Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 Jan 16.
Published in final edited form as: Neuroscience. 2003;116(1):19–22. doi: 10.1016/s0306-4522(02)00560-2

COCAINE-INDUCED PROLIFERATION OF DENDRITIC SPINES IN NUCLEUS ACCUMBENS IS DEPENDENT ON THE ACTIVITY OF CYCLIN-DEPENDENT KINASE-5

S D Norrholm a,*, J A Bibb b,c, E J Nestler c, C C Ouimet a, J R Taylor d, P Greengard b
PMCID: PMC4296576  NIHMSID: NIHMS648778  PMID: 12535933

Abstract

Repeated exposure to cocaine produces an enduring increase in dendritic spine density in adult rat nucleus accumbens. It has been shown previously that chronic cocaine administration increases the expression of cyclin-dependent kinase-5 in this brain region and that this neuronal protein kinase regulates cocaine-induced locomotor activity. Moreover, cyclin-dependent kinase-5 has been implicated in neuronal function and synaptic plasticity. Therefore, we studied the involvement of this enzyme in cocaine’s effect on dendritic spine density. Adult male rats, receiving intra-accumbens infusion of the cyclin-dependent kinase-5 inhibitor roscovitine or saline, were administered a 28-day cocaine treatment regimen. Animals were killed 24–48 h after the final cocaine injection and their brains removed and processed for Golgi–Cox impregnation. Our findings demonstrate that roscovitine attenuates cocaine-induced dendritic spine outgrowth in nucleus accumbens core and shell and such inhibition reduces spine density in nucleus accumbens shell of control animals. These data indicate that cyclin-dependent kinase-5 is involved in regulation of, as well as cocaine-induced changes in, dendritic spine density.

Keywords: psychostimulant abuse, dendritic morphology, synaptic plasticity

Long-term exposure to psychostimulant drugs produces enduring neuronal alterations in intracellular signaling pathways and structural changes in dendritic morphology (Nestler, 2001). For example, repeated exposure to cocaine increases spine density on dendrites of dopaminoceptive medium spiny neurons in the shell (but not core) division of nucleus accumbens (NAc) (Robinson and Kolb, 1999; Robinson et al., 2001) and increases the expression of several transcription factors that mediate gene expression in these neurons (Nestler, 2001). Recently, cyclin-dependent kinase-5 (Cdk5) was identified as a downstream target of the transcription factor ΔFosB which is persistently expressed in NAc of mice repeatedly treated with cocaine. Overexpression of ΔFosB increases Cdk5 expression and activity (Kelz et al., 1999; Bibb et al., 2001). Cdk5 is a protein serine/threonine kinase that regulates neurite outgrowth (Dhavan and Tsai, 2001) and modulates dopamine signaling (Bibb et al., 1999a). Interestingly, acute inhibition of Cdk5 activity in NAc potentiates the locomotor effects of chronic cocaine (Bibb et al., 2001). The purpose of the present study was to determine if this neuronal protein kinase is involved in cocaine-induced dendritic spine proliferation in rat NAc.

The potent Cdk5 inhibitor roscovitine (or vehicle) was bilaterally infused into NAc shell via ALZET mini-pump. The effect of a 4-week regimen of repeated i.p. injections of cocaine (or saline) on dendritic spine density was assessed by a modified Golgi staining technique. Detailed anatomical features along dendrites were easily discernible and dendritic spines were readily resolved (Fig. 1). Repeated cocaine administration increased dendritic spine density on medium spiny neurons in the NAc shell (16.2%, P=0.001) and core (16.7%, P=0.037) in control animals receiving ALZET mini-pump infusions of phosphate-buffered saline (PBS) (Fig. 2 panel a vs. b; panels e and f). This increase was blocked in both shell (P<0.001; Fig. 2 panel b vs. d) and core (P=0.011) when roscovitine, a potent Cdk5 inhibitor, was infused by ALZET mini-pump (Fig. 2, panels e and f). Interestingly, intra-accumbens infusion of roscovitine in animals not receiving i.p. cocaine injections reduced spine density in the shell (12.4%, P=0.014) (Fig. 2 panel a vs. c; panel e). There were no significant differences in mean dendritic length in any of the experimental conditions; this indicates that the observed changes in spine density do not result from dendritic lengthening or shortening. Together, these data demonstrate that the ability of chronic cocaine to increase NAc spine density is dependent on the activity of Cdk5.

