Substance P and cocaine employ convergent mechanisms to depress excitatory synaptic transmission in the rat nucleus accumbens in vitro

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Substance P and cocaine employ convergent mechanisms to depress

excitatory synaptic transmission in the rat nucleus accumbens in vitro

Kombian, Samuel B.; Ananthalakshmi, Kethireddy V. V.; Zidichouski, Jeffrey

A.; Saleh, Tarek M.

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SYNAPTIC MECHANISMS

Substance P and cocaine employ convergent mechanisms

to depress excitatory synaptic transmission in the rat

nucleus accumbens in vitro

Samuel B. Kombian,1Kethireddy V. V. Ananthalakshmi,1Jeffrey A. Zidichouski2,3and Tarek M. Saleh3

1_{Department of Applied Therapeutics, Faculty of Pharmacy, Kuwait University, PO Box 24923, Safat 13110, Kuwait} 2_{Institute of Nutrisciences and Health, National Research Council of Canada, Charlottetown, PE, Canada}

3_{Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, Canada}

Keywords: glutamate, N-methyl-d-aspartate, non-NMDA, non-opioid peptides, occlusion, synaptic transmission

Abstract

Substance P (SP) has been reported to produce effects on excitatory synaptic transmission in the nucleus accumbens (NAc) that are similar to those induced by cocaine. To address the question of whether SP serves as an endogenous mediator producing cocaine-like effects that are known to be D1-receptor-mediated, we tested the hypothesis that the effects of SP and cocaine on excitatory postsynaptic currents (EPSCs) in the NAc occlude one another. We report here that SP and SP5–11actions occlude the effect of cocaine

and vice versa. SP, SP5–11and cocaine all depressed evoked, non-N-methyl-d-aspartate (NMDA) receptor-mediated synaptic currents

in a concentration-dependent manner, with EC50values of 0.12, 0.17 and 8.3 lm, respectively. Although cocaine was the least potent, it

was most efficacious. SP, SP5–11 and cocaine all suppressed isolated NMDA receptor-mediated evoked EPSCs. SP5–11

(1 lm)-induced EPSC depression was blocked by the neurokinin-1 antagonist L732138 and by the D1-like receptor antagonist SCH23390. Pretreatment of slices with cocaine (30 lm) depressed the EPSC by 39.1% ± 4.8%. Application of SP or SP5–11(1 lm)

at the peak of the cocaine depressive effect on the EPSC did not produce any additional diminution of the response (5.7% ± 2.8%). In the reverse experiments, in which either SP or SP5–11was applied first, subsequent application of cocaine at the peak of the peptide’s effect

(30.3% ± 2.3%) produced a further but smaller depression (15.5% ± 3.6%) of the remaining EPSC. These data indicate that cocaine and SP produce similar effects on excitatory synaptic transmission in the NAc, and that their actions occlude one another. This suggests that SP may act like cocaine in its absence, and may be an endogenous trigger for the reward and behaviors associated with cocaine.

Introduction

The cellular and molecular mechanisms that underlie the use of psychogenic substances and the etiology of reward, leading to the development of addiction and drug dependency, have been intensively investigated for more than a quarter of a century. Recently, the Society for Neuroscience commissioned a special edition of review articles in the Journal of Neuroscience devoted solely to this pervasive problem. One of the articles (Kelley & Berridge, 2002) postulated that the human brain is endowed with the ability to derive pleasure from activities that are biologically important for survival (consumption of food) and facilitate reproduction (sex). They went on to conclude that humans have discovered drugs, such as cocaine and amphetamines, that tap into this potential to induce pleasure in their absence and act as surrogates for these natural, pleasurable stimuli. In addition to the acute effects of these exogenous psychogenic compounds, it has been shown that, long after exposure to such drugs, animals and neuronal

circuits involved in mediating these actions and ⁄ or effects remain primed to the experience (Kalivas & Duffy, 1993; Ungless et al., 2001; Beurrier & Malenka, 2002). Although it is now generally accepted that long-term neuro-adaptations are responsible for these changes [see the recent review by Thomas et al. (2008)], the question arising from the above is: what endogenous substances trigger and ⁄ or maintain these behaviors in the absence of exogenous psychogenic substances such as cocaine?

