptor expression [11] and electrophysiological evidence for a role for HVC in note sequencing [15] implicates this song region for possible involvement. Interestingly, cannabinoid reductions in the number of notes learned is similar in effect to imposing a period of syringeal paralysis late in the sensory-motor stage of song learning [8]. This raises the possibility of a common mechanism that may involve a practice effect on integration and storage of auditory and motor information generated during song rehearsal, and comparison of this information with patterns retrieved from memory. Because cannabinoid effects on memory processes in mammalian species appear to be specifically related to changes in activity in hippocampus and striatum [4,5], it is possible that identification of brain regions mediating cannabinoid effects on song learning will provide insight to the physiological substrate(s) for memorized vocal patterns, and mechanisms important for their storage.
Acknowledgements This work was supported by NIH grants DC02035 to F.J. and DA05986 and DA14693 to K.S.
217
References [1] I.B. Adams, B.R. Martin, Cannabis: pharmacology and toxicology in animals and humans, Addiction 91 (1996) 1585–1614. [2] A.J. Doupe, P.K. Kuhl, Birdsong and human speech: common themes and mechanisms, Annu. Rev. Neurosci. 22 (1999) 567–631. [3] M.R. Elphick, M. Egertova, The neurobiology and evolution of cannabinoid signalling, Phil. Trans. R. Soc. Lond. B Biol. Sci. 356 (2001) 381–408. [4] G.L. Gerdeman, J. Ronesi, D.M. Lovinger, Postsynaptic endocannabinoid release is critical to long-term depression in the striatum, Nat. Neurosci. 5 (2002) 446–451. [5] R.E. Hampson, S.A. Deadwyler, Cannabinoids reveal the necessity of hippocampal neural encoding for short-term memory in rats, J. Neurosci. 20 (2000) 8932–8942. [6] F. Johnson, K. Soderstrom, O. Whitney, Quantifying song bout production during zebra finch sensory-motor learning suggests a sensitive period for vocal practice, Behav. Brain Res. 131 (2002) 57–65. [7] R.G. Pertwee, Pharmacology of cannabinoid CB1 and CB2 receptors, Pharmacol. Ther. 74 (1997) 129–180. [8] C.L. Pytte, R.A. Suthers, Sensitive period for sensorimotor integration during vocal motor learning, J. Neurobiol. 42 (2000) 172–189. [9] C. Scharff, F. Nottebohm, A comparative study of the behavioral deficits following lesions of various parts of the zebra finch song system: implications for vocal learning, J. Neurosci. 11 (1991) 2896–2913. [10] N.L. Schramm, R.E. Egli, D.G. Winder, LTP in the mouse nucleus accumbens is developmentally regulated, Synapse 45 (2002) 213– 219. [11] K. Soderstrom, F. Johnson, CB1 cannabinoid receptor expression in brain regions associated with zebra finch song control, Brain Res. 857 (2000) 151–157. [12] K. Soderstrom, F. Johnson, The zebra finch CB1 cannabinoid receptor: pharmacology and in vivo and in vitro effects of activation, J. Pharmacol. Exp. Ther. 297 (2001) 189–197. [13] H. Steiner, T.I. Bonner, A.M. Zimmer, S.T. Kitai, A. Zimmer, Altered gene expression in striatal projection neurons in CB1 cannabinoid receptor knockout mice, Proc. Natl. Acad. Sci. USA 96 (1999) 5786–5790. [14] J.M. Sullivan, Cellular and molecular mechanisms underlying learning and memory impairments produced by cannabinoids, Learn. Mem. 7 (2000) 132–139. [15] E.T. Vu, M.E. Mazurek, Y.C. Kuo, Identification of a forebrain motor programming network for the learned song of zebra finches, J. Neurosci. 14 (1994) 6924–6934.
Short communication
Cannabinoid exposure alters learning of zebra finch vocal patterns Ken Soderstrom a , *, Frank Johnson b a
Department of Pharmacology and Toxicology, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA b Department of Psychology and Program in Neuroscience, Florida State University, Tallahassee, FL 32306, USA Accepted 18 February 2003
Abstract Using a well-established songbird model of juvenile vocal development, we have found that daily cannabinoid exposure at modest dosages alters sensory-motor vocal learning. Adult exposure did not change song that had already been learned. Our results demonstrate the potential for cannabinoid exposure to produce distinct effects during post-natal CNS development. 2003 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Learning and memory: pharmacology Keywords: Cannabinoid; Vocal learning; Post-natal CNS development
Similar to human language acquisition [2], vocal learning in song birds requires coordinated perception and motor production of memorized vocal patterns. Because these types of cognitive processes are influenced by cannabinoid receptor activation [3,14], cannabinoid signaling may be relevant to vocal development, a hypothesis supported by dense CB1 cannabinoid receptor expression in brain regions important for zebra finch song learning and production [11] and CB1-dependent reductions in singing behavior [12]. Here we show that daily cannabinoid exposure at modest dosages for 50 days alters learning of song by juvenile birds, while the same treatment in adult birds is without effect on the pattern of already-learned song. Subjects were male zebra finches raised in our breeding aviaries. Seven pairs of sibling or cross-fostered (2062 days of age) fledgling zebra finches were raised together by the same male tutor (so that the same song pattern was memorized) until 50 days of age. Animals were then housed singly in visual isolation and individuals from each pair were randomly assigned to receive either injections of the cannabinoid agonist WIN55212-2 (1 mg / kg, a transiently effective [2 h] dosage determined in prior experi*Corresponding author. Tel.: 11-252-816-2742; fax: 11-252-8163203. E-mail address: [email protected] (K. Soderstrom).