Fig. 1.

Fig. 1

Open in a new tab

Representative laser confocal photomicrograph of Golgi-impregnated medium spiny neuron from nucleus accumbens shell. Scale bar=10 µm.

Fig. 2.

Fig. 2

Open in a new tab

Representative laser confocal photomicrographs of dendritic processes from NAc shell of rats administered 4 week regimen of daily cocaine or saline injections while receiving intra-accumbens infusion of Cdk5 inhibitor roscovitine or PBS. Daily i.p. drug treatment is indicated above the panels and mini-pump perfusate is indicated to the left of each row in the figure (ROSC=roscovitine). Panels a–d illustrate dendritic segments from NAc shell after treatment with (a) saline + PBS, (b) cocaine + PBS, (c) saline + ROSC, or (d) cocaine + ROSC. Scale bar=10 µm. Panels e and f illustrate mean dendritic spine density in NAc (e) shell and (f) core of animals treated with chronic cocaine (Coc) or saline (Sal) while receiving intra-accumbens infusion of ROSC or PBS. *P<0.05 vs. PBS/Saline, +P<0.05 vs. PBS/Cocaine, n=5 animals per treatment group.

Previous studies have demonstrated that Cdk5 co-localizes with presynaptic proteins involved in exocytosis and synaptic transmission (Shuang et al., 1998; Fletcher et al., 1999) as well as post-synaptic D1 receptor-mediated second messenger cascades (Bibb et al., 1999a). Cdk5 co-localizes with DARPP-32 (Dopamine and cyclic AMP [cAMP] Regulated Phosphoprotein, Mr 32 kDa) in cell bodies and dendritic shafts in NAc (Bibb et al., 2001). We also examined whether Cdk5 is expressed presynaptically in NAc using laser confocal microscopy and fluorescently labeled secondary antibodies. Cdk5 and synapsin 1 were found to be co-localized to axon terminals in NAc (Fig. 3) indicating that Cdk5 is expressed both pre- and post-synaptically.

Fig. 3.

Fig. 3

Open in a new tab

NAc of adult male rat that has been double-immunostained for Cdk5 (red) and synapsin I (green). The distribution of Cdk5 is broader than that of synapsin I but most synapsin I-containing puncta also contain Cdk5 and are labeled yellow (arrows). Prominent colocalization (yellow) observed in many axon terminals demonstrates presynaptic expression of Cdk5. Scale bar=1 µm.

The data presented here demonstrate that infusion of the Cdk5 inhibitor roscovitine into NAc shell: (1) attenuates cocaine-induced elevations in dendritic spine density in NAc and (2) reduces spine density in the shell when administered to saline-injected controls. These two findings demonstrate a role for Cdk5 in increasing and maintaining dendritic spine density. Previous research has shown that the activity of Cdk5 is necessary for axonal migration and dendritic outgrowth during neurogenesis (Nikolic et al., 1996). This is the first evidence, however, suggesting that Cdk5 activity regulates the outgrowth and maintenance of spines in adult neurons in vivo. In addition, we report increased spine density in both NAc core and shell 1–2 days after 28-day cocaine exposure, whereas at 24–25 days post-treatment this effect was seen in the shell only (Robinson and Kolb, 1999; Robinson et al., 2001).

