Neuropharmacology xxx (2011) 1e8

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Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm

Cocaine sensitization does not alter SP effects on locomotion or excitatory synaptic transmission in the NAc of rats
Samuel B. Kombiana, *, Kethireddy V.V. Ananthalakshmia, Jeffrey A. Zidichouskib, c, Tarek M. Salehc
a

Department of Applied Therapeutics, Faculty of Pharmacy, Kuwait University, Kuwait Institute for Nutrisciences and Health, National Research Council of Canada, Charlottetown, PEI, Canada c Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PEI, Canada
b

a r t i c l e i n f o
Article history: Received 2 March 2011 Received in revised form 12 August 2011 Accepted 6 September 2011 Keywords: Substance abuse Cocaine Substance P Excitatory postsynaptic currents Synaptic plasticity Neuropeptides

a b s t r a c t
Substance P (SP) and cocaine employ similar mechanisms to modify excitatory synaptic transmission in the nucleus accumbens (NAc), a region implicated in substance abuse. Here we explored, using NAc slices, whether SP effects on these synaptic responses were altered in rats that have been sensitized to cocaine and whether SP could mimic cocaine in triggering increased locomotion in sensitized rats. Intraperitoneal (IP) injection of nave rats with cocaine (15 mg/kg) caused increased locomotion by 408.5 85.9% (n 5) which further increased by 733.1 157.8% (n 5) following a week of cocaine sensitization. A similar challenge with 10 mg/kg of SP after cocaine sensitization did not produce signicant changes in locomotion (170.6 61.0%; n 4). In contrast to cocaine, IP injection of rats with SP or SP5e11 (10e100 mg/kg) with or without phosphoramidon did not elicit changes in locomotion. In electrophysiological studies, both cocaine and SP depressed evoked NMDA and non-NMDA receptormediated excitatory synaptic currents (EPSCs) in slices obtained from nave rats. In slices derived from cocaine-sensitized rats, cocaine but not SP produced a more profound decrease in non-NMDA compared to NMDA responses. Similar to that in nave rats, cocaines effect on the EPSCs in these sensitized rats occluded those of SP. Thus, although SP and cocaine may employ similar mechanisms to depress EPSCs in the NAc, IP injection of SP does not mimic cocaine-induced hyperlocomotion indicating that not all of cocaines effects are mimicked by SP. 2011 Elsevier Ltd. All rights reserved.

1. Introduction The nucleus accumbens (NAc) is part of the mesolimbic dopaminergic system that is widely regarded as a critical region involved in substance use and abuse and undergoes plastic changes that may underlie substance abuse (Russo et al., 2010; Wolf, 2010; Wolf and Ferrario, 2010). The medium spiny neurons of the NAc have been shown to undergo neurochemical, molecular, structural and electrophysiological changes leading to the behaviors associated with exposure to cocaine and related psychostimulants (Beurrier and Malenka, 2002; Boudreau and Wolf, 2005; Pierce et al., 1996a, 1995; Robinson and Becker, 1986; Robinson and Kolb, 2004; Thomas et al., 2001). Endogenous non-opioid peptides, such as substance P (SP) and cholecystokinin which are both present in the NAc are reported to produce neurochemical, electrophysiological and behavioral effects that are similar to exogenous administration
* Corresponding author. Department of Applied Therapeutics, Faculty of Pharmacy, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait. Tel.: 965 2 498 6916; fax: 965 2 534 2807. E-mail address: kombian@hsc.edu.kw (S.B. Kombian). 0028-3908/$ e see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2011.09.008

or application of psychostimulants (Boix et al., 1992a,b; Hasenohrl et al., 1994; Hasenohrl et al., 1992; Kombian et al., 2009, 2003; Nikolaus et al., 1999a). In particular, SP causes an increase in extracellular dopamine concentration to produce its effects in the region; a mechanism similar to that reported for cocaine (Boix et al., 1992b; Kombian et al., 2009, 2003). Furthermore, evidence also suggest that SP may be involved in the development of addiction to other substances such as opioids (Commons and Serock, 2009). Repeated, intermittent contingent and non-contingent exposure to cocaine leads to enhanced and enduring increase in locomotor activity referred to as behavioral sensitization in rodents. This is believed to represent incentive-driven, drug-seeking behavior (Kalivas and Stewart, 1991; Robinson and Berridge, 1993, 2008). It has been reported and generally accepted that experience-based changes in glutamate transmission in the NAc underlies behavioral sensitization (Beurrier and Malenka, 2002; Boudreau and Wolf, 2005; Russo et al., 2010; Wolf and Ferrario, 2010), although other limbic regions such as the ventral tegmental area and the prefrontal cortex may also play critical roles in the glutamate plasticity that underlies behavioral sensitization (Borgland et al., 2004; Cornish and Kalivas, 2001; Kalivas and Nakamura, 1999;