Currently, overwhelming neurochemical and behavioral evidence indicates that cocaine produces its behavioral effects indirectly by increasing the levels of extracellular dopamine (DA) (Ritz et al., 1987; Kuhar et al., 1991; Kalivas & Duffy, 1993; Pierce & Kalivas, 1997; White & Kalivas, 1998; Kalivas & Volkow, 2005) in the mesolimbic DA system (Koob & Bloom, 1988; Everitt & Wolf, 2002; Picciotto & Corrigall, 2002; Weiss & Porrino, 2002). Additional evidence indicates that DA interacts with glutamate and c-aminobutyric acid (GABA) in the nucleus accumbens (NAc), and represents a critical component of this neural circuitry, which is involved in the behaviors associated with the repeated use of psychogenic compounds (Kalivas, 1995; Cornish et al., 1999;

Correspondence: Dr S. B. Kombian, as above. E-mail: kombian@hsc.edu.kw

Received 10 December 2008, revised 27 January 2009, accepted 11 February 2009

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Cornish & Kalivas, 2001; Everitt & Wolf, 2002; Dumont & Williams, 2004; Kalivas & Volkow, 2005).

One of the challenges facing researchers interested in this subject has been to identify an endogenous reinforcing trigger or mediator associated with pleasurable natural activities and the enduring effects of exogenous psychogenic substances such as cocaine, amphetamine, and related compounds. In this regard, substance P (SP), an 11 amino acid non-opioid peptide that is co-localized with GABA in medium spiny neurons of the NAc (Napier et al., 1995), has been shown to produce neurochemical and behavioral changes that are similar to those produced by cocaine (Iversen, 1982; Kalivas & Miller, 1984; Robinson & Becker, 1986; Kalivas & Stewart, 1991; Boix et al., 1992a; Huston & Hasenohrl, 1995; Schildein et al., 1998; Krasnova et al., 2000; Placenza et al., 2004). Electrophysiological evidence also showed that SP (Kombian et al., 2003) produced effects on excitatory synaptic transmission in the NAc that are similar to those of cocaine and amphetamine (Harvey & Lacey, 1996; Nicola et al., 1996). The neurochemical, behavioral and electrophysiological evidence led to the hypothesis that SP may serve as an endogenous psychogenic substance (Kombian et al., 2003; Kombian, 2004). Recent in vivo studies appear to support this hypothesis in some ways but not others. For example, Placenza et al. (2005) reported that activation of neurokinin-1 (NK1) receptors led to reinstatement of cocaine-seeking behavior, whereas blockade of these same receptors with putative NK1 receptor antagonists did not mimic the priming effects of cocaine on the drug-seeking behavior in rats. Despite this, it is still possible that appropriate changes in endogenous SP levels or activity may serve to trigger behaviors and ⁄ or maintain or retain memory of the behaviors usually triggered by exogenous psychogenic substances such as cocaine. This may be the case, because repeated or even a single cocaine exposure is known to trigger biochemical, cellular and behavioral changes that last from a few days up to 3 weeks after the last exposure (Kalivas & Duffy, 1993; Thomas et al., 2001; Ungless et al., 2001), and during this period, traces of the exogenous psychogenic compounds in plasma and ⁄ or the central nervous system may be negligible and non-pharmacological.

If this were to be the case, one would predict that the actions of SP and cocaine should be similar or convergent, and possibly occlude one another, as they may employ identical or convergent mechanisms to produce the effect. Here, we examined whether the effects of cocaine and SP on excitatory synaptic transmission in the NAc occlude one another, using the endogenous ligand and an active metabolite of the ligand, SP5–11 (Elliott et al., 1986; Boix et al., 1992b; Khan et al.,

1998). As the pharmacological effects of this truncated peptide are largely unknown, we determined its effects on excitatory synaptic responses in the NAc, and characterized its pharmacology, to determine whether they were the same as those of the endogenous ligand.

Materials and methods

International guidelines on the humane handling of animals as contained in the Canadian Council for Animal Care Guidelines were followed throughout this study, and the minimum number of animals necessary to produce the required results were used. All experiments involving cocaine were performed in Canada, under Health Canada license (File# 9639-SO219-1), and with strict adherence to use and storage requirements. Institutional guidelines for the use of animals were also followed. For experiments performed in Kuwait, male Sprague–Dawley rats were obtained from the Kuwait University Animal Resource Centre from a local breeding colony. In Canada, the

same species of male rats were purchased from Charles River Company. In both cases, rats (maximum of ﬁve per cage) were housed on a 12-h : 12-h dark ⁄ light cycle, and provided with food and water ad libitum.

Slice preparation

Parasagittal forebrain slices containing the NAc and the cortex were obtained using previously published techniques (Kombian et al., 2003). Briefly, rats (75–250 g) were anesthetized with halothane before decapitation. The brain was quickly removed from the rat, and placed in ice-cold (4C) artificial cerebrospinal fluid (ACSF), which was bubbled with 95% O2and 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, producing a

solution with an osmolarity of between 310 and 320 mOsm. Thin slices (350 lm in thickness) were cut in the ice-cold ACSF using Vibratome 1000 or 1500 series tissue slicers. Slices were incubated in ACSF (bubbled with 95% O2and 5% CO2) at room temperature, and

allowed to recover for at least 1 h before recording was commenced.

