Chronic Cocaine Prevents Depotentiation at Corticostriatal Synapses

ORIGINAL ARTICLES

Chronic Cocaine Prevents Depotentiation at Corticostriatal Synapses Diego Centonze, Cinzia Costa, Silvia Rossi, Chiara Prosperetti, Antonio Pisani, Alessandro Usiello, Giorgio Bernardi, Nicola B. Mercuri, and Paolo Calabresi Background: The advanced stages of addiction are characterized by compulsive drug-seeking and drug-taking behaviors despite the loss of the hedonic effect of drug consumption. A pathology of habit forming systems might underlie these features of addiction. Methods: We have compared use-dependent plasticity of corticostriatal synapses in saline- and cocaine-treated rats by means of single neuron electrophysiological recordings. Results: High-frequency stimulation of cortical afferents induced long-term potentiation (LTP) of corticostriatal synapses in treated and untreated animals. Saline- and acute– cocaine-treated rats, however, showed synaptic depotentiation in response to subsequent low-frequency stimulation of the same pathway, whereas chronic cocaine-treated animals were refractory to this process. Depotentiation was also absent in control slices bathed with cocaine, dopamine, or with the D1 receptor agonist SKF38393. The effect of cocaine on depotentiation was prevented by D1 but not D2 dopamine receptor antagonists and was mimicked by pharmacological inhibition of cyclin-dependent kinase 5, to enhance D1-receptor–associated intracellular signaling. Conclusions: These results provide the first evidence that cocaine blocks the reversal of LTP in brain circuits. This alteration might be important for the persistence of addictive behavior despite efforts to abstain. Key Words: Addiction, depotentiation, dopamine, electrophysiology, habit, LTP

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ompelling evidence indicates that addictive drugs produce their behavioral effects by abusing the molecular mechanisms of associative learning and reward (Berke and Hyman 2000; Robbins and Everitt 1999). Accordingly, cocaine exposure has been shown to induce synaptic plasticity in multiple neuronal systems of the reward circuitry. A single in vivo exposure to cocaine induces long-term potentiation (LTP) in dopamine neurons of the ventral tegmental area (Ungless et al 2001), whereas prolonged treatment induces long-term depression (LTD) in the nucleus accumbens (Thomas et al 2001). The first effect might be important for the early stages of the development of addiction (Ungless et al 2001), whereas the second one for the consolidation of behavioral sensitization to the drug (Thomas et al 2001). Synaptic adaptations in the dorsal part of the striatum underlie habit learning (Canales and Graybiel 2000; Jog et al 1999; Packard and Knowlton 2002) and might therefore be involved in the advanced stages of addiction, which are characterized by compulsive drug-taking behaviors despite serious negative consequences (Gerdeman et al 2003). Drug-induced habit learning consists of increasingly automatic motor actions particularly refractory to devaluation processes (Berke and Hyman 2000; Hyman and Malenka 2001; Robbins and Everitt 1999; Sutton et al 2003). In the striatum, therefore, and even when the outcome becomes undesirable, cocaine might consolidate synaptic adaptations responsible for habit formation. From the Clinica Neurologica (DC, SR, CP, AP, GB, NBM), Dipartimento di Neuroscienze, Università Tor Vergata; Centro Europeo per la Ricerca sul Cervello (CERC)/Fondazione Santa Lucia (DC, CC, SR, CP, AP, GB, NBM, PC), Rome; Behavioural Neuroscience Laboratory (AU), CEINGE - Biotecnologie Avanzate, Naples; Clinica Neurologica (CC, PC), Università di Perugia, Ospedale Silvestrini, Perugia, Italy. Address reprint requests to Diego Centonze, Clinica Neurologica, Dipartimento di Neuroscienze, Università Tor Vergata, Via Montpellier 1, 00133 Rome, Italy; E-mail: [email protected]. Received April 4, 2005; revised November 10, 2005; accepted November 28, 2005.

0006-3223/06/$32.00 doi:10.1016/j.biopsych.2005.11.018

In the attempt to clarify the physiological substrate of druginduced habit acquisition, we studied the effects of acute and chronic cocaine on LTP induction and maintenance at excitatory corticostriatal synapses. Long-term potentiation is a major candidate for the long-term effects of addictive drugs in the striatum, because, in vivo, it represents a synaptic substrate for rewardrelated habit learning (Reynolds et al 2001). This form of synaptic plasticity is induced in striatal spiny neurons both in vitro (Calabresi et al 1992, 2000b) and in vivo (Charpier and Deniau 1997; Reynolds et al 2001) after high-frequency stimulation (HFS) of glutamatergic corticostriatal terminals.