Please cite this article in press as: Kombian, S.B., et al., Cocaine sensitization does not alter SP effects on locomotion or excitatory synaptic transmission in the NAc of rats, Neuropharmacology (2011), doi:10.1016/j.neuropharm.2011.09.008

S.B. Kombian et al. / Neuropharmacology xxx (2011) 1e8 Regardless of the approach, animals were randomly assigned to control (salineinjected) or treatment groups (drug-treated). Each rat was then weighed on a daily basis to calculate the amount of drug or volume of saline to be injected. All drug solutions were then made to a concentration such that no animal received more than 1 ml of total uid via IP injection. The equivalent volume of saline was given to control rats. All rats were acclimatized to the recording environment (room and cage) for 2e3 h/day for 2 days prior to the experimental trials. On the rst two experimental days, each rat in control and drug-treated groups were injected with saline and locomotor activity was recorded. The activity on day two was used as the control against which drug treatment was compared. On day 3, rats in the control group received daily injections of saline while rats in the drug treatment group received the challenge dose of test drug (cocaine: 15 mg/kg or SP/SP5e11: 10e100 mg/kg). For sensitization with cocaine, rats then received a sensitizing daily dose of 30 mg/kg for 7 days while the control rats received equivalent volume of saline. For locomotor recording, the locomotor activity of each rat was recorded for 20e60 min as control (depending on the tracking approach), followed by a brief (no more than 5 min) interruption for drug injection and then recording was continued for 20 min to 2 h (depending on tracking approach). The video recording was continuous with manual pauses only for injections while for the automated approach (Versamax 4.10), the computer was programmed to run a primary session of 60 min and a secondary session of 120 min yielding a total run time/rat of 180 min. An automatic pause at the end of the primary session allowed for injections after which the secondary session was activated. The digital data collected above with the Versamax software was converted into an Excel-compatible format (using Versadat 3.02B software provided by the equipment manufacturer-AccuScan Instruments Inc, Columbus, OH, USA) for subsequent analyses. Locomotor activity on day 2 and at 20 min post-drug injection were used as control and peak drug effect. All digital video records were fragmented using a specialized, validated software that allowed for the horizontal activity to be quantied into distance/time. Activity was then cropped into 10 min epochs for comparison at different time points (see Results section). For the automated tracking system, the software provided data on horizontal, vertical and total distance programmed in 10 min epochs. To enable comparison with the video tracking data, horizontal activity/time was used to represent locomotor activity throughout this study. The activity of each animal at the end of 20 min i.e. between the 10th and 20th minutes on day two of recording was used to normalize the activity of each rat for comparison with others (see results). Analysis of locomotor activity for sensitization was done by an investigator that was blinded to the treatment groups. Furthermore, EPSCs were analyzed and quantied blindly prior to the sensitization data being decoded. 2.2. In vitro studies 2.2.1. Slice preparation Male Sprague-Dawley rats (100e250 g) were anesthetized using halothane before decapitation. The brain was quickly removed and placed in ice cold (4  C) articial cerebrospinal uid (ACSF) that was bubbled with 95% O2 and 5% CO2. The composition of the ACSF was 126 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 1.2 mM MgCl2, 2.4 mM CaCl2, 18 mM NaHCO3, and 11 mM glucose that was bubbled with a mixture of 95% O2 and 5% CO2 to yield a buffered solution with osmolarity between 310 and 320 mOsm. Parasagittal forebrain slices (350 mm in thickness) containing the nucleus accumbens and the cortex were cut at 4  C using previously published techniques (Kombian et al., 2003). The slices were cut with a Vibratome 1000 or 1500 series tissue slicer and were incubated in buffered ACSF at room temperature for 1 h to recover from trauma caused by the sectioning process. After at least 1 h recovery, a single slice was trimmed and transferred into a recording chamber and submerged in buffered ACSF and perfused at a ow rate of 2e3 ml/min at 29e31  C. Whole cell recordings were made using glass microelectrodes with a tip resistance between 4 and 10 MU. The composition of the internal recording solution was: potassium gluconate (135 mM), NaCl (8 mM), EGTA (0.2 mM), HEPES (10 mM), mg-ATP (2 mM) and GTP (0.2 mM). The pH of this solution was adjusted to 7.3 with KOH and the osmolarity to 270e280 mOsm. Synaptic responses were recorded using either Axopatch 1D or Multiclamp 700B amplier in voltage clamp mode. Cells were voltage clamped at a membrane potential of 80 mV and excitatory postsynaptic currents (EPSCs) were isolated in the presence of picrotoxin (50 mM) and evoked by placing bipolar tungsten stimulating electrodes at the prefrontal cortex-accumbens border. Input and access resistance of all cells were determined and monitored regularly throughout each experiment for stability by applying a 75 ms, 20 mV pulse, 150 ms after synaptic stimulation. In all reported cells, access resistance averaged about 25 MU which did not change by more than 15% throughout the experiment. Two successive synaptic responses were elicited at 30 s intervals, averaged and stored for off-line analysis. All data was acquired and analyzed using pClamp software (Clampex 8.2 or 9.2; Axon Instruments). All synaptic responses were sampled at a rate of 4e6 kHz and ltered at 1 or 10 kHz, digitized and stored for off-line analysis. Evoked glutamate-mediated EPSCs at the holding potential of 80 mV were almost exclusively non-NMDA receptor-mediated as previously reported (Kombian et al., 2009, 2003). To isolate NMDA receptor-mediated responses, CNQX (10 mM) was applied that abolished the response at 80 mV. The cell was then voltage clamped at between 50 mV and 40 mV and synaptic stimulation at this