ments [12]) or vehicle (1:1:18, DMSO /Alkamuls [Rhodia, Cranbury, NJ, USA] / PBS). Note that the affinity of WIN55212-2 for zebra finch brain membranes (Ki 563.3 nM [12]) is similar to that reported for mammalian species [7]. From 50 to 100 days of age, animals received daily treatments by i.m. injection into pectoralis of 50 ml 30 min prior to the beginning of 14-h light cycles as described previously [12]. Songs were recorded at adulthood (110 days of age) and 10 bouts from each animal were randomly selected for analysis by two independent observers blind to treatment. Data were pooled prior to analysis as described previously [6]. A second experiment examined effects of the same 50-day treatment regimen in groups of adult male zebra finches (n54). Treated birds learned significantly fewer notes than their vehicle-injected siblings (Fig. 1a). While treated birds developed mature, crystallized song patterns, their songs were significantly less well stereotyped than controls when scored according to a standard method [9] (Fig. 1b). Lower stereotypy was often associated with repetitive note production (compare Fig. 2a and b). Average note duration did not vary as a function of treatment (Table 1). Treatment differences were also not associated with changes in the volume of two song-control brain regions involved with the learning and production of song (the higher vocal center, HVC, and the robust nucleus of the archistriatum, RA, see Table 1). Treatment of adult birds had no effect
0165-3806 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0165-3806(03)00061-0
K. Soderstrom, F. Johnson / Developmental Brain Research 142 (2003) 215–217
216
Fig. 1. Effects of cannabinoid exposure during sensory-motor learning on song patterns produced at adulthood. Individuals from sibling pairs raised by the same adult tutor were randomly assigned to treatment (1 mg / kg WIN55212-2) or vehicle control groups (n57 pairs). Treatments were given from 50 to 100 days of age as described in the text. Songs were recorded at adulthood (110 days of age) and 10 bouts from each animal were randomly selected for analysis by two independent observers blind to treatment. Shown are means6S.E.M. (a) Cannabinoid-treated birds learned significantly fewer note types (mean55.660.5 vs. 7.860.3, *P50.025, paired t-test, two-tailed). (b) Songs of cannabinoid-treated birds were less well-stereotyped (mean scores5 0.5660.06 vs. 0.8160.02, **P50.004, paired t-test, two-tailed). (c) Mean note duration did not vary as a function of treatment (mean duration [ms]5177621 vs. 176613, p50.97, paired t-test, two-tailed).
Fig. 2. Representative adult song patterns produced by a sibling pair. (a) Vehicle-treated animal B379 and (b) WIN55212-2-treated animal B379. Shown are sonograms produced at 110 days of age. Introductory notes are indicated by ‘i’. Song notes are indicated alphabetically according to order of production by the control animal. A unique note type (with similarities to notes ‘h’ and ‘f’) produced by the WIN55212-2-treated animal is indicated by ‘*’. Evident are fewer note types and decreased stereotypy in the song pattern produced by the WIN55212-2-treated sibling.
Table 1 Effect of WIN55212-2 on song quality and volume of related brain regions Treatment group
Number of animals
HVC volume (mm 3 )
RA volume (mm 3 )
Stereotype score
Note duration (ms)
Notes produced
Juvenile VEH-Tx Juvenile WIN-Tx Adult VEH-Tx Adult WIN-Tx (Pre) Adult WIN-Tx (Post)
7 7 4 4 4
0.4360.04 0.3960.02 0.2660.06
0.2460.02 0.2460.01 0.2160.03
0.8160.02 0.5660.06**
176612 177621
7.860.3 5.660.5*
0.2660.02
0.1960.02
0.7560.07 0.7760.08
178621 179621
5.2560.75 5.2560.75
Shown are means6S.E.M., *P,0.05, **P,0.01, paired t-test, two-tailed. VEH-Tx550 daily vehicle injections, WIN-Tx550 daily WIN55212-2 injections. Juveniles were treated from 50 to 100 days of age. Adults were .90 days of age before initiation of treatment. Songs were recorded 10 days after treatments ended. Two animals with four-note songs contributed to low ‘Notes produced’ in Adult WIN-Tx group.