Methods and Materials Adult male Wistar rats (150 –250 g) were used for all the experiments. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Tor Vergata, Rome. Cocaine Treatment Rats were given intraperitoneal injections of either saline (.9% NaCl) or saline with cocaine (15 mg/kg). This dose of cocaine has been previously shown to induce both behavioral sensitization and changes in synaptic plasticity in the nucleus accumbens (Thomas et al 2001). Rats were housed with food and water ad libitum in a room with a 12-hour light/dark cycle and controlled (22°–23° C) temperature. Electrophysiological Experiments All the animals used for this study were killed by cervical dislocation. The saline- or cocaine-treated rats were killed 24 – 48 hours after the last intraperitoneal injection. Corticostriatal slices were prepared for electrophysiological recordings (Calabresi et al 1992, 2000b; Picconi et al 2003). Briefly, vibratome-cut coronal slices (200 –300 ␮m) were transferred to a recording chamber and submerged in a continuously flowing Krebs solution (35° C, 2–3 mL/min) gassed with 95% O2-5% CO2. The composition of the control solution was (in mmol/L): 126 NaCl, 2.5 KCl, 1.2 NaH2PO4, 2.4 CaCl2, 11 Glucose, 25 NaHCO3. Intracellular recording electrodes were filled with 2 mol/L KCl (30 – 60 mol/L⍀). Signals were recorded with the use of an BIOL PSYCHIATRY 2006;60:436 – 443 © 2006 Society of Biological Psychiatry

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D. Centonze et al Axoclamp 2A amplifier, displayed on a separate oscilloscope, and stored and analyzed on a digital system (pClamp 8, Axon Instruments, Union City, California). For synaptic stimulation, bipolar electrodes were used, located in the white matter between the cortex and the striatum to activate corticostriatal fibers. In distinct neurons, excitatory postsynaptic potentials (EPSPs) of similar amplitude were usually obtained with variable intensities of stimulation, mainly depending on the distance between the stimulating and recording sites. Magnesium ions were omitted from the medium to better disclose the N-methyl-D-aspartate (NMDA)–mediated component of the EPSP, and bicuculline (10 ␮mol/L) was added to block ␥-aminobuturic acid-A (GABAA)–mediated transmission. Under this experimental condition, HFS of corticostriatal fibers (3 trains, 3 sec duration, 100 Hz frequency, 20 sec interval) was used as an LTP-inducing protocol (Calabresi et al 1992, 2000b; Picconi et al 2003). Conversely, 10 min of low-frequency stimulation (LFS) (2 Hz) of corticostriatal fibers was applied to depotentiate LTP (Picconi et al 2003). Quantitative data on modifications of EPSPs are expressed as percentage of the controls, the latter representing the mean of responses recorded during a stable period (10 –20 min) before the repetitive (HFS) synaptic stimulation. To study miniature excitatory postsynaptic currents (mEPSCs), whole-cell patch-clamp recordings were performed. Electrodes (3–5 mol/L⍀) were filled with a solution containing (in mmol/L): K⫹-gluconate (125), NaCl (10), CaCl2, (1.0), MgCl2 (2.0), 1,2-bis (2-aminophenoxy) ethane-N,N,N,N-tetra-acetic acid (.5), HEPES (19), GTP (.3), and Mg-ATP (1.0), adjusted to pH 7.3 with KOH. Individual striatal neurons were visualized with a differential interference contrast (Nomarski) optical system, an Olympus BX50WI (Olympus Optical, Tokyo, Japan) non-inverted microscope with 40⫻ water immersion objective combined with an infrared filter, a monochrome CCD camera (COHU 4912), and a PC-compatible system for analysis of images and contrast enhancement (WinVision 2000, Delta Sistemi, Rome, Italy). Recording pipettes were advanced toward individual cells in the slice under positive pressure, whereas tight G⍀ seals were made by applying negative pressure. The membrane patch was then ruptured by suction and membrane current and potential monitored with an Axopatch 1D patch clamp amplifier and Clampex 8.1 software (Axon Instruments, Foster City, California). Whole-cell access resistances measured in voltage-clamp were in the range of 10 –30 mol/L⍀ before electronic compensation (60%– 80% was routinely used). Striatal spiny neurons were clamped at ⫺80 to ⫺85 mV, close to their resting membrane potential. The mEPSCs were recorded from striatal neurons in the presence of 10 ␮mol/L bicuculline and 1 ␮mol/L tetrodotoxin (TTX), to block GABAA-receptors and voltage-activated sodium channels, respectively. The mEPSCs were stored by using pClamp 8 (Axon Instruments) and analyzed offline with Mini Analysis 5.1 (Synaptosoft, Leonia, New Jersey) software. The detection threshold was set at twice the baseline noise. The fact that no false events would be identified was confirmed by visual inspection for each mEPSC. Values represent means ⫾ SEM. Only one cell per slice and ⬍6 neurons per animal were recorded. Only one neuron per rat was used for each type of experiment on synaptic plasticity. Unless otherwise specified, “n” refers to the number of cells. Student’s t test for unpaired and paired observations was used to compare two groups of means and analysis of variance was used for multiple comparisons.