Electrophysiological recording and data acquisition

A slice was trimmed, transferred into a recording chamber, and perfused while submerged at a ﬂow rate of 2–3 mL ⁄ min (29–31C) with ACSF that was bubbled with 95% O2and 5% CO2. ‘Blind patch’

recordings were performed in the conventional whole-cell mode, using glass microelectrodes with tip resistances ranging between 4.0 and 9.0 m. The internal recording solution had the following composition: potassium gluconate (135 mm), NaCl (8 mm), EGTA (0.2 mm), Hepes (10 mm), Mg-ATP (2 mm), and GTP (0.2 mm). pH and osmolarity were adjusted to 7.3 (with KOH) and 270–280 mOsm, respectively. Bipolar tungsten stimulating electrodes were positioned at the prefrontal cortex–accumbens border to evoke synaptic responses. Recordings were made using either Axopatch 1D or a Multiclamp 700B ampliﬁer in voltage clamp mode. Cells were voltage clamped at )80 mV (holding potential, Vh) close to their resting

potentials, and input and access (Ra) resistances of all cells were

determined and monitored regularly throughout each experiment by applying a 25–75 ms, 20 mV hyperpolarizing pulse. All cells reported in this study had Ravalues of 10–50 MX. Data from cells that showed

> 15% changes in Ra during the experiment were excluded from

further analysis.

All evoked synaptic responses [excitatory postsynaptic currents (EPSCs)] were routinely recorded in the presence of picrotoxin (50 or 100 lm) as inward currents at Vh of )80 mV, unless stated

otherwise. All cells had a graded evoked response to increasing stimulation intensity (ranging from 0.25 to 5.0 mA), and an intensity giving 50–60% of the maximum evoked synaptic response was used to evoke test responses. Each stored trace was an average of two successive synaptic responses elicited at 30 s intervals, yielding one averaged response per minute.

Sodium currents were recorded in voltage clamp mode using standard biophysical step protocols for activating sodium currents. Step-activated sodium currents were recorded in the presence of a solution containing tetraethylammonium (5 mm), cesium (Cs+_{; 1 mm)}

and cadmium (Cd2+; 100 lm). Series resistance was compensated at 80%. A protocol comprising a series of voltage steps ranging from )65 to +70 mV in 15 mV increments was applied to cells, and each step was preceded by a pre-pulse to )120 mV. Leak subtraction was applied to all currents to obtain the sodium current. All data were 1580 S. B. Kombian et al.

ª_{The Authors (2009). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd} European Journal of Neuroscience, 29, 1579–1587

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acquired using pClamp software (Clampex 8.2 or 9.2; Axon Instruments) at a sampling rate of 4–6.0 KHz, ﬁltered at 1 or 10 KHz, digitized, and stored for off-line analysis.

Analysis and statistics

For non-N-methyl-d-aspartate (NMDA) responses, EPSC amplitudes (at Vhof )80 or )100 mV) were measured from baseline to peak, and

taken as the synaptic strength at the chosen stimulus intensity. For isolated NMDA responses, the average duration of non-NMDA responses was determined, and the peak of the NMDA response was measured at 10 ms after this point, which was about 45 ms after the stimulus artefact. Responses were normalized by taking the mean of the last four or five responses prior to drug application and dividing the rest of the responses by this mean. These normalized values were then used for average plots. For these plots, all cells receiving the same treatment were aligned at the first minute of the first drug application, and averaged over the entire period. For peak drug effects, the last three synaptic responses (3 min) in the presence of a drug and the first two responses during washout were averaged. Percentage changes were then calculated by dividing this averaged peak response by the averaged baseline response multiplied by 100, )100. All values are given as mean ± standard error. One-way anova and post hoc tests, as indicated in Results, were used to compare different treatments (raw data or percentage changes) using SigmaStat (version 3.5) software (Systat Software Inc., San Jose, CA, USA). Differences between groups were taken as significant at a probability level of P = 0.05. Graphs were produced using SigmaPlot (version 9) (Systat Software Inc.), GraphPad Prism (version 3) (GraphPad Software, La Jolla, CA, USA), and CorelDraw (version 12) (Corel Corporation Inc., Ottawa, ON, Canada).

