Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2016 Jan 31.
Published in final edited form as: Neuropharmacology. 2014 Sep 16;89:113–121. doi: 10.1016/j.neuropharm.2014.09.001

Nucleus accumbens shell excitability is decreased by methamphetamine self-administration and increased by 5-HT2C receptor inverse agonism and agonism

Steven M Graves 1,*, Mary J Clark 3, John R Traynor 3, Xiu-Ti Hu 1, T Celeste Napier 1,2
PMCID: PMC4321813  NIHMSID: NIHMS628546  PMID: 25229719

Abstract

Methamphetamine profoundly increases brain monoamines and is a widely abused psychostimulant. The effects of methamphetamine self-administration on neuron function are not known for the nucleus accumbens, a brain region involved in addictive behaviors, including drug-seeking. One therapeutic target showing preclinical promise at attenuating psychostimulant-seeking is 5-HT2C receptors; however, the effects of 5-HT2C receptor ligands on neuronal physiology are unclear. 5-HT2C receptor agonism decreases psychostimulant-mediated behaviors, and the putative 5-HT2C receptor inverse agonist, SB 206553, attenuates methamphetamine-seeking in rats. To ascertain the effects of methamphetamine, and 5-HT2C receptor inverse agonism and agonism, on neuronal function in the nucleus accumbens, we evaluated methamphetamine, SB 206553, and the 5-HT2C receptor agonist and Ro 60-0175, on neuronal excitability within the accumbens shell subregion using whole-cell current-clamp recordings in forebrain slices ex vivo. We reveal that methamphetamine self-administration decreased generation of evoked action potentials. In contrast, SB 206553 and Ro 60-0175 increased evoked spiking, effects that were prevented by the 5-HT2C receptor antagonist, SB 242084. We also assessed signaling mechanisms engaged by 5-HT2C receptors, and determined that accumbal 5-HT2C receptors stimulated Gq, but not Gi/o. These findings demonstrate that methamphetamine-induced decreases in excitability of neurons within the nucleus accumbens shell were abrogated by both 5-HT2C inverse agonism and agonism, and this effect likely involved activation of Gq–mediated signaling pathways.

1. Introduction

Psychostimulants increase synaptic concentrations of monoamines and neuronal maladaptations can be a persistent consequence of repeated administration (for review, see (Kauer and Malenka, 2007)). Some maladaptations reflect changes in serotonin (5-HT) systems (Napier and Istre, 2008, Krasnova et al., 2010, Reichel et al., 2012) and 5-HT2C receptors (Rs) are likely involved. For example, chronic methamphetamine (meth) increases neuronal sensitivity in the limbic (ventral) pallidum to systemic administration of a 5-HT2A/2CR agonist (Napier and Istre, 2008). Moreover, agonists at these receptors attenuate cocaine-associated reinstatement (Grottick et al., 2000, Fletcher et al., 2002, Neisewander and Acosta, 2007, Burbassi and Cervo, 2008, Cunningham et al., 2011), self-administration (Grottick et al., 2000, Fletcher et al., 2002, Cunningham et al., 2011), as well as behavioral hyperactivity and sensitization (Grottick et al., 2000, Filip et al., 2004). 5-HT2CRs are constitutively active in rat brain (De Deurwaerdere et al., 2004), antagonists or inverse agonists with high affinity for 5-HT2A/2CRs nullify meth-induced neuronal sensitization (McDaid et al., 2007), and the putative 5-HT2CR inverse agonist SB 206553 (SB206) reduces meth-seeking and meth-evoked motor activity (Graves and Napier, 2012).

5-HT2CR inverse agonists and agonists exhibit similar behavioral effects in rats (Graves and Napier, 2012, Navailles et al., 2013). In contrast, neurochemical studies show that inverse agonists increase accumbal and striatal dopamine (De Deurwaerdere et al., 2004), whereas agonists have no effect, or decrease dopamine (Di Matteo et al., 1998, Willins and Meltzer, 1998, Di Matteo et al., 2000, Gobert et al., 2000). Discord also exists regarding 5-HT2CR-mediated function at the cellular level; inverse agonists decrease 5-HT2CR-linked second messengers (e.g., basal phospholipase (PL) PLC, PLA2 and Gi/o activity) (Berg et al., 1999, De Deurwaerdere et al., 2004, Berg et al., 2006, Berg et al., 2008a, Berg et al., 2008b, Labasque et al., 2010), but increase surface expression of 5-HT2CRs (Marion et al., 2004, Chanrion et al., 2008). However, after incubation with an inverse agonist, responses to serotonin are enhanced (Berg et al., 1998a, Marion et al., 2004, Chanrion et al., 2008), presumably due to the increased surface expression. Pleiotropic signaling further complicates 5-HT2CR function. The canonical pathway involves Gq stimulation (Cussac et al., 2002), yet the receptors are also reported to signal via Gi/o and G13 proteins as well as non-G protein coupled pathways (Berg et al., 1998b, Cussac et al., 2002, McGrew et al., 2002, De Deurwaerdere et al., 2004, Werry et al., 2005, Labasque et al., 2008, Labasque et al., 2010).

To advance understanding of 5-HT2CRs on meth-induced neuronal function and to provide insights into signaling mechanisms engaged in medium spiny neurons (MSNs) of the nucleus accumbens by these receptors, we used electrophysiological and biochemical approaches to evaluate 5-HT2CRs from rats trained to self-administer meth. Based in part on our prior demonstration that SB206 attenuates meth-seeking behavior (Graves and Napier, 2012), we hypothesized that acute 5-HT2CR inverse agonism opposes meth-induced adaptations in the nucleus accumbens shell and that 5-HT2CRs in the nucleus accumbens engage the canonical Gq pathway.

2.0 Materials and methods

2.1 Animals

Seventy-seven male Sprague-Dawley rats were purchased from Harlan (Indianapolis, IN). Subjects were housed in pairs, acclimated to the vivarium for 5 days, and handled a minimum of 3 times prior to the surgical procedures required for self-administration protocols. Food and water were provided ad libitum throughout the study. Rats were maintained in accordance with the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978) and with approval of the Rush University Institutional Animal Care and Use Committee. All efforts were made to minimize suffering and reduce the number of animals used.

2.2 Test Drugs

(+)-Methamphetamine HCl (meth; Sigma, St. Louis, MO) was dissolved in sterile saline. The stimulant was self-administered at 0.1mg/kg/0.1ml intravenously (iv). Ro 60-0175 (Ro), SB 206553 (SB206), and SB 242084 (SB242) were purchased from Tocris Bioscience (Ellisville, MO), dissolved in ddH20 as 1.0mM stock solutions, and added at appropriate concentrations to artificial cerebrospinal fluid (aCSF) for electrophysiological studies, or assay buffer for biochemical studies.

2.3 Intravenous catheter implantation

Isoflurane-anethetized rats (n=77) were instrumented as previously described (Graves and Napier, 2011) with custom built catheters constructed using silastic tubing (0.3mm i.d. x 0.64mm o.d.; Dow Corning Co., Midland, MI) and implanted into the right jugular vein. The distal end of the catheter extended to the mid-scapular region with a metal guide canulae (22 gauge; Plastics One Inc., Roanoke, VA) and anchored to a plastic mesh. Rats were allowed to recover for a minimum of 5 days prior to initiating self-administration procedures.

2.4 Self-Administration

Forty-one rats were trained to self-administer meth for 3hr/day for 14 days in operant chambers enclosed in ventilated, sound-attenuating cabinets (Med-Associates, St. Albans, VT). Each operant chamber contained two levers; the left lever was assigned as the “active” lever and the right lever was assigned as the “inactive” lever. Above each lever was a “cue” light, and located on the opposite wall was an “in-house” light. The cue light above the active lever was illuminated when the infusion pump was activated. The in-house light was subsequently illuminated for 20s, indicating a “time-out” period during which responses had no programmed consequences. Responding on the inactive lever had no programmed consequences. On protocol days 1–7, rats self-administered meth for 3hr/day on a fixed ratio (FR) 1 schedule of reinforcement; on days 8–14 rats self-administered on a FR5 for 3hr/day. The described paradigm was chosen based on our prior studies demonstrating stable and consistent self-administration, as well as the ability of mirtazapine (Graves and Napier, 2011) and SB206 (Graves and Napier, 2012) to attenuate meth-seeking behavior.