Pierce et al., 1996a; Vezina, 1993). Plastic changes include postsynaptic medium spiny cell dendritic branching, increases in the number and size of spines and AMPA receptor redistribution to the outer cell membrane surface and synapses (Boudreau et al., 2007; Russo et al., 2010; Wolf, 2010). This plasticity is reected as changes in the magnitude of non-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) in the NAc of cocaine exposed rodents as compared to nave ones (Beurrier and Malenka, 2002; Thomas et al., 2001). Dopamine, through its interaction with or via the modulation of excitatory amino acids and EPSCs, plays an important role in the phenomenon of behavioral sensitization (Anderson and Pierce, 2005; Harvey and Lacey, 1996; Kalivas, 1995; Nicola et al., 1996; Pierce et al., 1996a). Similar to cocaine, repeated dopamine application has been reported to cause an upregulation of AMPA receptors in NAc medium spiny cells which occluded with the synaptic scaling produced by prolonged blockade of excitatory activity with an AMPA receptor antagonist (Sun and Wolf, 2009). Thus, any substance that alters dopamine levels in the critical brain regions would be predicted to produce these bio-molecular changes and thus inuence the development of behavioral sensitization. Identifying endogenous substances with such an action will improve our understanding of the development of addiction and may lead to new targets or therapeutic options for substance abuse treatment and related psychiatric disorders. In this regard, SP has been reported to increase extracellular dopamine concentration in the NAc (Boix et al., 1992a,b; Cador et al., 1989; Placenza et al., 2004) and also shown to utilize dopamine as an intermediate to depress EPSCs in the NAc (Kombian et al., 2003). Furthermore, this effect of SP was reported to occlude the actions of cocaine (Kombian et al., 2009). Although chronic SP administration has been reported to produce certain behavioral modications (Hasenohrl et al., 1992; Krappmann et al., 1994), and mice lacking the NK1 receptor gene failed to show cocaine-induced reward and locomotor behavior (Murtra et al., 2000; Ripley et al., 2002), the question is whether SP can by itself induce behavioral sensitization similar to cocaine or interact with cocaine-induced behavioral sensitization and the accompanying plasticity in excitatory synaptic transmission. Given the reported interaction between cocaine and SP (Kraft et al., 2001a,b), we tested the hypothesis that SP can cause hyperlocomotion in rats and induce behavioral sensitization and the accompanying changes in excitatory synaptic transmission as those seen using cocaine. We sensitized rats to cocaine and tested if SP, given intraperitoneally (IP), could trigger enhanced locomotor activity and also tested if the excitatory synaptic effects of cocaine in sensitized rats occluded those of SP.
2. Materials and methods All experiments were performed in Kuwait using male Sprague-Dawley rats (100e250 g) obtained from the Kuwait University Animal Resource Centre from a local breeding colony, except those involving cocaine that were performed in Canada, under Health Canada license (File# 9639-SO219-1) and the United States. Institutional and international guidelines for the usage and humane handling of animals were followed throughout the study. A minimum number of rats was used to obtain the required results. Rats were housed (four per cage) on a 12 h:12 h dark/light cycle, and provided with food and water ad libitum. For in vivo experiments, rats were housed in individual cages throughout the study. 2.1. In vivo locomotor studies These studies were done using two different locomotor tracking approaches. The rst involved an automated locomotor and behavior tracking system (Versamax 4.10, AccuScan Instruments Inc, Columbus, OH, USA) that employed gridded photocells in standardized activity cages (see detailed description by Borgland et al., 2004; Kalivas and Duffy, 1993). The second approach involved digital video movie recording of rats in an activity cage. The resultant avi movie le was then defragmented into frames and saved. Using a custom-written script (ImageJ, NIH) a threshold was established for each image and the central co-ordinates of the centroid were saved. Using Microsoft Excel, the total distance traveled was determined.