Drugs were applied by dissolving them to the desired final concentration in the saline. Drugs were: bicuculline, butyrolactone I, cocaine, dopamine, L-sulpiride, roscovitine (Sigma, Italy); SCH23390, TTX (Tocris, Bristol, United Kingdom); SKF38393 (RBI, Italy). Conditioned Place Preference Experiments Cocaine-rewarding effects were evaluated in the conditioned place preference paradigm by using a biased procedure, as previously described (Acquas and Di Chiara 1994; Tzschentke 1998). Briefly, the protocol consists of three different phases: preconditioning, conditioning, and post-conditioning phases. During the preconditioning phase (days 1, 2, 3) the guillotine door separating the two compartments was kept lifted and each rat was given access to both compartments of the apparatus for 15 min. On day 3, the time spent by the rat in each compartment was recorded. The time recorded indicates the “unconditioned preference” of each rat for each compartment. In the conditioning phase (days 4 –10) the rats were administered cocaine 15 mg/kg and were then placed, for 25 min, in the “non-preferred” compartment. After an interval of about 4 hours, the rats were administered saline and placed in the other compartment. As a result of this conditioning phase, cocaine or saline were paired seven times to a specific compartment. During the post-conditioning phase (day 11), 24 hours after the last injection, the guillotine door was removed and the time spent by each rat in the drug-paired compartment was recorded during 15 min of observation. The difference in seconds between the time spent in the drug-paired compartment during the post-conditioning test and that spent in the preconditioning test was taken as a measure of the degree of conditioning induced by the drug.

Results Effects of Cocaine on Basic Properties of Striatal Neurons Resting membrane potential, input resistance, and pairedpulse ratio measured at 60 msec interstimulus interval were comparable in acute cocaine-, acute saline-, chronic cocaine-, and chronic saline-treated striatal neurons, which also exhibited similar tonic firing activity in response to the injection of depolarizing current (Figure 1). Corticostriatal LTP After Acute and Chronic Cocaine Exposure High-frequency stimulation induced LTP in both (1 day) saline- and (1 day) cocaine-treated striatal neurons. The amplitude and the time-course of corticostriatal LTP in these two groups of animals were similar (p ⬎ .05 at 5, 10, 30, 40, 60, and 90 min, not shown). Essentially identical results were also obtained with prolonged (7 days) saline or cocaine treatments (p ⬎ .05 at 5, 10, 30, 40, 60, and 90 min), suggesting that differences in LTP amplitude and duration could not account for the effects of chronic cocaine in the striatum (Figure 2A). In the four groups of animals, almost the totality (92%–96%) of the recorded cells underwent LTP after HFS in magnesium-free medium, whereas only a minority of neurons exhibited no potentiation and was therefore discarded. LFS-Induced Depotentiation in Control and Cocaine-Treated Rats Long-term potentiation is a flexible event, which can be erased by subsequent LFS of excitatory synapses (Bashir and Collingridge 1994; Huang and Hsu 2001; Picconi et al 2003; Sheng and Kim 2002). In physiological conditions, reversal of striatal LTP might function to “forget” maladaptive habits (Picconi www.sobp.org/journal

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D. Centonze et al injected controls. Low-frequency stimulation, however, produced different effects in the four experimental groups. Accordingly, it fully reversed LTP in rats receiving saline (3 and 5 days) treatment (n ⫽ 4/4 for both groups, and p ⬎ .05 compared with the respective pre-HFS values) and caused a partial block of depotentiation in the (3 days) cocaine-treated rats (n ⫽ 4/4, p ⬍ .05 compared with pre-HFS values) and a full blockade in the (5 days) cocaine-treated animals (n ⫽ 4/4 and p ⬎ .05 compared with post-HFS values) (Figure 3).

Figure 1. Cocaine treatment does not alter the basic properties of striatal cells. (A, B) The two graphs show that membrane potential and input resistance of striatal neurons are unaffected by both acute and chronic cocaine treatment. (C) Paired pulse ratio is also unaltered by cocaine. (D) Also the firing activity evoked by depolarizing current pulses is indistinguishable in chronic saline-treated (n ⫽ 7) and cocaine-treated (n ⫽ 7) neurons. The electrophysiological traces below are recorded from a salinetreated neuron (⫺84 mV) and a cocaine-treated neuron (⫺84 mV) after the injection of a depolarizing (800 pA) current pulse. EPSP, excitatory postsynaptic potential.