Thirty-six saline-yoked rats were used as controls. These subjects were implanted with jugular vein catheters as described for the meth self-administration protocol; 0.1ml infusions of saline were administered with cue and time out lights triggered according to the behavioral pattern of a meth self-administering rat (lever pressing had no programmed consequences). A total of 4 rats died after a behavioral test session (2 saline-yoked and 2 meth self-administering rats).

2.5 Electrophysiological experiments

On protocol days 15–18 (1–4 days after last behavioral test), rats were anesthetized with chloral hydrate (400mg/kg, ip) and transcardially perfused with 60mL of an ice cold, modified aCSF containing (in mM): 248 sucrose, 2.9 KCl, 2.0 MgSO4, 1.25 NaH2PO4, 10.0 glucose, 26.0 NaHCO3, 0.1 CaCl2, 3.0 kynurenic acid, and 1.0 ascorbic acid (225–235mOsm, pH=7.4). Forebrain coronal slices (300μm thick) containing the nucleus accumbens shell were cut with a vibrating microtome (Leica VT 1000S; Leica Microsystems Inc., Buffalo Grove, IL), and transferred to a holding chamber containing normal aCSF (in mM: 125 NaCl, 2.5 KCl, 1 MgCl2, 25 NaHCO3, 1.25 NaH2PO4, 15 glucose, and 2 CaCl2; 305–315mOsm, pH=7.4) with 1mM ascorbic acid at room temperature. A gas mixture of 95% O2/5% CO2 was constantly bubbled for a minimum of 45min prior to beginning patch clamp experiments. The nucleus accumbens shell was selected for study based on evidence for constitutively active 5-HT2CRs (Navailles et al., 2006) and its involvement in addiction (Di Chiara, 2002, Di Chiara et al., 2004). An early period of withdrawal was selected for study based on the ability of mirtazapine (Graves and Napier, 2011) and SB206 (Graves and Napier, 2012) to attenuate meth-seeking, and presence of psychostimulant-induced plasticity (Kourrich and Thomas, 2009) during this period.

Slices containing the nucleus accumbens shell were moved from the holding to the recording chamber which was perfused at a rate of 2ml/min by normal aCSF and held at 34°C. Neurons were visualized using an Olympus BX51WI upright microscope (Olympus Tokyo, Japan) and targeted under a 40X water-immersion objective, differential interference contrast, and infrared filter. The image from the microscope was enhanced using an IR-1400 DAGE-MIT (Michigan City, IN) camera and displayed on a computer monitor. Electrodes were pulled with a P-97 or P-1000 micropipette puller (Sutter Instruments, Novato, CA) to a resistance of 4–6MΩ and filled with a K+/gluconate-based internal solution (in mM: 0.1 EGTA, 120.0 K+ gluconate, 10.0 HEPES, 20.0 KCl, 2.0 MgCl2, 3.0 Na2ATP, and 0.3 NaGTP; 280–285mOsm, pH=7.3). Whole-cell current clamp recordings were obtained using a Multiclamp 700B (Molecular Devices, Instruments, Sunnyvale, CA). Signals were digitized by a Digidata 1320 A/D converter (Axon Instruments) and stored on-line using pClamp 9 software (Axon Instruments). All neurons included in the analyses were medium spiny neurons (MSNs) that met the criteria of a resting membrane potential more negative than −70mV and action potential peak greater than 60mV under basal conditions. MSNs were readily identifiable by their mid-sized soma and electrophysiological characteristics including hyperpolarized resting membrane potentials, latency before the first action potential at rheobase, and absence of Ih current (Wilson and Groves, 1980, Dong et al., 2006). The recording protocol consisted of 500ms pulses of current beginning at −0.5nA with increment steps of 50pA. Active and passive membrane properties were measured at the first action potential evoked by the rheobase (i.e., lowest current generating an action potential). Input resistance (Rin) was measured at −0.2nA current injection; data to construct the current-voltage curves were obtained at 400ms from the start of the current pulse.

MSN excitability was tested under one of the following conditions: (i) ascending concentration-response assessment of 0.1, 1.0, and 10.0μM of the 5-HT2C inverse agonist SB 206553 (SB206); (ii) 1.0μM of the 5-HT2C antagonist SB 242084 (SB242) followed by 1.0μM SB242 + 10.0μM SB206; (iii) ascending concentration-response assessment of 0.1, 1.0, and 10.0μM of the 5-HT2C agonist Ro 60-0175 (Ro); (iv) 1.0μM SB242 followed by 1.0μM SB242 + 10.0μM Ro. Prior to treatment, neurons were recorded while normal aCSF was perfused to establish a baseline (basal). Each drug concentration tested was perfused for at least 5min before collecting concentration-related data. Whole-cell pipette series resistance was less than 20MΩ, and compensation for bridge resistance was monitored throughout experiments.

Pilot studies were conducted with accumbal shell slices from untreated naïve rats to determine the following: (i) Baseline stability. This was accomplished by monitoring the active and passive membrane properties during 30 min of aCSF perfusion. For example, in neurons from naïve rats the resting membrane potential varied by less than 1% over a 30min period with continual aCSF perfusion (data not shown). (ii) Stability of responses to 5-HT2C ligands. This was accomplished by monitoring recordings during continual perfusion of 10.0μM of Ro or SB206. For example, in naïve rats the resting membrane potential varied by less than 1% with continual Ro perfusion (data not shown).

2.6 [35S]GTPγS binding

Ro-induced 5-HT2CR signaling mechanisms were assessed in nucleus accumbens tissue from rats that self-administered meth (n=8). The rats were euthanized one day after the last day of self-administration (i.e., protocol day 15) using rapid decapitation. The nucleus accumbens (including the core and shell subregions) was dissected and flash-frozen on dry ice. Tissue was homogenized in assay buffer (mM: 20.0 HEPES, 100.0 NaCl, 5.0 MgCl2, and 0.2 EGTA) and membrane preparations were made using a series of four centrifugations for 20min at 15,000rpm with intervening tissue homogenization in assay buffer. Tissue was incubated at 37°C after the second and third centrifugation for 15min to facilitate neurotransmitter dissociation from receptors. Protein concentrations were determined using BCA assay (Thermo Fisher Scientific, Rockford, IL) (Smith et al., 1985), and membranes were stored at −80°C until use. Samples were run in one of two conditions to determine Gq and Gi/o stimulation; protocols to determine Gq stimulation were modified from (Adlersberg et al., 2000). For detection of Gq stimulation, 10.0μg of tissue was preincubated with 0.1μM GDP at 30°C for 30min with either vehicle (basal) or drug after which 0.5nM [35S]GTPγS was added, and samples were incubated for an additional 60min at 30°C. Samples were passed through a Brandel harvester (Brandel, Gaithersburg, MD) and washed four times using wash buffer (in mM: 20.0 HEPES, 100.0 NaCl, and 5.0 MgCl2). For detection of Gi/o stimulation, the above protocol was altered by incubating with 30.0μM GDP and 0.1nM [35S]GTPγS. DTT was added to assay buffer at a concentration of 0.2mM prior. Tissue samples were assayed in triplicate. To determine the contribution of Gq vs. Gi/o stimulation to receptor agonism, samples were incubated with vehicle (basal), 10.0μM Ro, 10.0μM Ro + 1.0μM SB242, or 10.0μM DAMGO (μ-opioid receptor peptide agonist; used as a positive control for activation of Gi/o). Data are presented as the difference of the average concentration between treatment and basal condition, in pmols [35S]GTPγS bound/mg of protein.