The observed effects of the Cdk5 inhibitor roscovitine on spine density may reflect Cdk5 inhibition of several synaptic proteins expressed pre- and post-synaptically in NAc (Dhavan and Tsai, 2001). It is possible that Cdk5 is locally involved in spine outgrowth through an interaction with actin and its associated cytoskeletal proteins as in Cdk5-mediated axonal migration and dendritic morphogenesis (Humbert et al., 2000; Smith et al., 2001; Dhavan and Tsai, 2001). Presynaptically, Cdk5 binds to or phosphorylates several proteins that mediate vesicular exocytosis and synaptic transmission such as Munc-18, syntaxin 1A, and synapsin I (Matsubara et al., 1996; Fletcher et al., 1999). Thus, changes in Cdk5 activity within nerve terminals of NAc could alter neurotransmitter release that, in turn, could change spine density. Dendritic spines are highly responsive to fluctuations in dopaminergic and glutamatergic transmission (Meredith et al., 1995; Segal and Andersen, 2000; Robinson et al., 2001). Postsynaptically, Cdk5 can modulate D1/cAMP/PKA/DARPP-32 intracellular signaling and regulate NMDA receptor function (Bibb et al., 1999a,b; Li et al., 2001). Roscovitine may therefore impact cocaine-induced spine proliferation and normal spine maintenance by altering spine actin dynamics, intracellular signaling cascades, or glutamatergic and/or dopaminergic neurotransmission.

EXPERIMENTAL PROCEDURES

General methods were according to published procedures (Horger et al., 1999) and were performed in accordance with international guidelines for the ethical use of laboratory animals and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize animal suffering and the number of animals used. Male Sprague–Dawley (Camm, NJ, USA) rats with food and water available ad libitum were housed in pairs in a climate-controlled (12-h light/dark cycle) colony. Osmotic mini-pumps (ALZET model #2004, 0.25 µl/h) were surgically placed s.c. between the scapulae and were connected via plastic tubing (PE 60) to bilateral L-shaped cannulae (Plastics One, #3220PD) terminating in NAc. Stereotaxic coordinates for the NAc were anterior-posterior +1.7 mm from bregma, medial-lateral ±1.0 mm and dorsoventral −7.4 mm from dura (Paxinos and Watson 1986). Pumps were loaded with roscovitine (gift from Dr Laurent Meijer, Centre National de la Recherche Scientifique) or vehicle solution (10 mM sodium phosphate, pH 7.4, 0.9% NaCl, 50% dimethyl sulfoxide) and delivered 40 nM/day/side (microinfused in 0.5 µl over a 2-min period) into NAc shell for 28 days. The roscovitine dosage schedule was based on previous studies using intracerebral infusions of roscovitine and cAMP analogues (Punch et al., 1997; Bibb et al., 2001). Cocaine hydrochloride (15 mg/kg i.p., dissolved in 0.9% saline) was given daily.

Animals were perfused transcardially with 4% formaldehyde under deep anesthesia with sodium pentobarbital (60 mg/kg) 24–48 h after the last cocaine injection. Brains were prepared for Golgi impregnation according to previous methods (Norrholm and Ouimet, 2000).

Dendritic spines were counted on 1250× camera lucida images that included all spines observable in each focal plane occupied by the dendrite. Dendritic spines were counted along dendritic processes extending from the soma of fully impregnated medium spiny neurons in both shell and core of NAc (Fig. 1). Twenty dendritic segments (10 in each cerebral hemisphere; 50–100 µm in length) were examined in each NAc division for each rat (thus, a total of 100 dendrites were analyzed per NAc division per treatment condition). The 20 dendritic segments analyzed in each region of each animal represented three to five different neurons per cerebral hemisphere. When significant changes in dendritic spine density were observed, camera lucida images and the Zeiss CLSM measurement program were used to quantify dendritic length; this analysis was necessary to rule out changes in spine density resulting from increased or decreased dendritic length. Photomicrographs were obtained with helium-neon 633 laser and Zeiss 410 confocal laser scanning microscope. Experimenters were blind to treatment conditions as tissue slides were coded throughout the entire process of cell identification, counting, and subsequent data analysis. Data were analyzed using two-factor analysis of variance and Tukey post-hoc pairwise comparison tests (P<0.05).