et al 2003). Saline- and cocaine-treated rats significantly differed for their capability to undergo depotentiation after 10 min of LFS (2 Hz) of corticostriatal terminals. In fact, whereas LFS fully reversed LTP in the totality of (1 and 7 days) saline- and (1 day) acute cocaine-treated rats (n ⫽ 8 for each group, and p ⬎ .05 compared with respective pre-HFS values), it was unable to affect LTP amplitude in all the chronic (7 days) cocaine-treated animals (n ⫽ 12 and p ⬍ .001 compared with pre-HFS values) (Figure 2B). Unlike LTD, depotentiation does not affect basal synaptic responses but represents a reversal of synaptic strength from the potentiated state produced by LTP (Bashir and Collingridge 1994; Huang and Hsu 2001; Mulkey et al 1994; O’Dell and Kandel 1994). Accordingly, we found that, in the absence of prior induction of LTP, LFS failed to alter the amplitude of corticostriatal EPSPs both in acute and chronic saline-treated (n ⫽ 7/7 and p ⬎ .05 for both groups) and acute and chronic cocaine-treated animals (n ⫽ 6/6 and p ⬎ .05 for both groups) (Figure 2C). Time-Course for the Effect of Cocaine on Corticostriatal Synaptic Plasticity To investigate whether the cocaine-induced blockade of depotentiation was an all or none phenomenon, we also studied corticostriatal synaptic plasticity in rats receiving 3 or 5 days cocaine treatment. In both groups of animals, LTP amplitude was indistinguishable when compared with their respective salinewww.sobp.org/journal

Role of D1 Receptors in the Maintenance of Corticostriatal LTP The D1 class of dopamine receptors mediates many of the molecular effects of cocaine in the striatum (Berke and Hyman 2000; Schultz 2002) and is involved in both induction (Centonze et al 2003; Kerr and Wickens 2001) and maintenance of corticostriatal LTP (Picconi et al 2003). To investigate the possible role of dopamine and D1 receptors in the cocaine-induced inhibition of depotentiation, we used corticostriatal slices from (1 and 7 days) saline-treated rats. Pretreatment of these slices with 10 ␮mol/L cocaine (n ⫽ 6/6), 10 ␮mol/L dopamine (n ⫽ 6/7), or 3 ␮mol/L SKF38393, a D1 receptor agonist (n ⫽ 5/5), prevented LFS-induced depotentiation (p ⬍ .001 compared with pre-HFS values) without altering LTP induction (Figure 4A). The acute effects of bath application of cocaine were abolished by 10 ␮mol/L SCH23390 (n ⫽ 5/6), a D1 receptor antagonist, but not by L-sulpiride (3 ␮mol/L), a D2 receptor antagonist (n ⫽ 4/4) (Figure 4B). Notably, 10 ␮mol/L SCH23390

Figure 2. Chronic cocaine treatment prevents depotentiation at corticostriatal synapses. (A) High-frequency stimulation (HFS) induced long-term potentiation (LTP) in both saline- and cocaine-treated rats. (B) Synaptic depotentiation induced by low-frequency stimulation (LFS) was present in saline-treated rats (1 and 7 days, data pooled together) and in rats exposed to a single dose of cocaine, whereas it was lost in chronic cocaine-treated animals. Traces below the graph are excitatory postsynaptic potentials (EPSPs) recorded before HFS (pre), 10 min after HFS, and 30 min after LFS from saline and chronic cocaine-treated rats. (C) In saline-treated (1 and 7 days, data pooled together) and cocaine-treated rats, LFS failed to induce significant changes in the amplitude of corticostriatal EPSPs in the absence of a prior induction of LTP. In this figure and in the following ones, the resting membrane potential and input resistance of the recorded neurons were constant and ranged between ⫺83 ⫾ 5 mV and 48 ⫾ 22 mol/L⍀, respectively.

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Figure 3. Time-course for the effect of cocaine on corticostriatal synaptic plasticity. (A) Three days cocaine treatment partially prevents depotentiation at corticostriatal synapses. (B) Five days cocaine treatment fully blocks depotentiation. EPSP, excitatory postsynaptic potential; HFS, high-frequency stimulation; LFS, low-frequency stimulation.

but not 3 ␮mol/L L-sulpiride (n ⫽ 3/3, p ⬎ .05 compared with pre-LFS values, not shown), applied during the LFS protocol, also reversed the effects of cocaine (7 days) treatment on depoten-

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Figure 5. Stimulation of D1 receptors mediates the effects of chronic cocaine on corticostriatal depotentiation. (A) The graph shows that blockade of D1 receptors by 10 ␮mol/L SCH23390 reversed the effects of chronic cocaine on depotentiation. (B) Low-frequency stimulation (LFS) failed to induce significant changes in the amplitude of corticostriatal excitatory postsynaptic potentials (EPSPs) in the presence of the D1 receptor antagonist SCH23390 (10 ␮mol/L). Note that in the presence of this antagonist, high-frequency stimulation (HFS) failed to induce long-term potentiation in both saline-treated (1 and 7 days, data pooled together) and cocainetreated (7 days) rats.