2.7 Statistics

Number of infusions by rats trained to self-administer meth was analyzed using a one-way repeated measures (rm) ANOVA. To test for differences in the membrane properties between neurons from rats that self-administered meth and saline-yoked controls, Student’s t-tests were used, except for the current-spike and current-voltage (I–V) comparisons for which a mixed function two-way ANOVA was used with current as a repeated measure and treatment history as a non-repeated factor. Data assessing 5-HT2CR ligands on neuronal excitability were analyzed using two-way rmANOVA. Biochemical studies were analyzed by one-way rmANOVA. A Newman-Keuls was used post-hoc for all of the ANOVAs. Significance was met with α=0.05. Statistical outliers were operationally defined as measures outside of two standard deviations from the parameter mean and such outliers were excluded from analysis; less than 5% of collected data met outlier criteria.

3. Results

3.1 Meth self-administration and saline-yoked controls

Outcomes of the meth self-administration protocols are shown in Figure 1. Rats readily acquired the operant task, and when the reinforcement schedule was switched from FR1 to FR5 (day 8), active lever pressing increased to compensate for the more demanding reinforcement schedule. The number of inactive lever presses was consistently minimal, indicating that rats differentiated between the reinforced (active) and non-reinforced (inactive) levers. Finally, the number of infusions by rats self-administering meth was stable across the last 4 days of self-administration, similar to our previous reports (Graves and Napier, 2011, 2012). Average lifetime intake of meth was 17.4±1.7 mg/kg with an average of 1.6±0.2 mg/kg (n=39 rats) self-administered on the last day of operant procedures. Lever pressing by saline-yoked rats was minimal throughout behavioral testing with 5.6±1.4 and 4.2±1.0, active and inactive lever presses, respectively, on the last day of operant procedures (data not shown).

Figure 1. Behavioral data from rats trained to self-administer methamphetamine and saline-yoked controls.

Figure 1

Open in a new tab

Thirty-nine rats were trained to self-administer methamphetamine for 14 consecutive days at 0.1mg/kg/0.1ml infusion. On days 1–7, rats self-administered on a fixed ratio (FR) 1 schedule of reinforcement (i.e., one lever press per infusion) and on days 8–14, self-administration was switched to an FR5 schedule. Active and inactive lever presses are graphed according to the left Y-axis and infusions are graphed according to the right Y-axis. There was no difference in the number of infusions received over the last four days of self-administration (one-way repeated measures ANOVA; F(3,37)=1.85).

3.2 Consequences of meth self-administration and 5-HT2C receptor ligands on neuronal function in the nucleus accumbens shell

The intrinsic excitability of MSNs in the nucleus accumbens shell (assessed by evoked firing in response to somatic injection of depolarizing currents) was decreased in rats trained to self-administer meth (Figure 2A); no other active or passive membrane properties were altered by meth self-administration (Figure 2B; Table 1).

Figure 2. Methamphetamine self-administration significantly decreased evoked firing in accumbens shell medium spiny neurons.

Figure 2

Open in a new tab

(A) Medium spiny neurons (n=17) from rats (n=12) trained to self-administer (SA) methamphetamine (meth) showed a significant decrease in the number of evoked action potentials compared to neurons (n=16) from saline-yoked rats (n=13). Mixed function two-way ANOVA revealed a significant Treatment History effect (F(1,15,)=5.81), Current effect (F(7,100)=120.97), and Treatment History X Current interaction (F(7,100) =2.85); Newman-Keuls post-hoc analysis indicated significant differences between neurons from saline-yoked and rats trained to self-administer meth; *p<0.05, **p<0.01 meth vs. saline-yoked. Sample traces (left panel) from neurons of a saline-yoked (top) rat and a rat trained to SA meth (bottom) illustrate the change in evoked firing at 0.35nA current injection. (B) Meth self-administration did not alter the inward rectification. A mixed function two-way ANOVA revealed no Treatment History effect (F(1,14)=0.42), a significant Current effect (F(12,164)=242.46), and no Treatment History X Current interaction (F(12,164)=0.30); saline-yoked n=16 cells from 13 rats; meth self-administration n=17 cells from 12 rats.

Table 1.

Methamphetamine self-administration does not alter active or passive membrane properties in the nucleus accumbens shell.

Treatment history: Saline-Yoked Meth SA
Passive membrane properties
 RMP (mV) −79.9±0.9 −79.6±1.0
 Rin (MΩ) 84.4±6.3 81.7±7.0
Active membrane properties
 Rheobase (nA) 0.18±0.02 0.22±0.02
 AP threshold (mV) −36.8±1.4 −38.5±1.5
 AP amplitude (mV) 78.3±2.9 76.5±2.2
 AP half duration (msec) 1.54±0.05 1.59±0.05
 AHP amplitude (mV) 13.3±0.5 14.2±0.8
Open in a new tab

Neuronal membrane properties from saline-yoked rats (n=16 neurons from 13 rats) and rats trained to self-administer meth (Meth SA; n=17 neurons from 12) were compared using unpaired t-test; no differences were detected.

We have previously shown that the inverse agonist SB206 decreases meth-seeking and meth-induced motor hyperactivity (Graves and Napier, 2012). To determine if SB206 altered MSN function in a manner relevant to meth-induced plasticity, we assessed the effect of this inverse agonist on the intrinsic excitability of nucleus accumbens shell MSNs from rats trained to self-administer meth. SB206 (0.1, 1.0, and 10.0μM) increased the number of evoked action potentials (Figure 3A); similar observations were observed in MSNs from saline-yoked rats (Figure 3C). To determine receptor selectivity for the inverse agonist, SB206 was tested in the presence of the 5-HT2CR antagonist SB242. Co-perfusion of SB206 and SB242 prevented the changes in excitability in MSNs from rats trained to self-administer meth (Figure 3B) and in MSNs from saline-yoked controls (Figure 3D).

Figure 3. SB 206553 increased evoked firing in accumbens shell medium spiny neurons.

Figure 3

Open in a new tab

(A) Medium spiny neurons in the accumbens shell from rats trained to self-administer methamphetamine (meth) were repeatedly recorded while being perfused with aCSF (Basal), 0.1, 1.0, and 10.0μM of the 5-HT2CR inverse agonist SB 206553 (SB206) in ascending order. 1.0 and 10.0μM SB206 (but not 0.1μM) increased the number of evoked action potentials (n=16 neurons from 10 rats); two-way rmANOVA shows significant a Treatment effect (F(3,36)=11.93), Current effect (F(7,250)=56.55), and a Treatment X Current interaction (F(21,250)=3.98). The effect of SB206 on evoked firing was concentration dependent with 0.1 vs. 1.0μM significantly different at 0.35nA, 0.1 vs. 10.0μM from 0.20 to 0.40nA, and 1.0 vs. 10.0μM at 0.20 to 0.40nA (not illustrated). Sample traces (left panel) illustrate differences in spiking (0.35nA) at basal (top) and after 10.0μM SB206 (bottom); step protocol illustrated below. For illustrative purposes, data inset in figure A highlight the differences in evoked action potentials between basal and 10.0μM SB206. (B) Medium spiny neurons from rats trained to self-administer meth (n=12 neurons from 5 rats) were perfused with aCSF, 1.0μM SB 242084 (SB242; 5-HT2CR antagonist), and co-perfusion of 1.0μM SB242 + 10.0μM SB206 (5-HT2CR inverse agonist). Co-perfusion of the 5-HT2CR antagonist SB242 with SB206 prevented the inverse agonist-induced increases in action potential generation. Two-way rmANOVA revealed a significant Treatment effect (F(2,22)=3.87), a Current effect (F(7,154)=55.72), and no Treatment X Current interaction (F(14,154)=0.77). (C) The inverse agonist was also tested in saline-yoked controls; neurons (n=16 from 9 rats) were repeatedly recorded while being perfused with aCSF (Basal), 0.1, 1.0, and 10.0μM of the 5-HT2CR inverse agonist SB 206553 (SB206) in ascending order. 1.0 and 10.0μM SB 206 (but not 0.1μM) increased the number of evoked action potentials; two-way rmANOVA shows significant a Treatment effect (F(3,39)=3.75), Current effect (F(7,270)=54.33) and a Current X Treatment interaction (F(21,270)=1.68). The effect of SB206 on evoked firing did not show concentration-dependence (i.e., 0.1 vs. 1.0 vs. 10.0μM were not significantly different at any nA). Sample traces illustrate the differences in spiking (0.35nA) at basal (left) and after 10.0μM SB206 (right); step protocol illustrated on the bottom left. For illustrative purposes, data inset in figure A highlight the differences in evoked action potentials between basal and 10.0μM SB206. (D) The antagonist, SB242, also prevented SB206-induced effects in saline-yoked controls. Neurons (n=12 neurons from 5 rats) were perfused with aCSF, 1.0μM SB 242084 (SB242; antagonist), and co-perfusion of 1.0μM SB242 + 10.0μM SB 206553 (SB206; inverse agonist). Co-perfusion of the 5-HT2CR antagonist SB242 with SB206 prevented the inverse agonist-induced increases in action potential generation. Two-way rmANOVA revealed no significant Treatment effect (F(2,22)=0.22), a Current effect (F(7,149)=18.76), and no Treatment X Current interaction (F(14,149)=0.72). #p<0.05 and ###p<0.001 comparing basal vs. 1.0μM; **p<0.01, and ***p<0.001 comparing basal vs. 10.0μM.