Please cite this article in press as: Kombian, S.B., et al., Cocaine sensitization does not alter SP effects on locomotion or excitatory synaptic transmission in the NAc of rats, Neuropharmacology (2011), doi:10.1016/j.neuropharm.2011.09.008

S.B. Kombian et al. / Neuropharmacology xxx (2011) 1e8 holding potential revealed a slow rising, slowly decaying EPSC characteristic of the NMDA receptor-mediated response and which was subsequently abolished by DL-APV (100 mM), a selective NMDA receptor antagonist (Kombian et al., 2009). All other biophysical characteristics of these cells were similar to those previously reported (Kombian et al., 2003). 2.2.2. Data analysis and statistics Synaptic strength was measured as the peak current from baseline of each trace. For normalization, the peak currents of the last 3e5 traces taken every minute prior to drug application was averaged and used to normalize the entire responses in that experiment. These normalized values were then used for average plots and calculation of changes. All values are represented as mean standard error. In both in vivo and in vitro experiments, statistical differences between control and treated groups or cells were calculated using one way ANOVA followed by post-hoc t tests or multiple comparisons where appropriate using SigmaStat (Version 3.5) software (Systat Software Inc., San Jose, CA, USA). Signicance was set at a probability level of p 0.05. Graphs were prepared using SigmaPlot (Version 9; Systat Software Inc., San Jose, CA, USA) and CorelDraw (Version 12; Corel Corporation Inc., Ottawa, ON, Canada). 2.2.3. Chemicals and drugs SP, SP5e11, phosphoramidon, picrotoxin, DL-2-amino-5-phosphonovalerate (DL-APV) were obtained from Sigma (Natick, MA, USA) and desired stock solutions were made by dissolving them in the solvents suggested by the drug manufacture and stored at 20  C. 6,7-cyanoquinoxaline-2,3-dione (CNQX) was purchased from RBI (Natick, MA, USA) and dissolved in dimethylsulfoxide. Cocaine hydrochloride was purchased from Sigma (Natick, MA, USA) and the required stock solution was prepared fresh everyday by dissolving in puried water. For in vitro studies, all drugs were diluted to the desired nal concentrations as indicated in the results and gures and bath perfused. All other routine laboratory salts for buffers and solvents were obtained from Sigma (Natick, MA, USA).

compared to day 2 activity, n 4, Fig. 2). After cocaine sensitization, these animals were subsequently challenged with SP (10 mg/kg, IP) and the increase in locomotor activity was only 170.6 61.0% (p > 0.05 compared to day 2; n 4, Fig. 2). Similar to the above, but in a converse experiment to test if cocaine interacted with SP, we attempted to sensitize rats to SP. Since SP has been reported to cross the blood brain barrier (see review by Banks and Kastin, 1985) and SP as well as the SP active analogs are reported to produce neurochemical and some behavioral effects

3. Results 3.1. Cocaine and SP effects on locomotor activity The horizontal locomotor activity was monitored in rats to assess the effects of cocaine and SP. Throughout each experiment, all precautions were taken to ensure that animals were acclimatized to the recording environment a day before experiments commenced and at least for 20e60 min on each experimental day or session. Fig. 1A shows averaged plots (n 7 rats each) of typical timedependent decrease in locomotion (horizontal activity) during the rst hour of placement in the activity cage. A cocaine challenge (15 mg/kg; IP) in control (nave animals) elicited an increase in horizontal activity, which peaked at about 20 min (1972.8 425/ 10 min) and then declined back to baseline in about 20e30 min. By contrast animals that received a once daily sensitizing dose of cocaine (30 mg/kg; IP for 7 days) exhibited a much larger increase in locomotor activity also peaking at 20 min (3991.9 1164.4/10 min) but lasted for about 60e80 min. This enhanced and long lasting locomotor activity after repeated intermittent injections of cocaine is referred to as behavioral sensitization. Because the peak locomotor activity after the test cocaine challenge (15 mg/kg) occurred at about 20 min in both control and sensitized rats, the horizontal activity in the second (10 min) epoch post-cocaine challenge was taken as peak activity and used for comparison with other treatments. For normalization to baseline, the activity in the last 10 min epoch prior to any drug challenge was taken as control activity for the day and the same value for day 2 was taken for cross-day comparison. In subsequent experiments, when the test dose of cocaine (15 mg/kg) was given to the drug-treated group on day 3, it caused an increase in horizontal activity of 408.5 85.9% above the baseline activity observed on day 2 (p < 0.05, n 5; Fig. 1B). After 7 days of sensitization, the same test dose of cocaine now caused a signicantly larger increase in locomotor activity to 733.1 157.8% over baseline (p < 0.05 compared to days 2 and 3 activities, Tukeys test; n 5; Fig. 1B). To test if SP could trigger the same enhanced locomotor response after sensitization to cocaine, another group of animals was challenged with cocaine on day 3 and the increase in locomotion was 377.4 113.6% over day 2 activity (p < 0.05