Chemicals and solutions

All drugs were bath perfused at the ﬁnal concentrations indicated, obtained by dissolving aliquots of stock in the ACSF. Most stock solutions were prepared in the solvents suggested by the manufacturer, and stored at )20C. All laboratory salts were purchased from Sigma Company (Germany and USA). Tetraethylammonium, Cs+, tetrodo-toxin, Cd2+_{, SP and SP}

5–11, dl-2-amino-5-phosphonovalerate and

picrotoxin were also obtained from Sigma, and stock solutions were made by dissolving them in water. 6,7-Cyanoquinoxaline-2,3-dione (CNQX) was obtained from RBI (Natick, MA, USA), and dissolved in dimethylsulfoxide. Cocaine hydrochloride was purchased from Sigma (Natick, MA, USA), and the stock solution was prepared fresh every day by dissolving in water.

Results

The results were obtained from whole-cell recordings in 130 neurons located in the core region of the rostral NAc. The majority of cells in this region (95%) are medium spiny GABAergic neurons (Pennartz et al., 1994), and these characteristically exhibited very negative resting membrane potentials ranging from )72 to )90 mV. The other passive and active membrane properties of these cells were similar to those previously reported (Kombian et al., 2003). Five cells were discarded because they displayed synaptic and active membrane properties that were markedly different from those of the rest. These cells had low resting potentials, high capacitance, high input resistance, and low spike thresholds with synaptic responses that often had action potentials riding on them. These probably

corresponded to the large soma aspiny cholinergic interneurons, which make up about 5% of the neuronal population of the NAc (Pennartz et al., 1994).

Cocaine, SP and SP5–11all suppress excitatory synaptic

transmission in the NAc

Under control conditions, stimulation of the cortico-accumbens afferents results in a mixed response mediated by glutamate and GABA (Pennartz & Kitai, 1991). Most of the experiments reported here were performed with cells voltage clamped near their resting potential. At our routine holding potential of )80 mV, currents mediated by both of these transmitters were inward. In order to study pure glutamate-mediated excitatory synaptic responses, picrotoxin (50 or 100 lm) was present in the perfusion medium throughout or prior to recording evoked responses. The inward synaptic response under this condition was occasionally veriﬁed to be a pure, (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propanoate ⁄ kainate (non-NMDA) glutamate receptor response by applying CNQX (10 lm), which completely abolished the evoked currents (Fig. 1A; n = 7). As previously reported (Kombian et al., 2003), SP caused a largely irreversible inhibition of the evoked non-NMDA receptor-mediated EPSCs. For example, 1 lm SP caused 33.4% ± 4.8% depression (n = 7; paired t-test; P = 0.001; Figs 1B and 2A). Similar to SP, 1 lm SP5–11also irreversibly depressed the EPSCs (32.8% ± 5.0%; n = 8;

paired t-test; P = 0.001; Figs 1C and 2A). Unlike the peptides, cocaine (30 lm) caused a reversible depression of the EPSC amplitude (42.3% ± 4.7%; n = 10; paired t-test; P = 0.001; Figs 1D and 2A). The effects of cocaine and of both peptides on EPSCs were all concentration-dependent (Fig. 2B). SP at concentrations < 10 nm produced no detectable synaptic depression, whereas the maximum synaptic depression of 42.8% ± 10.1% (n = 3) was recorded with 10 lm SP. This resulted in a calculated )log EC50(pD2) of 6.9 ± 0.29.

SP5–11, on the other hand, produced a maximum depression of

32.8% ± 5.0% (n = 8) at 1 lm, with a calculated )log EC50 of

6.8 ± 0.44. Cocaine had no detectable effect on the EPSC amplitude at concentrations below 100 nm, but caused a maximal suppression of 65.7% ± 5.0% (n = 7) at 100 lm, with a calculated )log EC50 of

5.1 ± 0.24 (Fig. 2B). These pD2 values were equivalent to the

following EC50 values: SP, 0.12 lm; SP5–11, 0.17 lm; and cocaine,

8.3 lm.

To study pure NMDA receptor-mediated responses, we used two isolation protocols: a combination of biophysical and pharmacological protocols, and a purely biophysical protocol (Fig. 3). In the combination protocol, CNQX (10 lm) was used to abolish the non-NMDA component recorded at )80 mV. Subsequently, the membrane potential was depolarized up to )50 or )40 mV, and the synaptic response was then triggered (Fig. 3A). The responses recorded under this condition were shown to be pure NMDA-mediated responses, as they were sensitive to blockade by dl-2-amino-5-phosphonovalerate (100 lm; n = 3) applied at the end of some of the experiments. In the purely biophysical protocol, non-NMDA synaptic responses were ﬁrst elicited by stepping the holding membrane potential from )80 to )100 mV prior to synaptic stimulation. Fifty milliseconds after this response, the holding potential was stepped from )100 to )20 mV, and it was held there for about 150 ms to stabilize the membrane; the NMDA receptor-mediated synaptic response was then triggered, and the membrane was returned to the routine holding level of )80 mV (Fig. 3B).