Agonism at the 5-HT2CR attenuates psychostimulant-mediated behaviors in rats (Grottick et al., 2000, Fletcher et al., 2002, Filip et al., 2004, Frankel and Cunningham, 2004, Neisewander and Acosta, 2007, Burbassi and Cervo, 2008, Cunningham et al., 2011). We therefore assessed the effects of the 5-HT2CR agonist Ro on MSN function in the nucleus accumbens shell. Ro increased the number of evoked action potentials in both MSNs from both rats trained to self-administer meth (Figure 4A) and saline-yoked controls (Figure 4C), similar to findings with the inverse agonist SB206 (Figure 3A&C). To confirm selectivity for the agonist, we tested the ability of the 5-HT2CR antagonist SB242 to occlude Ro-induced changes in MSN excitability using ex vivo slices from rats trained to self-administer meth. Comparable to the selectivity seen with SB206, SB242 prevented Ro-induced increased evoked action potential generation (Figure 4B). These data revealed that both agonism and inverse agonsim have the same effect on accumbens shell intrinsic excitability and that this effect opposed that induced by meth self-administration. Moreover, 5-HT2C receptor ligands increased the input resistance in a dose-dependent manner in neurons from both saline-yoked rats and rats trained to self-administer meth (Supplementary Table 1), suggesting altered K+ conductances by 5-HT2CR ligands.

Figure 4. Ro 60-0175 increased evoked firing in accumbens shell medium spiny neurons.

Figure 4

Open in a new tab

(A) Medium spiny neurons in the nucleus accumbens shell from rats trained to self-administer methamphetamine (meth) were repeatedly recorded while being perfused with aCSF (Basal), 0.1, 1.0, and 10.0μM of the 5-HT2CR agonist Ro 60-0175 (Ro) in ascending order. 1.0 and 10.0μM Ro (but not 0.1μM) increased the number of evoked action potentials (n=14 neurons from 8 rats); the effect of Ro on evoked firing was concentration dependent; 0.1 vs. 1.0 μM was significantly different at 0.2nA, 0.1 vs. 10.0μM from 0.15 to 0.3nA, and 1.0 vs. 10.0μM at 0.20nA (not illustrated). Two-way rmANOVA shows a significant Treatment effect (F(3,30)=6.57), a Current effect (F(7,210)=87.14), and Treatment X Current interaction (F(21,210)=2.14). Sample traces (left panel) illustrate the difference in spiking (0.35nA) at basal (top) and after 10.0μM Ro (bottom); step protocol illustrated on the bottom left. For illustrative purposes, data inset in figure A highlight the differences in evoked action potentials between basal and 10.0μM Ro. (B) Neurons from rats trained to self-administer meth (n=12 neurons from 4 rats) were perfused with aCSF, 1.0μM SB 242084 (SB242; antagonist), and co-perfusion of 1.0μM SB242 + 10.0μM Ro 60-0175 (Ro). Co-perfusion of the 5-HT2CR antagonist SB242 with Ro prevented the agonist-induced increases in action potential generation in accumbens shell medium spiny neurons from rats trained to self-administer meth. Two-way rmANOVA revealed no Treatment effect (F(2,22=0.52), a Current effect (F(7,148)=38.068), and no Treatment X Current interaction (F(14,148)=0.80). (C) Medium spiny neurons in the accumbens shell from saline-yoked rats (n=15 neurons from 5 rats) were repeatedly recorded while being perfused with aCSF (Basal), 0.1, 1.0, and 10.0μM of the 5-HT2CR agonist Ro 60-0175 (Ro) in ascending order. Bath application of Ro (1.0 and 10.0μM, but not 0.1μM) increased the number of evoked action potentials; two-way rmANOVA shows significant Treatment effect (F(3,36)=9.74), Current effect (F(7,248)=85.75), and Treatment X Current interaction (F(21,248)=2.57). The effect of Ro on evoked firing was concentration-dependent; significant differences between 0.1 and 1.0 μM occurred from 0.15 to 0.2 and 0.3 to 0.4nA, differences between 0.1 and 10.0μM occurred from 0.15 to 0.4nA, and between 1.0 and 10.0μM at 0.25nA(for sake of clarity, these points of significance are not illustrated on the graph. Sample traces illustrate the difference in spiking (0.35nA) at basal (left) and after 10.0μM Ro (right); step protocol illustrated on the bottom left. For illustrative purposes, data inset in figure C highlight the differences in evoked action potentials between basal and 10.0μM Ro. #p<0.05 and ###p<0.001 comparing basal vs. 1.0μM; *p<0.05, **p<0.01, and ***p<0.001 comparing basal vs. 10.0μM.

3.3 5-HT2C receptor mediated activation of Gq proteins but not Gi/o proteins

5-HT2CRs are reported to signal through a variety of pathways including, but not limited to, Gi/o and Gq (De Deurwaerdere et al., 2004). [35S]GTPγS binding assays were used to evaluate 5-HT2CR-linked coupling to different G proteins in nucleus accumbens tissue harvested one day after the last self-administration session (i.e., protocol day 15). Two experimental conditions were tested, one conducive to detecting Gq and one for detecting Gi/o activation (refer to Methods). Incubation of 10.0μM Ro (5-HT2CR agonist) stimulated [35S]GTPγS binding under Gq (Figure 5A) but not Gi/o (Figure 5B) conditions. Stimulation under Gq conditions was attenuated by co-incubation of 1.0μM of the 5-HT2CR antagonist SB242 (Figure 5A). We tested DAMGO (10.0μM), a full agonist at μ-opioid receptors which couple to Gi/o but not Gq proteins, as a negative control for Gq activation and positive control for Gi/o activation. DAMGO did not increase [35S]GTPγS binding under Gq conditions (Figure 5A), but did so under Gi/o conditions (Figure 5B). These experiments demonstrated that 5-HT2CRs in the nucleus accumbens from rats trained to self-administer meth activated Gq, but did not stimulate Gi/o proteins at detectable levels.

Figure 5. Stimulation of 5-HT2C receptors activated Gq, but not Gi/o proteins in rats trained to self-administer meth.