Fig. 1. Repeated cocaine injections lead to enhanced and prolonged locomotor activity in sensitized rats. A: An average horizontal activityetime plot in two groups of rats (n 7 each), one saline-injected (control) and the other cocaine-injected (30 mg/kg/d for 7 days). Note that in both groups the initial activity level (primary session) was quite high which then declined with time. Although not shown here, upon daily repeats of this regimen, the initial activity levels also declined such that by the third day, activity level was minimal by 20 min indicating that the animals habituated to their environment. In both groups, immediately after the 6th 10 min epoch, a cocaine challenge (15 mg/kg given IP) resulted in an increase in activity that peaked at 20 min post-injection and then declined slowly toward baseline activity. B: Normalized horizontal activity (normalized to activity on day 2) of rats showing the experimental protocol and activity over several experimental days in control rats (saline-injected; n 5) and cocaine-sensitized rats (n 5). A test (control) cocaine response (15 mg/kg) was elicited in the sensitized group on day 3. This was followed by 7 days of daily, IP cocaine (30 mg/kg) injections then another test (cocaine challenge) on day 13 with 15 mg/kg. The control animals received equivalent volumes of daily saline injections. In this and Fig. 2, asterisk (*) represents statistical signicance at p < 0.05 (ANOVA) compared to day 2 level and # represents signicance compared to day 3.

Please cite this article in press as: Kombian, S.B., et al., Cocaine sensitization does not alter SP effects on locomotion or excitatory synaptic transmission in the NAc of rats, Neuropharmacology (2011), doi:10.1016/j.neuropharm.2011.09.008

S.B. Kombian et al. / Neuropharmacology xxx (2011) 1e8

Fig. 2. Substance P does not induce enhanced locomotor activity in cocaine-sensitized rats. Normalized horizontal activity of rats (normalized to activity on day 2) in control (saline-injected; n 5) and cocaine-sensitized (n 4) following the protocol in Fig. 1B except that the 13th day challenge was with SP.

when given IP in microgram per kg doses (Boix et al., 1992a,b, 1993), we used this route for SP administration in this study. We did however, choose higher doses (mg/kg) with the aim of possibly eliciting robust and reproducible responses. After the initial acclimatization to the test environment, the horizontal activity of each group of rats was recorded during the primary session and these produced the predictable trend of an initial high level of activity, which then declined to be negligible after 20e30 min. Each group, then received an IP injection of saline, SP or SP5e11 at doses of 10 mg/kg or 100 mg/kg of body weight prior to the secondary session. Neither SP (n 4e8) nor SP5e11 (n 4e8) at 10 mg/kg or 100 mg/kg produced a signicant change in horizontal activity throughout the 120 min secondary session (Fig. 3A). Since SP is known to be metabolized by endopeptidase enzymes, we reasoned that protecting the peptides with the endopeptidase inhibitor, phosphoramidon (Chiba and Misawa, 1994; Damas et al., 1996; Kumar et al., 2000), would improve the probability of eliciting an enhanced locomotor response. However, similar to the above results, concurrent administration of the peptides with phosphoramidon (5 mg/kg; not shown) did not alter or improve the ability of either peptide to elicit enhanced locomotor activity although 3 mg/kg intravenous injection has been reported to produce SP-dependent effects in other systems (Chiba and Misawa, 1994). Phosphoramidon (5 mg/kg) by itself, did not elicit a change in locomotor activity in a group of rats that was challenged with it alone although there was a slight increase between 60 and 100 min post-challenge which was absent in the group that subsequently received SP (Fig. 3B). 3.2. SP and cocaine effects on EPSCs in cocaine-sensitized rats Some of the results contained here are based on work in a previous report (Kombian et al., 2009) on SP, SP5e11 and cocaine in nave rats that was conducted at the same time of this study. Our previous work showed that SP and its active analog SP5e11 produced effects similar to cocaine on excitatory synaptic responses recorded in the NAc (Kombian et al., 2009, 2003). The actions of SP and cocaine on the EPSCs were mutually occlusive (Kombian et al., 2009). We therefore examined if these interactions were altered in cocainesensitized rats by testing their effects on EPSCs recorded from cells in slices obtained from rats that had been sensitized to cocaine

Fig. 3. Substance P does not induce enhanced locomotor activity in nave rats. A: An average horizontal activityetime plot in three groups of rats (n 4e8 for each group), one group received no injections, another group received saline injections and the third group received an IP injection of SP (100 mg/kg) after the primary session. Note that in all groups the initial activity level (primary session) was quite high which then declined with time. B: An average activityetime plot showing that pretreatment of rats with an endopeptidase inhibitor, phosphoramidon (5 mg/kg) neither caused a change in locomotor activity nor improved the action of SP (100 mg/kg; n 4 per group).