In the biophysical recording protocol, SP (1 lm) depressed the non-NMDA component (at )100 mV) by 33.4% ± 4.8% (n = 7), an effect that was not signiﬁcantly different from that found under routine

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recording conditions, when cells were held at )80 mV throughout the experiment (27.4% ± 2.1%; n = 5; F1,10= 0.53; P = 0.48; Fig. 2C

and D). In these same cells, SP (1 lm) also depressed the NMDA component (see measurement criteria in Materials and methods) by 31.7% ± 4.5% (n = 7), an effect that was not signiﬁcantly different from that recorded using the combination protocol to isolate this component (34.7% ± 3.6%; n = 5; F1,10= 0.26; P = 0.62; Fig. 2C

and D). Similar results were obtained with 1 lm SP5–11(Fig. 3D). For

example, this truncated SP analog suppressed the non-NMDA component (isolated via the biophysical protocol) by 39.8% ± 5.8% (n = 6), as compared with the 32.8% ± 5.0% reduction (n = 8; F1,12= 1.04; P = 0.33; Fig. 3D) recorded routinely at )80 mV. The

effect of SP5–11on the NMDA component was 32.6% ± 6.7% (n = 6),

as compared with 36.6% ± 6.7% (n = 5; F1,9= 0.21; P = 0.66;

Fig. 3D), for the biophysical and combination isolation, respectively. These results indicate that SP and the truncated analog SP5–11produce

nearly identical effects on excitatory synaptic transmission in the NAc.

Because the two isolation protocols for NMDA responses produced similar results, and the combined isolation (pharmacological and biophysical) produced more stable, longer-lasting recordings and unambiguous NMDA responses, this protocol was employed through-out the rest of this study. Similar to its effect on non-NMDA receptor-mediated responses, cocaine (30 lm) also depressed the NMDA component by 47.8% ± 4.5% (n = 4; paired t-test; P = 0.001; Fig. 4). Unlike the non-NMDA receptor current suppression, this effect did not appear to substantially recover during the approximately 10–15-min washout period used in this study. Thus, SP, SP5–11and

cocaine all suppressed both non-NMDA and NMDA receptor-mediated responses in the NAc.

SP5–11-induced EPSC depression is NK1 receptor-mediated

and DA D1-like receptor-mediated

We have previously studied and characterized the pharmacology of the actions of cocaine (Nicola et al., 1996) and SP (Kombian et al., 2003) on excitatory synaptic responses in the NAc. To determine whether the truncated SP analog has identical pharmacological actions to those of cocaine and SP, we examined whether the EPSC depressant effects

Fig. 2. Substance P (SP)-induced, SP5–11-induced and cocaine-induced excitatory postsynaptic current (EPSC) depressions are concentration depen-dent. (A) Bar graph summarizing the effect of 1 lm SP, 1 lm SP5–11and 30 lm cocaine on the EPSC amplitude. In this ﬁgure and in all other ﬁgures, *P £ 0.05, compared to control, and the n-value above each group of bars indicates the number of cells that received the same concentration of the compound. (B) Concentration–response curves generated by applying different concentrations of the three different compounds. Each point on the graph has an n-value of 3–10 cells.

Fig. 1. Substance P (SP), a truncated analog of SP (SP5–11) and cocaine all depress excitatory postsynaptic currents (EPSCs) recorded in the nucleus accumbens (NAc). (A) Sample EPSCs recorded in an NAc cell at a holding potential of )80 mV in control, after 6–8 min of bath application of selected concentrations of each compound, and following 10–15-min washout of the applied compound. Finally, a 2–4-min application of 6,7-cyanoquinoxaline-2,3-dione (CNQX) completely abolished the response. (B) Normalized and averaged time–effect plot for SP (1 lm), SP5–11 (1 lm; C), and cocaine (30 lm; D). In contrast to the reversible effect of cocaine, the effects of SP and SP5–11did not recover for the duration of washout (10–20 min).

1582 S. B. Kombian et al.

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observed above involved the same receptor(s) as the effects of SP and cocaine. First, we tested whether, like that induced by SP, SP5–11

-induced non-NMDA receptor-mediated EPSC depression was

medi-ated through the NK1 receptors, by pretreating slices with the NK1 receptor selective antagonist L732138 (10 lm) (MacLeod et al., 1993; Kombian et al., 2003). Although L732138 did not produce a signif-icant change in this response (15.5% ± 18.0%; n = 5; paired t-test; P = 0.39), it blocked the ability of 1 lm SP5–11 to depress this

response (2.8% ± 2.8%; n = 5; F2,12= 0.65; P = 0.54; Fig. 5). As the

effects of cocaine and SP on EPSCs have been reported to be mediated by DA through D1-like receptors (Harvey & Lacey, 1996; Nicola et al., 1996; Kombian et al., 2003), we tested whether the SP5–11effect

on synaptic transmission was also DA-dependent. Bath application of SCH23390 (30 lm) did not produce a signiﬁcant change in EPSC amplitude (10.7% ± 4.4%; n = 5; paired t-test; P = 0.06), but blocked the ability of 1 lm SP5–11 to depress the response (9.7% ± 3.7%;

n = 5; F2,12= 0.08; P = 0.92; Fig. 5). These results indicate that, like

the endogenous peptide, the truncated analog works through NK1 and DA D1-like receptors to suppress EPSCs in the NAc.