Figure 5

Open in a new tab

Membrane preparations were made from the nucleus accumbens of rats trained to self-administer methamphetamine (n=4). [35S]GTPγS binding was used to detect Gq (A) and Gi/o (B) stimulation separately under two distinct assay conditions (refer to methods). (A) The 5-HT2CR agonist Ro 60-0175 (Ro, 10.0μM) increased [35S]GTPγS binding under Gq conditions (i.e., low GDP and high [35S]GTPγS concentrations) in accumbens tissue from meth-exposed rats, indicating that 5-HT2CRs were coupled to Gq proteins. Co-incubation of 1.0μM SB 242084 (5-HT2C antagonist; SB242) nullified the ability of Ro to stimulate [35S]GTPγS binding. Incubation with 10.0μM DAMGO, an agonist for Gi/o-linked μ-receptor, did not stimulate [35S]GTPγS binding under Gq conditions, verifying the absence of measurable Gi/o activity under such assay conditions. One-way repeated measures ANOVA (F(3,8)=7.33) with Newman-Keuls post-hoc reveals significant difference between Ro vs. Basal, Ro vs. Ro+242, and Ro vs. DAMGO (**p<0.01 for each comparison). (B) Under conditions to detect Gi/o stimulation (i.e., moderate GDP and low [35S]GTPγS concentrations), Ro (10.0μM) produced no stimulation whereas DAMGO (10.0μM) increased [35S]GTPγS stimulation, which served as a positive control. One-way repeated measures ANOVA (F(2,9)=40.49); Newman-Keuls post-hoc revealed significant difference between DAMGO vs. Basal and DAMGO vs. Ro (***p<0.001 for each comparison).

4. Discussion

In the current study, we determined the effects of meth self-administration and 5-HT2CR ligands on the excitability of MSNs in the nucleus accumbens shell. We revealed that: (i) self-administration of meth (even at doses that were less than 2mg/kg/day) was sufficient to decrease intrinsic excitability. (ii) The 5-HT2CR inverse agonist, SB206, and the agonist, Ro, increased excitability. (iii) Inverse agonist and agonist actions on accumbal excitability and signal transduction were blocked by a 5-HT2CR antagonist. (iv) 5-HT2CRs engaged Gq but not Gi/o heterotrimeric G proteins.

The decrease in evoked spiking induced by meth in MSNs of the nucleus accumbens shell parallels decreased excitability measured in vivo from meth-sensitized rats with more protracted times of forced abstinence (i.e., 17–25 days) (Brady et al., 2003, 2005). Repeated cocaine also produces similar effects. Ex vivo studies in cocaine-sensitized mice after short (1–3 days) and protracted (10–14 days) periods of forced abstinence (Kourrich and Thomas, 2009, Kourrich et al., 2012), cocaine-sensitized rats (Zhang et al., 1998) and mice trained to self-administer cocaine (Mu et al., 2010) after short periods of forced abstinence all show decreased excitability. These reports suggest a common and enduring ability of psychostimulants to decrease nucleus accumbens shell excitability. The psychostimulant-mediated effects on evoked firing likely reflect changes in intrinsic excitability that are distinct from changes in synaptic excitability. For example, AMPA receptor trafficking to the cell surface is not changed in the accumbens 1 day following repeated cocaine (Boudreau and Wolf, 2005), acute amphetamine (Nelson et al., 2009), acute meth (Herrold et al., 2013), or 21 days following repeated administration of amphetamine (Nelson et al., 2009), or 14 d following repeated meth (Herrold et al., 2013). However, this interpretation is not definitive, as the biochemical assays for AMPA receptors did not delineate the nucleus accumbens shell and core, and these subregions are differentially altered in rodents chronically exposed to cocaine (Kourrich and Thomas, 2009).

It is unclear which ion channels underlie meth-induced reductions in MSN evoked spiking as none of the measured membrane properties were significantly altered by a history of meth in the current study. Investigations of cocaine-mediated effects indicate several ion channel candidates. For example, during early withdrawal from repeated non-contingent cocaine, rats show reductions in high voltage-voltage activated Ca2+ channels (Zhang et al., 2002, Hu et al., 2004), voltage-sensitive Na+ channels (Zhang et al., 1998) and enhancements of voltage-gated K+ conductance (Hu et al., 2004), all of which diminish excitability.

We have observed that 5-HT2CR inverse agonism reduces meth-seeking behaviors (Graves and Napier, 2012) and several laboratories have shown that 5-HT2CR agonism attenuates cocaine-seeking (Grottick et al., 2000, Fletcher et al., 2002, Neisewander and Acosta, 2007, Burbassi and Cervo, 2008, Cunningham et al., 2011). The current electrophysiological studies complement these behavioral findings by demonstrating that 5-HT2CR agonism and inverse agonism have the opposite effects on accumbens shell excitability than does meth self-administration. These observations support the idea that augmenting 5-HT2CR activity may be a strategy to oppose psychostimulant-induced pathology. However, it is important to note that the effects of 5-HT2CR ligands on intrinsic excitability were similar for MSNs from saline-yoked controls and rats trained to self-administer meth. This suggests that overall 5-HT2CR function in the accumbens shell was not altered by meth self-administration per se and that the previously reported behavioral effects of 5-HT2CR agonism and inverse agonism may reflect actions on the downstream consequences of meth-induced changes. For example, MSNs in the accumbens shell provide GABAergic inputs to the ventral pallidum (Heimer et al., 1987, Zahm and Heimer, 1988, Zahm, 1989, Zahm and Heimer, 1990, Heimer et al., 1991, Zahm and Brog, 1992, Groenewegen et al., 1993), a region known to regulate psychostimulant-mediated behaviors including seeking (McFarland and Kalivas, 2001, Tang et al., 2005, Li et al., 2009). With decreased excitability of MSNs by meth self-administration, the ventral pallidum should be disinhibited, and targeting 5-HT2CRs in the accumbens shell could serve to mitigate this disinhibition and help to restore balance in the mesolimbic circuit. While further study is necessary to fully ascertain the engaged circuit, 5-HT2CR pharmacotherapy may still provide palliative care for meth addiction even though the ligands don’t directly target the specific cellular maladaptations imposed by meth. This notion is supported by the finding that these 5-HT2CR ligands increased input resistance (Supplementary Table 1), and that this parameter was unchanged by meth self-administration (Table 1). Thus, 5-HT2CRs may compensate for meth-induced changes, but do so through a mechanism independent of meth-induced pathology.

We observed that the inverse agonist SB206 exhibited an “agonist-like” effect on accumbal neuronal activity, i.e., SB206 increased excitability similar to that induced by the 5-HT2CR agonist, Ro. Studies with HEK cells and dissociated mouse cortical neurons demonstrate inverse agonist-dependent trafficking of constitutively active 5-HT2CRs from intracellular pools to the membrane surface (Marion et al., 2004, Chanrion et al., 2008), and enhanced responses to a subsequent 5-HT challenge (Berg et al., 1998a, Marion et al., 2004, Chanrion et al., 2008). In our study, binding of 5-HT2CRs by SB206 may have resulted in a redistribution of intracellular, constitutively active receptors into the neuronal membrane, leading to increased agonist-independent 5-HT2CR signaling and an “agonist-like” action. In vitro studies assessing changes in receptor trafficking typically use incubation periods of 30min to 1hr. Marion et al. (2004), demonstrated that a two-fold increase in surface expression of constitutively active 5-HT2CRs occurs within 30min using HEK 293 cells (shorter time periods were not assessed). This profile contrasts other molecular studies in recombinant systems wherein inverse agonists attenuate basal activity of second messenger systems that are activated by the homotypic agonist (Berg et al., 1999, De Deurwaerdere et al., 2004, Berg et al., 2006, Berg et al., 2008a, Labasque et al., 2010). In vivo microdialysis studies in rodents provide a correlate to these oppositional effects wherein striatal and accumbal dopamine levels are increased by SB206 (De Deurwaerdere et al., 2004), and decreased or not changed by 5-HT2CR agonists (Di Matteo et al., 1998, Willins and Meltzer, 1998, Di Matteo et al., 2000, Gobert et al., 2000, Lucas and Spampinato, 2000, De Deurwaerdere et al., 2004). In vivo extracellular dopamine concentrations in the striatum and accumbens are dependent on activity of the substantia nigra pars compacta and ventral tegmental area, respectively; whereas our investigations were focused on the intrinsic excitability of MSNs in the nucleus accumbens shell, and these different outcome perspectives complicate direct comparison between the two different experimental approaches. Moreover, the pharmacodynamics of receptors on midbrain dopaminergic neurons may not necessarily translate to other neuronal populations. For example, metabotropic glutamate receptors, muscarinic receptors, and noradrenergic α1 receptors couple to Gq proteins, which increase excitability of many types of neurons (Valenti et al., 2002, Mathie, 2007), can inhibit dopaminergic neurons (Fiorillo and Williams, 1998, 2000, Paladini et al., 2001, Paladini and Williams, 2004).