(1e7 days post-cocaine challenge; see above). Non-NMDA receptormediated glutamate responses and NMDA responses were pharmacologically isolated and examined. Similar to previously reported data derived from nave animals, 1 mM SP and its truncated active analog, SP5e11 both suppressed the evoked non-NMDA receptormediated response by 29.2 2.0% (p < 0.05, n 5) and 37.3 5.5% (p < 0.05, n 7; Fig. 4A) respectively and the NMDA component by 34.7 5.1% (p < 0.05, n 5) and 32.6 6.8% (p < 0.05, n 6; Fig. 5A) respectively. These effects yielded an estimated non-NMDA/NMDA depression ratio of w0.85 for SP and w1.2 for SP5e11. Because of the almost identical effects of SP and SP5e11 on the EPSCs, these data sets were pooled to produce an average depression of 35.0 3.4% (p < 0.05, n 11) for non-NMDA and 33.6 3.8% (p < 0.05, n 11) for NMDA, yielding a ratio of 1.1. This close to unity ratio indicated that the peptides suppressed the non-NMDA and NMDA components to nearly the same extent in these nave animals. As a result of their almost identical effects on EPSCs, SP5e11, which appears to be more stable was used throughout the remainder of the study and in the gures to represent the actions for both peptides. In a similar cohort of nave rats, cocaine (30 mM) depressed the non-NMDA response by 31.6 4.5% (p < 0.05, n 5; Fig. 4A) and the NMDA component by 49.4 8.9% (p < 0.05, n 3; Fig. 5A) yielding a non-NMDA/NMDA ratio of 0.65 suggesting a preferential depression of NMDA responses.

Please cite this article in press as: Kombian, S.B., et al., Cocaine sensitization does not alter SP effects on locomotion or excitatory synaptic transmission in the NAc of rats, Neuropharmacology (2011), doi:10.1016/j.neuropharm.2011.09.008

S.B. Kombian et al. / Neuropharmacology xxx (2011) 1e8

Fig. 4. Effects of substance P or its truncated active analog and cocaine on evoked non-NMDA receptor-mediated EPSC in nave (saline-injected) and cocaine-sensitized cells. A: Averaged time-effect plots for SP5e11 (n 7) and cocaine (n 5) in nave rats superimposed for comparison. Right panel is the summary bar graph showing these effects. B: Averaged time-effect plots for SP5e11 (n 4) and cocaine (n 6) in cocaine-sensitized rats superimposed for comparison. Right panel is the summary bar graph showing these effects. In this and in all the remaining gures, the asterisk (*) represents statistical signicance at p < 0.05 compared to control (ANOVA).

Fig. 5. Effects of substance P or its truncated active analog and cocaine on evoked NMDA receptor-mediated EPSC in nave (saline-injected) and cocaine-sensitized cells. A: Averaged time-effect plots for SP5e11 (n 3) and cocaine (n 3) in nave rats superimposed for comparison. Right panel is the summary bar graph showing these effects. B: Averaged time-effect plots for SP5e11 (n 5) and cocaine (n 4) in cocainesensitized rats superimposed for comparison. Right panel is the summary bar graph showing these effects.

When these same experiments were repeated, recordings made from cells in rats that had been sensitized to cocaine (see Figs. 1 and 2), SP5e11 suppressed the non-NMDA component by 39.1 3.8% (p < 0.05, n 4; Fig. 4A) and the NMDA component by 36.1 9.6% (p < 0.05, n 5; Fig. 5A) again yielding an estimated nonNMDA/NMDA ratio of approximately 1.0. In a similar cohort of cocaine-sensitized rats, cocaine (30 mM) suppressed the non-NMDA component by 57.3 9.1% (p < 0.05, n 6; Fig. 4A) and the NMDA component by 39.6 6.3% (p < 0.05, n 4; Fig. 5A) yielding a nonNMDA/NMDA ratio of nearly 1.5. The above effect of cocaine on the non-NMDA response in sensitized rats is almost two times (1.8) the effect observed on these same responses recorded from nave rats (31.6 4.5%). This, however, was not the case for the NMDA receptor, where cocaines effects were nearly similar in magnitude. The observed enhanced effect of cocaine on the non-NMDA receptor-mediated response but not the NMDA receptor-mediated response is consistent with other reports showing that there is an increased cell surface receptor expression (upregulation) of AMPA receptors (see review by Wolf and Ferrario, 2010). This enhanced response in nave versus sensitized rats was not observed for SP5e11. To further investigate any possible SP and cocaine interaction following sensitization, we performed occlusion experiments on both the NMDA and non-NMDA receptor-mediated responses. Similar to results we previously reported (Kombian et al., 2009),

cocaine occluded SP5e11 effects on both these glutamate-mediated excitatory responses (Fig. 6A). In sensitized rats, a similar occlusion was also observed. For example, cocaine (30 mM) depressed the non-NMDA responses in a cohort by 48.7 3.7% (n 6; p < 0.05 compare to baseline; Fig. 6B and D). In four (4) of these cells for which we were able to make long lasting recording, subsequent application of 1 mM SP5e11 produced a depression of only 6.0 8.2% (p > 0.05 compared to peak cocaine effect, Fig. 6B and D). For the NMDA component, cocaine produced a depression of 39.6 6.3% (p < 0.05 compared to control, n 4) and subsequent perfusion with 1 mM SP5e11 in 3 of these cells in the presence of cocaine produced a depression of 13.5 7.2% (p > 0.05% compared to peak cocaine effect, n 3; Fig. 6C and D). These occlusion experiments show that the interaction between SP and cocaine on excitatory synaptic responses does not appear to be altered by repeated exposure to cocaine. 4. Discussion This study was conducted to determine whether SP, a non-opioid neuropeptide with synaptic actions similar to cocaine would mimic the behavioral effects of cocaine and to verify if the reported synaptic effects of SP in nave animals was altered in animals that had been repeatedly exposed to and therefore sensitized to cocaine. Here, we found that SP did not produce enhanced locomotor activity