tiation, providing stronger support to the idea that abnormally active D1 receptors mediate the action of chronic cocaine on this synaptic phenomenon (n ⫽ 4/5, p ⬍ .01 compared with pre-LFS values) (Figure 5A). Consistent with previous reports (Calabresi et al 2000b; Centonze et al 2003; Kerr and Wickens 2001), in both control and chronic cocaine-treated rats, SCH23390 (10 ␮mol/L) blocked LTP induction when applied during HFS (n ⫽ 5/5, p ⬎ .05 for both groups) and failed to induce synaptic depression by LFS in non-potentiated synapses (n ⫽ 5/5, p ⬎ .05 for both groups) (Figure 5B).

Figure 4. Stimulation of D1 receptors mediates the effects of cocaine on corticostriatal synaptic plasticity. (A) Bath application of 10 ␮mol/L cocaine, 10 ␮mol/L dopamine, or 3 ␮mol/L SKF38393 failed to affect the induction of long-term potentiation but prevented corticostriatal depotentiation by low-frequency stimulation (LFS). Traces below the graph are excitatory postsynaptic potentials (EPSPs) recorded before high-frequency stimulation (HFS) (pre), 10 min after HFS, and 30 min after LFS from cocaine-treated slices. (B) Bath application of 10 ␮mol/L SCH23390 but not of 3 ␮mol/L L-sulpiride blocked the effects of 10 ␮mol/L cocaine on corticostriatal depotentiation. Traces below the graph are EPSPs recorded before HFS (pre), 10 min after HFS, and 30 min after LFS from slices bathed in cocaine plus SCH23390, and from slices bathed in cocaine plus L-sulpiride.

Effects of Cocaine on Striatal Basal Excitatory Transmission The cocaine-induced disruption of depotentiation might be attributed to changes in glutamate receptor expression and/or function. As an index of basal glutamate transmission, therefore, we measured mEPSCs in chronic (7 days) saline- and cocaineinjected rats. In the dorsal striatum, mEPSC frequency and amplitude did not differ in the two groups of rats (n ⫽ 5 for both groups, 10 different rats, p ⬎ .05 for both parameters), suggesting that the cocaine-induced blockade of depotentiation was independent of a direct effect of the drug on glutamate receptors (Figure 6). This conclusion was also supported by measuring the input-output relationships obtained in striatal neurons (n ⫽ 6, four different rats) before and during the application of a dose of cocaine (10 ␮mol/L) able to fully prevent depotentiation. The synaptic responses to stimulation intensities of progressively www.sobp.org/journal

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D. Centonze et al corticostriatal LTP (p ⬎ .05 for both compounds, as compared with control solution at 5, 10, 30, 40, 60, and 90 min post-HFS) but fully prevented its depotentiation by LFS (p ⬍ .01 and n ⫽ 5/5 for both inhibitors). Resting membrane potential, input resistance, and paired-pulse ratio measured at 60 msec interstimulus interval were unchanged in the recorded neurons before and during the application of either cdk5 inhibitors (Figure 8). Cocaine-Induced Conditioned Place Preference To investigate whether the administration regimen of cocaine able to block depotentiation might produce addictive behavior in the intact animal, we tested the rewarding properties of 7 days 15 mg/kg cocaine treatment in a conditioned place preference paradigm, a simple and robust behavioral test for studying the rewarding properties of drugs with dependence liability on humans (Acquas and Di Chiara 1994; Tzschentke 1998). Our results, obtained for each rat (n ⫽ 7 rats for both saline and cocaine groups) as the difference between post-conditioning and preconditioning time spent in drug-paired compartment, indicated that chronic cocaine administration induced a significant (p ⬍ .05) conditioned place preference in the treated rats (Figure 9).

Discussion Figure 6. Miniature excitatory postsynaptic potentials (mEPSCs) in chronic saline- and cocaine-treated striatal neurons. (A) Electrophysiological traces are examples of miniature glutamate-mediated synaptic currents recorded in striatal neurons of saline-treated (left) and cocaine-treated (right) rats. (B, C) The graphs on the left show the cumulative distributions of inter-event intervals and amplitudes of mEPSCs recorded from a saline- and a cocainetreated neuron. The graphs on the right show that both frequency and amplitude of mEPSCs were similar in saline- and cocaine-treated neurons.