Technical differences between microdialysis studies and the current electrophysiological experiments may also contribute to the divergent outcomes. The microdialysis studies employed halothane anesthesia, which reduces K+ conductance (Elliott et al., 1992, Mathie, 2007), a parameter consistently altered by 5-HT2CR activation (North and Uchimura, 1989, Stevens et al., 1992, Hu et al., 1998, Di Giovanni et al., 1999, Speake et al., 2004, Xiang et al., 2005, Weber et al., 2008), and a decreased K+ conductance was likely involved in the current studies given the 5-HT2CR-dependent increased input resistance. Alternatively, SB206 may be acting as an agonist in MSNs (and inverse agonist in dopamine neurons regulating dopamine release). Ligand efficacy is system dependent (for review, see (Kenakin, 2001)) and the pharmacodynamic actions of the tested ligands may be different depending on the neurons (i.e. system) tested. For example, in MSNs 5-HT2CRs ligands increased excitability (current study) but the pharmacodynamic actions of these same ligands may have distinct effects in dopamine neurons, potentially contributing to the differences observed between our electrophysiological studies and the previously discussed microdialysis studies. Given the complex pharmacology of SB206, it is important to interpret outcomes in lieu of the preparation and neuronal system studied.

Our biochemical assessments demonstrated that 5-HT2CRs in the nucleus accumbens stimulated Gq but not Gi/o and confirmed receptor selectivity for the ability of 5-HT2CRs to engage Gq proteins. We demonstrated that SB242 (a potent 5HT2CR antagonist in most systems studied (Bromidge et al., 1997, Kennett et al., 1997)) prevented 5-HT2CR inverse agonist and agonist-induced changes in excitability confirming receptor specificity for these parameters. Thus, the observed alterations in neuronal excitability are selective for 5-HT2CRs and likely a consequence, at least in part, of Gq activation.

In summary, this study revealed that 5-HT2CRs in the nucleus accumbens engage Gq but not Gi/o proteins. This study also demonstrated that contingently-administered meth decreased the intrinsic excitability of MSNs in the nucleus accumbens shell and this effect was acutely opposed by both the inverse agonist SB 206553 and agonist Ro 60-0175. These findings are consistent with behavioral observations where both compounds attenuate psychostimulant-seeking (Grottick et al., 2000, Fletcher et al., 2002, Burbassi and Cervo, 2008, Graves and Napier, 2012). As the accumbal agonist and inverse agonist effects were also seen in saline-yoked controls, our work implicate an involvement of meth-induced changes in brain regions that may be downstream to the nucleus accumbens in the reported behavioral actions of 5-HT2CR agonists on psychostimulant–seeking.

Supplementary Material

1

Supplementary Table 1. Input resistance

2

Highlights.

  • Methamphetamine self-administration decreased excitability of accumbens shell neurons.

  • 5-HT2C receptor agonism increased accumbens shell excitability

  • 5-HT2C receptor inverse agonism increased accumbens shell excitability.

  • 5-HT2C receptors in the accumbens engage Gi/o but not Gq proteins.