Cocaine-induced, SP-induced and SP5–11-induced EPSC

depression are mutually occlusive

Previous work on cocaine and SP has indicated that both of these compounds modulate excitatory synaptic transmission in the NAc by

Fig. 3. Substance P (SP) and SP5–11 depress both N-methyl-d-aspartate (NMDA) and non-NMDA receptor-mediated responses to a similar extent. NMDA receptor-mediated responses were recorded using two protocols. (A) A combination of pharmacological and biophysical protocols was used, whereby the non-NMDA response was first recorded at the routine holding potential of )80 mV. Subsequently, the selective non-NMDA receptor antagonist 6,7-cyanoquinoxaline-2,3-dione (CNQX) was applied, and abolished the response. The potential was then depolarized to a holding potential of between )40 and )50 mV to reveal the NMDA component. The insert includes sample traces of non-NMDA and NMDA responses alone and superimposed. (B) In the purely biophysical protocol, both the non-NMDA and NMDA responses were recorded simultaneously by first hyperpolarizing the cell to )100 mV from the holding potential of )80 mV. The membrane was then stepped to a new holding potential of )20 mV, and the same stimulus was repeated; this then evoked a combination of non-NMDA and NMDA responses with different kinetics. The insert below is a representative trace showing the two recordings. (C) Normalized and averaged time–effect plots of the effect of 1 lm SP on the two synaptic components recorded in B (see Materials and methods for quantification of the NMDA component). (D) A summary bar graph showing the effect of SP (1 lm) and SP5–11(1 lm) on the NMDA and non-NMDA receptor-mediated responses recorded using the two different protocols. EPSC, excitatory postsynaptic current. *P £ 0.05, compared with control.

Fig. 4. Cocaine depresses N-methyl-d-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSCs). (A) Normalized and averaged time– effect plots for the effects of cocaine (30 lm) on the NMDA-receptor mediated EPSCs recorded using the combination protocol. Following a 10-min washout of cocaine, dl-2-amino-5-phosphonovalerate (dl-APV) (100 lm) application abolished the remaining response. (B) A summary bar graph showing the effects of 30 lm cocaine (n = 4) and dl-APV (n = 3) on the NMDA receptor-mediated responses. *P £ 0.05, compared with control.

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increasing the levels of extracellular DA, which then activates presynaptic D1-like receptors to suppress EPSC amplitude (Harvey & Lacey, 1996; Nicola et al., 1996; Kombian et al., 2003). This has led to the suggestion that SP may serve as an endogenous psychogenic peptide (Kombian et al., 2003; Kombian, 2004). If this were to be the case, one would predict that the effect of SP on EPSCs would be blunted in the presence of cocaine and vice versa. To test the hypothesis that the effects of SP and cocaine effects on excitatory synaptic transmission are mutually occlusive, we per-formed experiments whereby the effects of SP (1 lm) on EPSCs in the presence of cocaine (30 lm) were tested. We chose these concentrations because 1 lm peptide produced the most consistent optimal synaptic depression, and 30 lm cocaine has been used in previous studies and shown to be non-anesthetic. In eight cells, the application of 30 lm cocaine resulted in a peak EPSC depression of 39.1% ± 4.8% (n = 8) (Fig. 6A and C). This concentration of cocaine has previously been shown to produce synaptic depression through a non-anesthetic mechanism (Nicola et al., 1996), and this was conﬁrmed in this study, when no effect on tetrodotoxin-sensitive sodium currents was observed with 30 lm cocaine (6.2% ± 4.2%; n = 6; P = 0.1; paired t-test). When SP (1 lm; n = 6) or SP5–11

(1 lm; n = 2) was subsequently applied at the time of this peak effect, neither peptide produced any further depression in the EPSC

amplitude (5.7% ± 2.8%; n = 6; paired t-test; P = 0.54 as compared with the peak cocaine effect; Fig. 6A and C). When the converse experiments, whereby SP (n = 4) was applied ﬁrst, it produced a peak EPSC suppression of 27.0% ± 3.8% (n = 4; Fig. 6B). When cocaine (30 lm) was subsequently applied at the time of the peak effect, it produced a further suppression of 16.7% ± 3.4% (n = 4; paired t-test; P = 0.03 as compared with the peak effect of SP; Fig. 6B and C), yielding a combined depression of 38.7% ± 3.5% (n = 4; F1,12= 0.03; P = 0.87 as compared with the effect of cocaine

alone). Similar to the SP action described above, 1 lm SP5–11

(n = 2) also produced a suppression that partially occluded cocaine’s effects.