In the present study, we have provided evidence that chronic cocaine exposure is associated with altered corticostriatal synaptic plasticity. In particular, cocaine failed to affect the induction and the amplitude of LTP, whereas it favored its maintenance by blocking the process of depotentiation. The cocaine-treatment regimen able to prevent the reversal of LTP had positive reinforcing properties, as indicated by the induction of a conditioned place preference in the treated rats.

increased strength, in fact, were remarkably similar in the absence and in the presence of cocaine. Bath application of cocaine also failed to affect resting membrane potential, input resistance, and paired-pulse ratio measured at 60 msec interstimulus interval (Figure 7). Involvement of Cyclin-Dependent Kinase 5 in Corticostriatal Depotentiation We have already described that the D1-receptor– dependent inhibition of corticostriatal depotentiation is mimicked by the adenylyl cyclase activator forskolin and by inhibitors of protein phosphatases, to enhance the protein kinase A (PKA)catalyzed phosphorylation of dopamine and cyclic adenosine 3’,5’-monophosphate–regulated phosphoprotein 32 kDa (DARPP32) on threonine 34 (Thr34) and the resulting inhibition of protein phosphatase-1 (PP-1, Picconi et al 2003). The D1/PKA/ Thr34DARPP-32/PP-1 intracellular signaling is negatively regulated by the activity of cyclin-dependent kinase 5 (cdk5), which converts DARPP-32 into an inhibitor of PKA (Bibb et al 1999; Nishi et al 2000). Accordingly, intrastriatal blockade of cdk5 mimics some behavioral and molecular effects of D1 receptor stimulation and of chronic exposure to cocaine (Bibb et al 2001). Therefore, to confirm that the blockade of LTP erasing processes is a major synaptic correlate of the molecular action of cocaine in the striatum, we decided to investigate the effects of two cdk5 inhibitors (roscovitine and butyrolactone I) on corticostriatal synaptic plasticity. As with cocaine, roscovitine (10 ␮mol/L, n ⫽ 5/5) and butyrolactone I (10 ␮mol/L, n ⫽ 4/4) each failed to affect baseline EPSP and the amplitude and the duration of www.sobp.org/journal

Figure 7. Bath application of cocaine does not alter the synaptic and intrinsic properties of striatal cells. (A) In a representative neuron, the synaptic responses to stimulation intensities of progressively increased strength were remarkably similar before and after the application of 10 ␮mol/L cocaine (left). Also paired-pulse ratio was unaffected by cocaine application (right). (B) The two graphs show that membrane potential and input resistance of striatal neurons are unaffected by bath application of 10 ␮mol/L cocaine.

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Figure 8. Inhibition of cyclin-dependent kinase 5 (cdk5) mimics the effects of cocaine treatment on corticostriatal depotentiation. (A) Pharmacological blockade of cdk5 by either butyrolactone I (10 ␮mol/L) or roscovitine (10 ␮mol/L) failed to alter both induction and maintenance of high-frequency stimulation (HFS)-induced corticostriatal long-term potentiation. (B) Bath application of either roscovitine or butyrolactone I inhibited low-frequency stimulation (LFS)-induced corticostriatal depotentiation. (C) Both cdk5 inhibitors failed to alter resting membrane potential, input resistance, and paired pulse ratio of the recorded neurons.

Together, these observations support the conclusion that the synaptic effects of cocaine observed in the present study represent a part of the neuronal adaptations mediating addiction; however, other parallel changes involving several transmitter systems and receptors must occur in the brain in response to cocaine consumption, accounting for the complex behavioral effects induced by the drug. In this respect, it should be noted that the stimulation of both D1 and D2 dopamine receptors is necessary for behavioral sensitization to cocaine, whereas only the D1 receptor is involved in the cocaine-induced blockade of depotentiation. Receptor and Post-Receptor Mechanisms of Cocaine-Induced Inhibition of Depotentiation The effects of chronic cocaine on depotentiation were likely mediated by the stimulation of D1 receptors. This conclusion is mainly based on the observation that the effects of chronic cocaine treatment on this synaptic phenomenon were prevented after pharmacological inhibition of D1 but not of D2 receptors during LFS. In addition, SKF38393, a D1 receptor agonist, also blocked depotentiation, whereas SCH23390, a D1 receptor antagonist, but not L-sulpiride, a D2 receptor antagonist, prevented the effects of acute application of cocaine in slices. A main limitation of these observations is that the effects of SCH23390 were studied only during application of this compound in slices. Although the inhibition of D1 receptors in vivo during chronic cocaine treatment would in principle demonstrate in a more straightforward manner the involvement of these receptors in the effects of cocaine on depotentiation, these experiments are hardly feasible. Blockade of striatal D1 receptors in vivo, in fact, prevents the expression of corticostriatal LTP, therefore making subsequent exploration of depotentiation impossible (Picconi et al 2004). We have shown that depotentiation was prevented by phar-