Please cite this article in press as: Kombian, S.B., et al., Cocaine sensitization does not alter SP effects on locomotion or excitatory synaptic transmission in the NAc of rats, Neuropharmacology (2011), doi:10.1016/j.neuropharm.2011.09.008

S.B. Kombian et al. / Neuropharmacology xxx (2011) 1e8

Fig. 6. Cocaines effects on NMDA and non-NMDA receptor-mediated EPSCs occlude those of SP in cells obtained from nave (saline-injected) and cocaine-sensitized rats. A: Summary bar graph showing that, in cells obtained from nave (saline-injected) rats, SP5e11 (1 mM) no longer depresses the NMDA (n 4) or non-NMDA (n 6) components of the EPSC after the cells have been pretreated with cocaine (30 mM). B & C: Averaged time-effect plot generated from cells obtained from cocaine-sensitized rats showing that cocaine (30 mM) occludes the effect of SP5e11 (1 mM) on the non-NMDA and the NMDA components respectively. Insert in both panels are representative traces taken from the control period (left), in the presence of cocaine alone (middle) and the combined presence of cocaine and SP (right). D: Summary bar graph as in A showing the occlusive effect of cocaine on SP5e11 on both EPSC components (n 3e6) in cocaine-sensitized rats.

in either nave rats or those that had been sensitized to cocaine. Furthermore, the excitatory synaptic inhibitory effects of SP were not altered by cocaine sensitization. As shown here and widely reported in the literature, cocaine administration to rats causes increased locomotor activity that is enhanced and prolonged after repeated administration producing a phenomenon called behavioral or psychomotor sensitization which is thought to represent a drug-seeking behavior (see reviews by Kalivas and Stewart, 1991; Robinson and Becker, 1986; Wolf and Ferrario, 2010). This behavioral sensitization is reported to be associated with neurochemical, electrophysiological and structural changes in the mesolimbic dopamine system, particularly in the NAc and ventral tegmental area (VTA) (Borgland et al., 2004; Cornish and Kalivas, 2001; Kalivas and Duffy, 1993; Pierce et al., 1996b; Russo et al., 2010; Sun and Wolf, 2009; Wolf, 2010; Wolf and Ferrario, 2010). Electrophysiological changes include alteration in a-amino3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and NMDA receptor-mediated EPSCs (Beurrier and Malenka, 2002; Thomas et al., 2001; Schilstrom et al., 2006; Mameli et al., 2011). Both these changes were shown to be due to increased surface expression and localization of these receptors during and/or following cocaine exposure or sensitization (Churchill et al., 1999; Ferrario et al., 2010; Sun et al., 2008, 2005; Schilstrom et al., 2006; Mameli et al., 2011). Cocaines actions on excitatory synaptic responses and subsequent behavioral sensitization are known to be mediated primarily by dopamine (Nicola et al., 1996; Ritz et al., 1987; Tilley and Gu, 2008) and dopamine has been shown in NAc neuronal cultures to produce similar changes in AMPA receptor (AMPAR) trafcking as cocaine (Sun et al., 2008; Sun and Wolf, 2009) and to mediate cocaines effects on NMDA receptor redistribution in the VTA (Schilstrom et al., 2006). Thus, the long-term alterations in AMPA- and NMDA receptor-mediated transmission induced by cocaine in vivo may be caused in part by long-term changes in dopamine receptor signaling. If dopamine receptor signaling is that critical to cocaines effects, then it stands to reason that substances such as SP that alter or affect dopamine transmission will alter cocaines effects and vice versa. Indeed, SP has been reported to modulate cocaine-evoked dopamine overow in the rat striatum (Kraft et al., 2001a,b). Our study therefore sought to determine any interaction between cocaine and SP, a neuropeptide known to alter dopamine levels (Boix et al., 1992a,b; Elliott et al., 1986) and to produce synaptic (Kombian et al., 2009, 2003) and behavioral (Nikolaus et al., 1999a; Placenza et al., 2004) effects that are similar to those of dopamine and cocaine. Our results on the effect of acute cocaine challenge on locomotor activity in rats that had been repeatedly exposed to cocaine for a week, showed that rats were sensitized to cocaine. The corresponding enhanced cocaine-induced suppression of non-NMDA receptor but not NMDA receptor-mediated responses in the NAc conrmed that the sensitization to cocaine was successfully achieved. However, neither SP nor its truncated active metabolite SP5e11 had a signicant effect on locomotor activity. Furthermore, these peptides did not produce an enhanced suppression of nonNMDA receptor-mediated EPSCs in cocaine-sensitized animals. Thus, despite previous reports that SP-induced behavioral changes in rats (Nikolaus et al., 1999a; Placenza et al., 2004), we did not detect any SP- or SP5e11einduced changes in locomotion in nave and sensitized rats. This may have been due to the chosen IP route of administration of the peptides raising the possibility that adequate CNS concentration may not have been achieved. This, however is unlikely as both SP and SP5e11 are reported to cross the blood brain barrier and thus, should achieve signicant levels in the CNS (Banks and Kastin, 1985). For example similar IP injections of SP or its analogs in the microgram concentrations (50e250 mg/kg), have been reported to alter dopamine concentrations in the NAc (Boix et al., 1992a, 1993, 1994) possibly leading to their behavioral