Fig. 5. Depression of excitatory postsynaptic currents (EPSCs) induced by a truncated analog of substance P (SP5–11) is blocked by neurokinin-1 (NK1) and dopamine receptor antagonists. (A) Normalized and averaged time–effect plots in obtained from seven cells that were pretreated with SCH23390 (30 lm) prior to SP5–11(1 lm) application. (B) A bar graph summarizing the effect of SP5–11 in the control (n = 7), in the presence of an NK1 receptor antagonist (L732138, 10 lm, n = 5), and in the presence of a dopamine D1-like receptor antagonist (SCH23390, 30 lm, n = 7). *P £ 0.05, compared with control.

Fig. 6. Cocaine and substance P (SP)-induced excitatory postsynaptic current (EPSC) depression are mutually occlusive. (A) A normalized averaged time– effect plot obtained from six cells, showing the effect of 30 lm cocaine on evoked EPSCs, and that the subsequent application of 1 lm SP caused no additional synaptic depression. (B) A normalized averaged time–effect plot obtained from four cells, showing the effect of 1 lm SP on evoked EPSCs, and that the subsequent application of 30 lm cocaine caused additional synaptic depression. (C) A summary bar graph depicting the effects of cocaine and SP, alone and in combination, on EPSC amplitude (*P £ 0.05, compared with control; **P < 0.05, compared with peptide effects).

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Discussion

The results of this study show that SP and its truncated metabolically active analog SP5–11 produce similar effects on excitatory synaptic

responses recorded in the NAc, with nearly identical potencies and efﬁcacies. By contrast, cocaine reversibly depresses these same responses with superior efﬁcacy but lower potency than the peptides. Finally, our results show that the actions of the peptides and cocaine are mutually occlusive, suggesting that they employ a convergent mechanism to depress EPSCs in the NAc (Fig. 7).

We previously reported that SP, through activation of presynaptic NK1 receptors, caused the release of DA, which in turn increased the levels of extracellular adenosine, resulting in synaptic depression of fast, non-NMDA receptor-mediated EPSCs in the NAc (Kombian et al., 2003). Here, we report that the active metabolite of SP, SP5–11,

depresses EPSCs in the NAc by a similar mechanism, although in this case we did not test the involvement of adenosine. The almost

identical potency, efﬁcacy and irreversibility of SP and SP5–11actions

and the blockade of SP5–11effects by the same concentrations of NK1

and DA D1-like receptor antagonists support this position. Further-more, the effects of the endogenous peptide or its metabolite on the NMDA receptor-mediated response were also the same, regardless of the method used to record this response. In contrast to the irreversible effects of SP and SP5–11, the effect of cocaine on the non-NMDA

receptor-mediated fast EPSCs was reversible, but it was less potent by about two log units (EC50 values approximately 0.1 vs. 10 lm).

However, cocaine was more efﬁcacious, producing a maximum depression of up to approximately 70% at 100 lm, a concentration that may have a local anesthetic effect (Dunwiddie et al., 1988). The observed effects of cocaine in this study are similar to those already found in other in vitro studies (Harvey & Lacey, 1996; Nicola et al., 1996). The concentration of 30 lm, a non-local anesthetic concentra-tion (Dunwiddie et al., 1988; Nicola et al., 1996), produced an effect (40%) that was slightly greater than the maximum depression produced by a 1 lm concentration of either SP or SP5–11(30%). At

the peak of the cocaine (30 lm)-induced depression of EPSCs, subsequent application of 1 lm of either SP or SP5–11 failed to

produce any additional depression of EPSCs. However, when the converse experiment was performed, whereby one of the peptides was applied ﬁrst, subsequent application of cocaine at the time of their peak effect produced a further depression of EPSCs that was signiﬁcantly different from that at the peptide peak. The combined magnitude of the peptide-induced depression and that caused by cocaine in the presence of the peptides was approximately 40%, an effect that was not different from that produced by the test concentration (30 lm) of cocaine alone, indicating that the peptides partially occluded the cocaine effect at their maximal concentration. These results suggest that the peptides and cocaine employ a convergent pathway to suppress synaptic responses in the NAc (Fig. 7).