Acknowledgments

Work supported by the Daniel F. and Ada L. Rice foundation and USPHSGs DA015760 to TCN, DA024923 to SMG and TCN, and MH083754 to JRT. The authors thank Gregory Ruber for his excellent technical assistance.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Adlersberg M, Arango V, Hsiung S, Mann JJ, Underwood MD, Liu K, Kassir SA, Ruggiero DA, Tamir H. In vitro autoradiography of serotonin 5-HT(2A/2C) receptor-activated G protein: guanosine-5′-(gamma-[(35)S]thio)triphosphate binding in rat brain. Journal of neuroscience research. 2000;61:674–685. doi: 10.1002/1097-4547(20000915)61:6<674::AID-JNR11>3.0.CO;2-F. [DOI] [PubMed] [Google Scholar]
  2. Berg KA, Dunlop J, Sanchez T, Silva M, Clarke WP. A conservative, single-amino acid substitution in the second cytoplasmic domain of the human Serotonin2C receptor alters both ligand-dependent and -independent receptor signaling. The Journal of pharmacology and experimental therapeutics. 2008a;324:1084–1092. doi: 10.1124/jpet.107.131524. [DOI] [PubMed] [Google Scholar]
  3. Berg KA, Harvey JA, Spampinato U, Clarke WP. Physiological and therapeutic relevance of constitutive activity of 5-HT 2A and 5-HT 2C receptors for the treatment of depression. Progress in brain research. 2008b;172:287–305. doi: 10.1016/S0079-6123(08)00914-X. [DOI] [PubMed] [Google Scholar]
  4. Berg KA, Maayani S, Clarke WP. Interactions between effectors linked to serotonin receptors. Annals of the New York Academy of Sciences. 1998a;861:111–120. doi: 10.1111/j.1749-6632.1998.tb10181.x. [DOI] [PubMed] [Google Scholar]
  5. Berg KA, Maayani S, Goldfarb J, Scaramellini C, Leff P, Clarke WP. Effector pathway-dependent relative efficacy at serotonin type 2A and 2C receptors: evidence for agonist-directed trafficking of receptor stimulus. Molecular pharmacology. 1998b;54:94–104. [PubMed] [Google Scholar]
  6. Berg KA, Navailles S, Sanchez TA, Silva YM, Wood MD, Spampinato U, Clarke WP. Differential effects of 5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxyl]-5-pyridyl]carbamoyl]-6-trifluoromethyli ndone (SB 243213) on 5-hydroxytryptamine(2C) receptor-mediated responses. The Journal of pharmacology and experimental therapeutics. 2006;319:260–268. doi: 10.1124/jpet.106.104448. [DOI] [PubMed] [Google Scholar]
  7. Berg KA, Stout BD, Cropper JD, Maayani S, Clarke WP. Novel actions of inverse agonists on 5-HT2C receptor systems. Molecular pharmacology. 1999;55:863–872. [PubMed] [Google Scholar]
  8. Boudreau AC, Wolf ME. Behavioral sensitization to cocaine is associated with increased AMPA receptor surface expression in the nucleus accumbens. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2005;25:9144–9151. doi: 10.1523/JNEUROSCI.2252-05.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brady AM, Glick SD, O’Donnell P. Changes in electrophysiological properties of nucleus accumbens neurons depend on the extent of behavioral sensitization to chronic methamphetamine. Annals of the New York Academy of Sciences. 2003;1003:358–363. doi: 10.1196/annals.1300.026. [DOI] [PubMed] [Google Scholar]
  10. Brady AM, Glick SD, O’Donnell P. Selective disruption of nucleus accumbens gating mechanisms in rats behaviorally sensitized to methamphetamine. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2005;25:6687–6695. doi: 10.1523/JNEUROSCI.0643-05.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bromidge SM, Duckworth M, Forbes IT, Ham P, King FD, Thewlis KM, Blaney FE, Naylor CB, Blackburn TP, Kennett GA, Wood MD, Clarke SE. 6-Chloro-5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-indoline (SB-242084): the first selective and brain penetrant 5-HT2C receptor antagonist. Journal of medicinal chemistry. 1997;40:3494–3496. doi: 10.1021/jm970424c. [DOI] [PubMed] [Google Scholar]
  12. Burbassi S, Cervo L. Stimulation of serotonin2C receptors influences cocaine-seeking behavior in response to drug-associated stimuli in rats. Psychopharmacology (Berl) 2008;196:15–27. doi: 10.1007/s00213-007-0916-7. [DOI] [PubMed] [Google Scholar]
  13. Chanrion B, Mannoury la Cour C, Gavarini S, Seimandi M, Vincent L, Pujol JF, Bockaert J, Marin P, Millan MJ. Inverse agonist and neutral antagonist actions of antidepressants at recombinant and native 5-hydroxytryptamine2C receptors: differential modulation of cell surface expression and signal transduction. Molecular pharmacology. 2008;73:748–757. doi: 10.1124/mol.107.041574. [DOI] [PubMed] [Google Scholar]
  14. Cunningham KA, Fox RG, Anastasio NC, Bubar MJ, Stutz SJ, Moeller FG, Gilbertson SR, Rosenzweig-Lipson S. Selective serotonin 5-HT(2C) receptor activation suppresses the reinforcing efficacy of cocaine and sucrose but differentially affects the incentive-salience value of cocaine- vs. sucrose-associated cues. Neuropharmacology. 2011;61:513–523. doi: 10.1016/j.neuropharm.2011.04.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Cussac D, Newman-Tancredi A, Duqueyroix D, Pasteau V, Millan MJ. Differential activation of Gq/11 and Gi(3) proteins at 5-hydroxytryptamine(2C) receptors revealed by antibody capture assays: influence of receptor reserve and relationship to agonist-directed trafficking. Molecular pharmacology. 2002;62:578–589. doi: 10.1124/mol.62.3.578. [DOI] [PubMed] [Google Scholar]
  16. De Deurwaerdere P, Navailles S, Berg KA, Clarke WP, Spampinato U. Constitutive activity of the serotonin2C receptor inhibits in vivo dopamine release in the rat striatum and nucleus accumbens. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2004;24:3235–3241. doi: 10.1523/JNEUROSCI.0112-04.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Di Chiara G. Nucleus accumbens shell and core dopamine: differential role in behavior and addiction. Behavioural brain research. 2002;137:75–114. doi: 10.1016/s0166-4328(02)00286-3. [DOI] [PubMed] [Google Scholar]
  18. Di Chiara G, Bassareo V, Fenu S, De Luca MA, Spina L, Cadoni C, Acquas E, Carboni E, Valentini V, Lecca D. Dopamine and drug addiction: the nucleus accumbens shell connection. Neuropharmacology. 2004;47(Suppl 1):227–241. doi: 10.1016/j.neuropharm.2004.06.032. [DOI] [PubMed] [Google Scholar]
  19. Di Giovanni G, De Deurwaerdere P, Di Mascio M, Di Matteo V, Esposito E, Spampinato U. Selective blockade of serotonin-2C/2B receptors enhances mesolimbic and mesostriatal dopaminergic function: a combined in vivo electrophysiological and microdialysis study. Neuroscience. 1999;91:587–597. doi: 10.1016/s0306-4522(98)00655-1. [DOI] [PubMed] [Google Scholar]
  20. Di Matteo V, Di Giovanni G, Di Mascio M, Esposito E. Selective blockade of serotonin2C/2B receptors enhances dopamine release in the rat nucleus accumbens. Neuropharmacology. 1998;37:265–272. doi: 10.1016/s0028-3908(98)00014-8. [DOI] [PubMed] [Google Scholar]
  21. Di Matteo V, Di Mascio M, Di Giovanni G, Esposito E. Acute administration of amitriptyline and mianserin increases dopamine release in the rat nucleus accumbens: possible involvement of serotonin2C receptors. Psychopharmacology. 2000;150:45–51. doi: 10.1007/s002130000420. [DOI] [PubMed] [Google Scholar]
  22. Dong Y, Green T, Saal D, Marie H, Neve R, Nestler EJ, Malenka RC. CREB modulates excitability of nucleus accumbens neurons. Nature neuroscience. 2006;9:475–477. doi: 10.1038/nn1661. [DOI] [PubMed] [Google Scholar]
  23. Elliott JR, Elliott AA, Harper AA, Winpenny JP. Effects of general anaesthetics on neuronal sodium and potassium channels. General pharmacology. 1992;23:1005–1011. doi: 10.1016/0306-3623(92)90278-r. [DOI] [PubMed] [Google Scholar]
  24. Filip M, Bubar MJ, Cunningham KA. Contribution of serotonin (5-hydroxytryptamine; 5-HT) 5-HT2 receptor subtypes to the hyperlocomotor effects of cocaine: acute and chronic pharmacological analyses. The Journal of pharmacology and experimental therapeutics. 2004;310:1246–1254. doi: 10.1124/jpet.104.068841. [DOI] [PubMed] [Google Scholar]
  25. Fiorillo CD, Williams JT. Glutamate mediates an inhibitory postsynaptic potential in dopamine neurons. Nature. 1998;394:78–82. doi: 10.1038/27919. [DOI] [PubMed] [Google Scholar]
  26. Fiorillo CD, Williams JT. Selective inhibition by adenosine of mGluR IPSPs in dopamine neurons after cocaine treatment. Journal of neurophysiology. 2000;83:1307–1314. doi: 10.1152/jn.2000.83.3.1307. [DOI] [PubMed] [Google Scholar]
  27. Fletcher PJ, Grottick AJ, Higgins GA. Differential effects of the 5-HT(2A) receptor antagonist M100907 and the 5-HT(2C) receptor antagonist SB242084 on cocaine-induced locomotor activity, cocaine self-administration and cocaine-induced reinstatement of responding. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology. 2002;27:576–586. doi: 10.1016/S0893-133X(02)00342-1. [DOI] [PubMed] [Google Scholar]