BIOL PSYCHIATRY 2006;60:436 – 443 441 macological blockade of cdk5 by roscovitine or butyrolactone I, an effect that might explain why intrastriatal injection of one of these inhibitors markedly potentiates cocaine-induced locomotor sensitization, a behavioral correlate of addiction (Bibb et al 2001). Not surprisingly, increased cdk5 activity has recently been found to attenuate cocaine-mediated dopamine signaling in the striatum (Takahashi et al 2005). The development of behavioral sensitization to cocaine is coupled with upregulation of the dopamine D1-receptor associated signaling (Berke and Hyman 2000; Nestler et al 1996), implying loss of depotentiation as a plausible mechanism for the synaptic effects of repeated exposure to cocaine. Accordingly, it has been shown that blockade of D1 receptors prevents locomotor sensitization to psychostimulants (Crawford et al 1997) and favors depotentiation in the hippocampus (Otmakhova and Lisman 1998) and the striatum (Picconi et al 2003; present study). Pharmacological blockade of cdk5 might interfere with corticostriatal depotentiation by enhancing the D1-receptor–initiated intracellular pathway. Accordingly, the PKA-mediated phosphorylation of DARPP-32 at Thr34 is a critical requirement for the blockade of corticostriatal depotentiation (Picconi et al 2003); DARPP-32, however, can be phosphorylated at Thr75 by cdk5, an event which attenuates the D1/PKA/Thr34DARPP-32/PP-1 pathway by converting DARPP-32 into an inhibitor of PKA (Bibb et al 1999). Therefore, by blocking cdk5 activity, roscovitine and butyrolactone I might prevent corticostriatal depotentiation by enhancing the PKA/Thr34DARPP-32 signaling. Consistent with this hypothesis, intrastriatal injections of roscovitine have been reported to enhance the PKA-mediated phosphorylation of DARPP-32 at Thr34 (Bibb et al 2001). In addition, cdk5 inhibitors have also been shown to potentiate the effects of dopamine D1 receptors stimulation on NMDA receptor-mediated excitatory synaptic transmission in the striatum (Chergui et al 2004). The inhibition of cdk5, however, might interfere with NMDAdependent corticostriatal synaptic plasticity through another mechanism. It has been recently reported, in fact, that cdk5 also phosphorylates the N-terminal domain of the postsynaptic density protein PSD-95, a postsynaptic scaffolding protein that links NMDA receptors to the cytoskeleton and signaling molecules (Morabito et al 2004). The use of the recently generated mice with point mutations at Thr75 of DARPP-32 will help to clarify this issue (Svenningsson et al 2003).

Figure 9. Cocaine-induced conditioned place preference. Drug-induced conditioned place preference was tested in seven chronic (7 days) salinetreated and cocaine-treated (7 days, 15 mg/kg) rats. The results are expressed as change of the time spent in drug-paired side ⫾ SEM, calculated as the difference in the time spent during the post-conditioning phase versus preconditioning phase in the less preferred drug-paired compartment. Two-tailed unpaired t test indicated a significant conditioned place preference in the cocaine treated rats (*p ⬍ .05).

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442 BIOL PSYCHIATRY 2006;60:436 – 443 Different Forms of Synaptic Plasticity in the Striatum High-frequency stimulation of corticostriatal terminals induces either LTD or LTP of excitatory transmission. Both forms of activity-dependent synaptic plasticity have been found in the striatum in vivo (Charpier and Deniau 1997; Reynolds and Wickens 2000; Reynolds et al 2001) and in vitro (Akopian et al 2000; Partridge et al 2000) in the absence of any manipulation of external medium, indicating that they represent alternative physiological responses to repetitive activation of corticostriatal synapses; however, in our in vitro experiments, LTP was studied after the removal of magnesium ions from the bathing solution, a procedure that prevents the voltage-dependent blockade of NMDA receptors. As reported (Calabresi et al 2000b; Centonze et al 2003), in fact, this experimental approach aims at minimizing the substantially unpredictable appearance of LTP over LTD after HFS (Akopian et al 2000; Partridge et al 2000) and at favoring NMDA receptor-dependent corticostriatal depotentiation (Picconi et al 2003). Depotentiation is a recently described synaptic phenomenon that interferes with the late phase of LTP (Bashir and Collingridge 1994; Huang and Hsu 2001). Although both depotentiation and LTD reduce the strength of synaptic transmission, the two phenomena are remarkably different. In the striatum, in fact, LTD induction requires HFS of corticostriatal terminals, is independent of NMDA receptor stimulation, and is favored by D1receptor activation (Centonze et al 2001). In contrast, depotentiation is the reversal of a previously induced LTP, requires LFS, is NMDA-receptor dependent, and is blocked by D1-receptor stimulation (Picconi et al 2003; present study). The different mechanisms and physiological roles of LTD and depotentiation are also supported by the evidence that chronic cocaine facilitates LTD in the ventral striatum (Thomas et al 2001) but prevents depotentiation in the dorsal striatum (present study). A previous study showed that methamphetamine-induced behavioral sensitization was associated with the promotion of LTP over LTD formation in the striatum (Nishioku et al 1999). These data, taken together with the results of the present study, indicate that both induction and maintenance of striatal LTP are involved in the behavioral effects of drugs of abuse. Functional Implications The automated or habitual nature of persistent drug consumption in addicted individuals suggests that control over drug-seeking behavior might gradually devolve to a habit system in the brain (Gerdeman et al 2003; Robbins and Everitt 1999). Synaptic plasticity in the dorsal striatum is involved in a form of learning in which stimulus-response associations or habits are incrementally acquired (Packard and Knowlton 2002) and behavioral and molecular data converge in indicating that adaptations in striatal function occur during addiction (Berke and Hyman 2000). Here we have described that chronic cocaine treatment interferes with the process of synaptic depotentiation at corticostriatal synapses, an effect attributable to the stimulation of dopamine D1-receptor associated signaling. Notably, loss of corticostriatal depotentiation also underlies the motor complications of chronic L-DOPA therapy in parkinsonian rats (Picconi et al 2003), indicating that drug addiction and L-DOPA–induced dyskinesias share common features. In this line, a pathology of habit forming system has been hypothesized as the basis of both drug addiction (Berke and Hyman 2000; Gerdeman et al 2003; Robbins and Everitt 1999) and L-DOPA–induced dyskinesias (Calabresi et al 2000a; Graybiel et al 2000), and evidence exists that parkinsonian patients tend to take amounts of L-DOPA far www.sobp.org/journal