Immunocytochemistry was performed as previously described (Bibb et al., 2001) with a monoclonal antibody against synapsin I and a polyclonal antibody against Cdk5 (C-8, Santa Cruz Biotechnologies, Inc., Santa Cruz, CA, USA).

Acknowledgements

We thank Valyphone Phantharagsny, Victoria Stewart, Charles Badland, Amanda Clark, and Martin Knowles for their technical assistance. This work was supported by NARSAD young investigators award (J.A.B.) and grants from the National Institutes on Drug Abuse (E.J.N., J.R.T., and P.G.) and Mental Health (P.G.).

Abbreviations

Cdk5

cyclin-dependent kinase-5

Coc

cocaine

DARPP-32

dopamine and cyclic AMP [cAMP] regulated phosphoprotein, Mr 32 kDA

NAc

nucleus accumbens

PBS

phosphate buffered saline

ROSC

roscovitine

Sal

saline

REFERENCES

  1. Bibb JA, Chen J, Taylor JR, Svenningson P, Nishi A, Snyder GL, Yan Z, Sagawa ZK, Ouimet CC, Nairn AC, Nestler EJ, Greengard P. Effects of chronic exposure to cocaine are regulated by the neuronal protein Cdk5. Nature. 2001;410:376–380. doi: 10.1038/35066591. [DOI] [PubMed] [Google Scholar]
  2. Bibb JA, Nishi A, O’Callaghan JP, Ule J, Lan M, Snyder GL, Horiuchi A, Saito T, Hisanaga T, Czernik AJ, Nairn AC, Greengard PG. Phosphorylation of Protein Phosphatase Inhibitor-1 by Cdk5. J Biol Chem. 1999b;276:14490–14497. doi: 10.1074/jbc.M007197200. [DOI] [PubMed] [Google Scholar]
  3. Bibb JA, Snyder GL, Nishi A, Yan Z, Meijer L, Fienberg AA, Tsai L, Kwon YT, Girault J, Czernik AJ, Huganir RL, Hemmings HC, Nairn A, Greengard P. Phosphorylation of DARPP-32 by Cdk5 modulates dopamine signalling in neurons. Nature. 1999a;402:669–671. doi: 10.1038/45251. [DOI] [PubMed] [Google Scholar]
  4. Dhavan R, Tsai LH. A decade of CDK5. Nat Rev Mol Cell Biol. 2001;2:749–759. doi: 10.1038/35096019. [DOI] [PubMed] [Google Scholar]
  5. Fletcher AI, Shuang R, Giovannucci DR, Zhang L, Bittner MA, Stuenkel EL. Regulation of exocytosis by cyclin-dependent kinase 5 via phosphorylation of Munc18. J Biol Chem. 1999;274:4027–4035. doi: 10.1074/jbc.274.7.4027. [DOI] [PubMed] [Google Scholar]
  6. Horger BA, Iyasere CA, Berhow MT, Messer CJ, Nestler EJ, Taylor JR. Enhancement of locomotor activity and conditioned reward to cocaine by brain-derived neurotrophic factor. J Neurosci. 1999;19:4110–4122. doi: 10.1523/JNEUROSCI.19-10-04110.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Humbert S, Dhavan R, Tsai L. p39 activates cdk5 in neurons, and is associated with the actin cytoskeleton. J Cell Sci. 2000;113:975–983. doi: 10.1242/jcs.113.6.975. [DOI] [PubMed] [Google Scholar]
  8. Kelz MB, Chen J, Carlezon WB, Jr, Whisler K, Gilden L, Beckmann AM, Steffen C, Zhang YJ, Marotti L, Self DW. Expression of the transcription factor delta fos B in the brain controls sensitivity to cocaine. Nature. 1999;401:272–276. doi: 10.1038/45790. [DOI] [PubMed] [Google Scholar]