K. Soderstrom, F. Johnson / Developmental Brain Research 142 (2003) 215–217
on song region morphology or on the structure of alreadylearned song patterns, demonstrating that the effects of cannabinoid treatment were specific to the learning and development of song in juveniles (Table 1). Altered vocal learning in juveniles may be the result of cannabinoid effects on neural development, mechanisms of learning, or both. For example, our findings are consistent with developmental changes in CNS gene expression in CB1 deficient mice [13], and underscore the importance of appreciating neurochemical differences between developing and mature animals (cf. Ref. [10]). Cannabinoid-effected reductions in the number of notes learned and repetitive note production may be independent effects on processes related to memory and timing, both of which are wellknown to be influenced by cannabinoid agonists [1]. In the case of note timing, a combination of distinct high-density CB1 receptor expression [11] and electrophysiological evidence for a role for HVC in note sequencing [15] implicates this song region for possible involvement. Interestingly, cannabinoid reductions in the number of notes learned is similar in effect to imposing a period of syringeal paralysis late in the sensory-motor stage of song learning [8]. This raises the possibility of a common mechanism that may involve a practice effect on integration and storage of auditory and motor information generated during song rehearsal, and comparison of this information with patterns retrieved from memory. Because cannabinoid effects on memory processes in mammalian species appear to be specifically related to changes in activity in hippocampus and striatum [4,5], it is possible that identification of brain regions mediating cannabinoid effects on song learning will provide insight to the physiological substrate(s) for memorized vocal patterns, and mechanisms important for their storage.
Acknowledgements This work was supported by NIH grants DC02035 to F.J. and DA05986 and DA14693 to K.S.
217
References [1] I.B. Adams, B.R. Martin, Cannabis: pharmacology and toxicology in animals and humans, Addiction 91 (1996) 1585–1614. [2] A.J. Doupe, P.K. Kuhl, Birdsong and human speech: common themes and mechanisms, Annu. Rev. Neurosci. 22 (1999) 567–631. [3] M.R. Elphick, M. Egertova, The neurobiology and evolution of cannabinoid signalling, Phil. Trans. R. Soc. Lond. B Biol. Sci. 356 (2001) 381–408. [4] G.L. Gerdeman, J. Ronesi, D.M. Lovinger, Postsynaptic endocannabinoid release is critical to long-term depression in the striatum, Nat. Neurosci. 5 (2002) 446–451. [5] R.E. Hampson, S.A. Deadwyler, Cannabinoids reveal the necessity of hippocampal neural encoding for short-term memory in rats, J. Neurosci. 20 (2000) 8932–8942. [6] F. Johnson, K. Soderstrom, O. Whitney, Quantifying song bout production during zebra finch sensory-motor learning suggests a sensitive period for vocal practice, Behav. Brain Res. 131 (2002) 57–65. [7] R.G. Pertwee, Pharmacology of cannabinoid CB1 and CB2 receptors, Pharmacol. Ther. 74 (1997) 129–180. [8] C.L. Pytte, R.A. Suthers, Sensitive period for sensorimotor integration during vocal motor learning, J. Neurobiol. 42 (2000) 172–189. [9] C. Scharff, F. Nottebohm, A comparative study of the behavioral deficits following lesions of various parts of the zebra finch song system: implications for vocal learning, J. Neurosci. 11 (1991) 2896–2913. [10] N.L. Schramm, R.E. Egli, D.G. Winder, LTP in the mouse nucleus accumbens is developmentally regulated, Synapse 45 (2002) 213– 219. [11] K. Soderstrom, F. Johnson, CB1 cannabinoid receptor expression in brain regions associated with zebra finch song control, Brain Res. 857 (2000) 151–157. [12] K. Soderstrom, F. Johnson, The zebra finch CB1 cannabinoid receptor: pharmacology and in vivo and in vitro effects of activation, J. Pharmacol. Exp. Ther. 297 (2001) 189–197. [13] H. Steiner, T.I. Bonner, A.M. Zimmer, S.T. Kitai, A. Zimmer, Altered gene expression in striatal projection neurons in CB1 cannabinoid receptor knockout mice, Proc. Natl. Acad. Sci. USA 96 (1999) 5786–5790. [14] J.M. Sullivan, Cellular and molecular mechanisms underlying learning and memory impairments produced by cannabinoids, Learn. Mem. 7 (2000) 132–139. [15] E.T. Vu, M.E. Mazurek, Y.C. Kuo, Identification of a forebrain motor programming network for the learned song of zebra finches, J. Neurosci. 14 (1994) 6924–6934.