D. Centonze et al beyond those needed to treat their motor disabilities (Lawrence et al 2003). The evidence that cocaine facilitates LTP consolidation but not its induction is in tune with the observation that psychostimulants enhance reward-based memory in animals even when administered after the training episode (White 1988), as also predicted by the delayed activation of dopamine neurons during associative learning trials (Hollerman and Schultz 1998; Schultz 2002). On the basis of these observations, theories of striatal reinforcement learning originally proposed that an appropriate activation of corticostriatal synapses does not induce LTP but sets up a “tag” that marks the synapses as eligible for long-lasting modification by a subsequent signal arising from dopamine system (Schultz 1998, 2002). The recent electrophysiological data on corticostriatal synaptic plasticity, however, contributed to partially revise this model, because it is now clear that repetitive activation of corticostriatal synapses does induce LTP, which is, however, highly susceptible to disruption (depotentiation) unless the dopamine D1-receptor signal is activated. Concluding Remarks Addictive drugs might constitute rewards in their own right and/or might amplify the dopamine responses to natural rewards (Schultz 1998, 2002). Whereas the first possibility seems to occur in classical structures of the reward circuitry (ventral tegmental area and nucleus accumbens, where cocaine induces per se synaptic plasticity [Thomas et al 2001; Ungless et al 2001]), the second is likely to take place in the dorsal striatum, where cocaine does not induce LTP but prevents its extinction (present study). It should be noted, however, that our results, like those reported in many other physiological studies (Thomas et al 2001; Ungless et al 2001), rather than representing the synaptic correlate of cocaine exposure, might reflect the effects of a possible stress-cocaine interaction brought about by the repeated intraperitoneal injections of the drug. Experiments with less stressful delivery of the drug might help to clarify this point. Understanding the molecular and physiological correlates of the addictive behavior is essential for the development of effective strategies against drug abuse (Nestler 2002). We thank M. Tolu for his technical assistance. This work was supported by a grant from Ministero della Salute, Ricerca Finalizzata (to DC) and by a CNR Biotecnologie grant and a Telethon grant (to PC). Acquas E, Di Chiara G (1994): D1 receptor blockade stereospecifically impairs the acquisition of drug-conditioned place preference and place aversion. Behav Pharmacol 5:555–569. Akopian G, Musleh W, Smith R, Walsh JP (2000): Functional state of corticostriatal synapses determines their expression of short- and long-term plasticity. Synapse 38:271–280. Bashir ZI, Collingridge GL (1994): An investigation of depotentiation of longterm potentiation in the CA1 region of the hippocampus. Exp Brain Res 100:437– 443. Berke JD, Hyman SE (2000): Addiction, dopamine, and the molecular mechanisms of memory. Neuron 25:515–532. Bibb JA, Chen J, Taylor JR, Svenningsson P, Nishi A, Snyder GL, et al (2001): Effects of chronic exposure to cocaine are regulated by the neuronal protein Cdk5. Nature 410:376 –380. Bibb JA, Snyder GL, Nishi A, Yan Z, Meijer L, Fienberg AA, et al (1999): Phosphorylation of DARPP-32 by Cdk5 modulates dopamine signalling in neurons. Nature 402:669 – 671. Calabresi P, Giacomini P, Centonze D, Bernardi G (2000a): Levodopa-induced dyskinesia: A pathological form of striatal synaptic plasticity? Ann Neurol 47(4 Suppl 1):S60 –S68.

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