  9. Li BS, Sun MK, Zhang L, Takahashi S, Ma W, Vinade L, Kulkarni AB, Brady RO, Pant HC. Regulation of NMDA receptors by cyclin-dependent kinase 5. Proc Natl Acad Sci USA. 2001;98:12742–12747. doi: 10.1073/pnas.211428098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Matsubara M, Kusubata M, Ishiguro K, Uchida T, Titani K, Taniguchi H. Site-specific phosphorylation of synapsin I by mitogen-activated protein kinase and Cdk5 and its effects on physiological functions. J Biol Chem. 1996;271:21108–21113. doi: 10.1074/jbc.271.35.21108. [DOI] [PubMed] [Google Scholar]
  11. Meredith GE, Ypuma P, Zahm DS. Effects of dopamine depletion on the morphology of medium spiny neurons in the shell and core of the rat nucleus accumbens. J Neurosci. 1995;15:3808–3820. doi: 10.1523/JNEUROSCI.15-05-03808.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Nestler EJ. Molecular basis of long-term plasticity underlying drug addiction. Nat Rev Neurosci. 2001;2:119–128. doi: 10.1038/35053570. [DOI] [PubMed] [Google Scholar]
  13. Nikolic M, Dudek H, Kwon YT, Ramos YF, Tsai LH. The cdk5/p35 kinase is essential for neurite outgrowth during neuronal differentiation. Genes Dev. 1996;10:816–825. doi: 10.1101/gad.10.7.816. [DOI] [PubMed] [Google Scholar]
  14. Norrholm SD, Ouimet CC. Repeated fluoxetine administration prevents age-associated dendritic spine proliferation in juvenile rat hippocampus. Brain Res. 2000;883:205–215. doi: 10.1016/s0006-8993(00)02909-7. [DOI] [PubMed] [Google Scholar]
  15. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. Orlando, FL: Academic Press; 1986. [Google Scholar]
  16. Punch L, Self D, Nestler EJ, Taylor JR. Opposite modulation of opiate withdrawal behaviors upon microinfusion of a protein kinase A inhibitor versus activator into the locus coeruleus or periaquiductal gray. J Neurosci. 1997;17:8528–8535. doi: 10.1523/JNEUROSCI.17-21-08520.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Robinson TE, Gorny G, Mitton E, Kolb B. Cocaine self-administration alters the morphology of dendrites and dendritic spines in the nucleus accumbens and neocortex. Synapse. 2001;39:257–266. doi: 10.1002/1098-2396(20010301)39:3<257::AID-SYN1007>3.0.CO;2-1. [DOI] [PubMed] [Google Scholar]
  18. Robinson TE, Kolb B. Alterations in the morphology of dendrites and dendritic spines in the nucleus accumbens and prefrontal cortex following repeated treatment with amphetamine or cocaine. Eur J Pharmacol. 1999;11:1598–1604. doi: 10.1046/j.1460-9568.1999.00576.x. [DOI] [PubMed] [Google Scholar]
  19. Segal M, Andersen P. Dendritic spines shaped by synaptic activity. Curr Opin Neurobiol. 2000;10:582–586. doi: 10.1016/s0959-4388(00)00123-9. [DOI] [PubMed] [Google Scholar]
  20. Shuang R, Zhang L, Fletcher A, Groblewski GE, Pevsner J, Steunkel EL. Regulation of Munc-18/syntaxin 1A interaction by cyclin-dependent kinase 5 in nerve endings. J Biol Chem. 1998;273:4957–4966. doi: 10.1074/jbc.273.9.4957. [DOI] [PubMed] [Google Scholar]
  21. Smith DS, Greer PL, Tsai LH. Cdk5 on the brain. Cell Growth Differ. 2001;12:277–283. [PubMed] [Google Scholar]

RESOURCES