Cocaine is known to have two effects on neurons: a local anesthetic effect (Warnick et al., 1971; Ritchie & Green, 1990), and a non-local anesthetic effect (Ritz et al., 1987). These actions are concentration-dependent, such that, at lower concentrations, only the non-local anesthetic effect, which is inhibition of the DA transporter, is observed (Ritz et al., 1987; Tilley & Gu, 2008), whereas at higher concentra-tions, both of these effects may be present. Previous in vitro studies have revealed that the cocaine-induced synaptic depressant effect in the NAc is indirect, through DA acting on D1-like receptors, as the effect could be blocked or reversed by SCH23390, a DA D1-like antagonist (Harvey & Lacey, 1996; Nicola et al., 1996), although another group has reported the involvement of D2-like receptors in DA-induced synaptic depression in the NAc (O’Donnell & Grace, 1994). The fact that SP (Kombian et al., 2003) and its active metabolite SP5–11both have effects on EPSCs that are blocked by

SCH23390 suggests that these peptides and cocaine may employ a common mechanism to depress excitatory synaptic transmission. Moreover, pretreatment of NAc slices with cocaine prevented the peptides from producing further synaptic depression, strongly sug-gesting that cocaine and these peptides employ identical mechanisms to depress EPSCs in the NAc. The point of convergence is therefore DA, after which point the second messenger system(s) are common. Thus, whereas cocaine blocks the DA transporter (Ritz et al., 1987), increasing extracellular DA levels, SP may act to release DA from the presynaptic terminal (DeBelleroche & Gardiner, 1983). This increased extracellular DA then activates presynaptic D1-like receptors, ulti-mately leading to a decrease in EPSC amplitude (Harvey & Lacey, 1996; Nicola et al., 1996). We have shown that, as in the signal transduction pathways reported for cocaine (White & Kalivas, 1998;

Fig. 7. A schematic diagram showing the possible point of convergence of the effects of cocaine and substance P (SP) on excitatory synaptic transmission in the nucleus accumbens (NAc) and subsequent behavior. DA, dopamine; DAT, DA transporter; NK1, neurokinin-1.

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White et al., 1998; Nestler, 2001; White, 2002), the SP effect on EPSCs is mediated by cAMP-dependent and protein kinase C-dependent pathways (Matowe et al., 2007). Cyclic AMP, protein kinase A and protein kinase C have been reported to couple the DA receptors to their effectors (Berke & Hyman, 2000).

In conclusion, we have shown that SP, and one of its active metabolites, SP5–11, produce similar effects to those of cocaine on

evoked EPSCs in the NAc in vitro. The question is, how relevant is this in vitro finding to in vivo cocaine effects and their behavioral consequences or sequelae? Recent in vivo evidence indicates that some of the behavioral effects of cocaine can be induced by SP or its active analogs, whereas others cannot. For example, in one report, although endogenous SP did not mimic the cocaine-induced priming of drug-seeking behavior in rats, it was able to elicit the reinstatement of the drug-seeking behavior primed by cocaine (Placenza et al., 2004, 2005). In other studies, knockout of NK1 receptors in mice failed to alter cocaine-induced reward and locomotor behaviors (Murtra et al., 2000; Ripley et al., 2002). These conflicting findings on the role of SP in cocaine-induced behaviors may suggest a complex action of this peptide in vivo, whereby the pharmacological agonism and antago-nism and molecular biology knockout experiments may be con-founded by compensatory mechanisms operational in these in vivo experiments. A possible explanation for the reported inability of SP to replicate all of the behavioral effects of cocaine is that some of these effects of cocaine may be related to the efficacy of synaptic modulation rather than potency. As reported in this study, cocaine, although less potent, was more efficacious than both SP and its active metabolite in inhibiting excitatory synaptic transmission recorded in the crucial NAc region. Thus, the results of these in vitro experiments strongly suggest that SP can produce the same effects as cocaine, and this raises the possibility that, in an appropriate enabling in vivo situation, this peptide would produce the effects associated with cocaine use. The continuing challenge, then, is to identify the conditions under which SP may replicate all of the effects of cocaine.

Acknowledgements

This work was supported by Kuwait University grant no. PT01 ⁄ 06 to S. B. Kombian. Work in T. M. Saleh’s laboratory was supported by an AIF (grant no. 189266).

Abbreviations

ACSF, artiﬁcial cerebrospinal ﬂuid; CNQX, 6,7-cyanoquinoxaline-2,3-dione; DA, dopamine; EPSC, excitatory postsynaptic current; GABA, c-aminobutyric acid; NAc, nucleus accumbens; NK1, neurokinin-1; NMDA, N-methyl-d-aspartate; Ra, access resistance; SP, substance P; Vh, holding potential.

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