Please cite this article in press as: Kombian, S.B., et al., Cocaine sensitization does not alter SP effects on locomotion or excitatory synaptic transmission in the NAc of rats, Neuropharmacology (2011), doi:10.1016/j.neuropharm.2011.09.008

S.B. Kombian et al. / Neuropharmacology xxx (2011) 1e8

effects (Hasenohrl et al., 1994; Oitzl et al., 1990). In this study, we used milligram doses (10e100 mg/kg) of SP as well as the relatively more stable SP analog but did not detect any changes in locomotor activity in either nave or in cocaine-sensitized rats. Not even the inhibition of endopeptidases with phosphoramidon produced any signicant changes in locomotor responses. Given that peripherally administered SP produces central effects (Hasenohrl et al., 1994; Oitzl et al., 1990), our results suggest that monitoring locomotor activity is not the most sensitive way to detect central or behavioral effects of SP and its analogs since reported central effects of SP monitored other behavioral paradigms or indicators (e.g. rearing, snifng, head and limb movement) which may be better indicators of cocaine-induced psychomotor sensitization (see review by Wolf and Ferrario, 2010). Although SP-induced might have been detected if given intracranially into the NAc or VTA (Nikolaus et al., 1999b; Placenza et al., 2004; Schildein et al., 1998), others have reported that they could not detect changes in locomotion when SP was injected directly into the NAc (Kalivas and Miller, 1984). Thus, in contrast to cocaine, SP or its active analog, given IP, lacked an effect on locomotion in nave and cocaine-sensitized rats. Furthermore, their effects on evoked glutamate-mediated EPSCs were not altered in slices obtained from cocaine-sensitized rats. The magnitude of non-NMDA and NMDA receptor-mediated EPSC suppression by SP and/or SP5e11 was no different in nave compared to cocaine-sensitized rats. The lack of enhanced depression of nonNMDA and/or NMDA receptor-mediated responses by SP in cocainesensitized rats tends to lend support to the in vivo locomotor studies, especially in the cocaine-sensitized group. This nding suggests that although SP elevates extracellular dopamine levels like cocaine does, the exact mechanism underlying the augmentation may be critical to causing addiction or producing the behaviors associated with addiction. For example, cocaine enhances extracellular dopamine by blocking the dopamine transporter (Ritz et al., 1987) while SP does so mainly by triggering dopamine release (DeBelleroche and Gardiner, 1983; Mantyh et al., 1984; West and Michael, 1991). Thus, it appears that repeated exposure to cocaine may cause plastic changes (see reviews by Russo et al., 2010; Wolf, 2010; Wolf and Ferrario, 2010) to the dopamine transporter leading to enhanced levels of extracellular dopamine (Akimoto et al., 1989; Kalivas and Duffy, 1990; Pettit et al., 1990; Pierce and Kalivas, 1997) while the release process may remain unchanged. This may lead to the same magnitude of SPs effect on extracellular dopamine levels regardless of previous cocaine experience. This aplastic effect or action of SP and its analogs, may have pathophysiologic and clinical implications in the future development of SP-based drugs for the management of cocaine addiction and related psychological conditions. Disclosure/conict of interest All the authors declare that there is no potential or known conict of interest as it relates to the subject matter of this manuscript. Acknowledgments We wish to thank Dr Peter Kalivas of the Medical University of South Carolina (MUSC) for according Dr Kombian the opportunity to train with his group in MUSC and providing the facilities and support for training in behavioral sensitization. Thanks also to Dr Alejandro Pacchioni who provided the hands-on training on the use of the automated animal tracking system during Dr Kombians visit to MUSC. We nally wish to thank Dr John Bradley for his help in the video analysis. This work was supported by Kuwait University Research Administration (KURA) Grant # PT01/06.

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Please cite this article in press as: Kombian, S.B., et al., Cocaine sensitization does not alter SP effects on locomotion or excitatory synaptic transmission in the NAc of rats, Neuropharmacology (2011), doi:10.1016/j.neuropharm.2011.09.008