  28. Frankel PS, Cunningham KA. m-Chlorophenylpiperazine (mCPP) modulates the discriminative stimulus effects of cocaine through actions at the 5-HT2C receptor. Behav Neurosci. 2004;118:157–162. doi: 10.1037/0735-7044.118.1.157. [DOI] [PubMed] [Google Scholar]
  29. Gobert A, Rivet JM, Lejeune F, Newman-Tancredi A, Adhumeau-Auclair A, Nicolas JP, Cistarelli L, Melon C, Millan MJ. Serotonin(2C) receptors tonically suppress the activity of mesocortical dopaminergic and adrenergic, but not serotonergic, pathways: a combined dialysis and electrophysiological analysis in the rat. Synapse. 2000;36:205–221. doi: 10.1002/(SICI)1098-2396(20000601)36:3<205::AID-SYN5>3.0.CO;2-D. [DOI] [PubMed] [Google Scholar]
  30. Graves SM, Napier TC. Mirtazapine alters cue-associated methamphetamine seeking in rats. Biological psychiatry. 2011;69:275–281. doi: 10.1016/j.biopsych.2010.09.032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Graves SM, Napier TC. SB 206553, a putative 5-HT2C inverse agonist, attenuates methamphetamine-seeking in rats. BMC neuroscience. 2012;13:65. doi: 10.1186/1471-2202-13-65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Groenewegen HJ, Berendse HW, Haber SN. Organization of the output of the ventral striatopallidal system in the rat: ventral pallidal efferents. Neuroscience. 1993;57:113–142. doi: 10.1016/0306-4522(93)90115-v. [DOI] [PubMed] [Google Scholar]
  33. Grottick AJ, Fletcher PJ, Higgins GA. Studies to investigate the role of 5-HT(2C) receptors on cocaine- and food-maintained behavior. The Journal of pharmacology and experimental therapeutics. 2000;295:1183–1191. [PubMed] [Google Scholar]
  34. Heimer L, Zaborszky L, Zahm DS, Alheid GF. The ventral striatopallidothalamic projection: I. The striatopallidal link originating in the striatal parts of the olfactory tubercle. The Journal of comparative neurology. 1987;255:571–591. doi: 10.1002/cne.902550409. [DOI] [PubMed] [Google Scholar]
  35. Heimer L, Zahm DS, Churchill L, Kalivas PW, Wohltmann C. Specificity in the projection patterns of accumbal core and shell in the rat. Neuroscience. 1991;41:89–125. doi: 10.1016/0306-4522(91)90202-y. [DOI] [PubMed] [Google Scholar]
  36. Herrold AA, Persons AL, Napier TC. Cellular distribution of AMPA receptor subunits and mGlu5 following acute and repeated administration of morphine or methamphetamine. Journal of neurochemistry. 2013;126:503–517. doi: 10.1111/jnc.12323. [DOI] [PubMed] [Google Scholar]
  37. Hu S, Wang S, Gibson J, Gilbertson TA. Inhibition of delayed rectifier K+ channels by dexfenfluramine (Redux) The Journal of pharmacology and experimental therapeutics. 1998;287:480–486. [PubMed] [Google Scholar]
  38. Hu XT, Basu S, White FJ. Repeated cocaine administration suppresses HVA-Ca2+ potentials and enhances activity of K+ channels in rat nucleus accumbens neurons. Journal of neurophysiology. 2004;92:1597–1607. doi: 10.1152/jn.00217.2004. [DOI] [PubMed] [Google Scholar]
  39. Kauer JA, Malenka RC. Synaptic plasticity and addiction. Nature reviews Neuroscience. 2007;8:844–858. doi: 10.1038/nrn2234. [DOI] [PubMed] [Google Scholar]
  40. Kenakin T. Inverse, protean, and ligand-selective agonism: matters of receptor conformation. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2001;15:598–611. doi: 10.1096/fj.00-0438rev. [DOI] [PubMed] [Google Scholar]
  41. Kennett GA, Wood MD, Bright F, Trail B, Riley G, Holland V, Avenell KY, Stean T, Upton N, Bromidge S, Forbes IT, Brown AM, Middlemiss DN, Blackburn TP. SB 242084, a selective and brain penetrant 5-HT2C receptor antagonist. Neuropharmacology. 1997;36:609–620. doi: 10.1016/s0028-3908(97)00038-5. [DOI] [PubMed] [Google Scholar]
  42. Kourrich S, Klug JR, Mayford M, Thomas MJ. AMPAR-independent effect of striatal alphaCaMKII promotes the sensitization of cocaine reward. J Neurosci. 2012;32:6578–6586. doi: 10.1523/JNEUROSCI.6391-11.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Kourrich S, Thomas MJ. Similar neurons, opposite adaptations: psychostimulant experience differentially alters firing properties in accumbens core versus shell. J Neurosci. 2009;29:12275–12283. doi: 10.1523/JNEUROSCI.3028-09.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Krasnova IN, Justinova Z, Ladenheim B, Jayanthi S, McCoy MT, Barnes C, Warner JE, Goldberg SR, Cadet JL. Methamphetamine self-administration is associated with persistent biochemical alterations in striatal and cortical dopaminergic terminals in the rat. PloS one. 2010;5:e8790. doi: 10.1371/journal.pone.0008790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Labasque M, Meffre J, Carrat G, Becamel C, Bockaert J, Marin P. Constitutive activity of serotonin 2C receptors at G protein-independent signaling: modulation by RNA editing and antidepressants. Molecular pharmacology. 2010;78:818–826. doi: 10.1124/mol.110.066035. [DOI] [PubMed] [Google Scholar]
  46. Labasque M, Reiter E, Becamel C, Bockaert J, Marin P. Physical interaction of calmodulin with the 5-hydroxytryptamine2C receptor C-terminus is essential for G protein-independent, arrestin-dependent receptor signaling. Molecular biology of the cell. 2008;19:4640–4650. doi: 10.1091/mbc.E08-04-0422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Li X, Li J, Peng XQ, Spiller K, Gardner EL, Xi ZX. Metabotropic glutamate receptor 7 modulates the rewarding effects of cocaine in rats: involvement of a ventral pallidal GABAergic mechanism. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology. 2009;34:1783–1796. doi: 10.1038/npp.2008.236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Lucas G, Spampinato U. Role of striatal serotonin2A and serotonin2C receptor subtypes in the control of in vivo dopamine outflow in the rat striatum. Journal of neurochemistry. 2000;74:693–701. doi: 10.1046/j.1471-4159.2000.740693.x. [DOI] [PubMed] [Google Scholar]
  49. Marion S, Weiner DM, Caron MG. RNA editing induces variation in desensitization and trafficking of 5-hydroxytryptamine 2c receptor isoforms. The Journal of biological chemistry. 2004;279:2945–2954. doi: 10.1074/jbc.M308742200. [DOI] [PubMed] [Google Scholar]
  50. Mathie A. Neuronal two-pore-domain potassium channels and their regulation by G protein-coupled receptors. The Journal of physiology. 2007;578:377–385. doi: 10.1113/jphysiol.2006.121582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. McDaid J, Tedford CE, Mackie AR, Dallimore JE, Mickiewicz AL, Shen F, Angle JM, Napier TC. Nullifying drug-induced sensitization: behavioral and electrophysiological evaluations of dopaminergic and serotonergic ligands in methamphetamine-sensitized rats. Drug and alcohol dependence. 2007;86:55–66. doi: 10.1016/j.drugalcdep.2006.05.014. [DOI] [PubMed] [Google Scholar]
  52. McFarland K, Kalivas PW. The circuitry mediating cocaine-induced reinstatement of drug-seeking behavior. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2001;21:8655–8663. doi: 10.1523/JNEUROSCI.21-21-08655.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. McGrew L, Chang MS, Sanders-Bush E. Phospholipase D activation by endogenous 5-hydroxytryptamine 2C receptors is mediated by Galpha13 and pertussis toxin-insensitive Gbetagamma subunits. Molecular pharmacology. 2002;62:1339–1343. doi: 10.1124/mol.62.6.1339. [DOI] [PubMed] [Google Scholar]
  54. Mu P, Moyer JT, Ishikawa M, Zhang Y, Panksepp J, Sorg BA, Schluter OM, Dong Y. Exposure to cocaine dynamically regulates the intrinsic membrane excitability of nucleus accumbens neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2010;30:3689–3699. doi: 10.1523/JNEUROSCI.4063-09.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Napier TC, Istre ED. Methamphetamine-induced sensitization includes a functional upregulation of ventral pallidal 5-HT2A/2C receptors. Synapse. 2008;62:14–21. doi: 10.1002/syn.20460. [DOI] [PubMed] [Google Scholar]
  56. Navailles S, Lagiere M, Roumegous A, Polito M, Boujema MB, Cador M, Dunlop J, Chesselet MF, Millan MJ, De Deurwaerdere P. Serotonin2C ligands exhibiting full negative and positive intrinsic activity elicit purposeless oral movements in rats: distinct effects of agonists and inverse agonists in a rat model of Parkinson’s disease. The international journal of neuropsychopharmacology/official scientific journal of the Collegium Internationale Neuropsychopharmacologicum. 2013;16:593–606. doi: 10.1017/S1461145712000417. [DOI] [PubMed] [Google Scholar]
  57. Navailles S, Moison D, Ryczko D, Spampinato U. Region-dependent regulation of mesoaccumbens dopamine neurons in vivo by the constitutive activity of central serotonin2C receptors. Journal of neurochemistry. 2006;99:1311–1319. doi: 10.1111/j.1471-4159.2006.04188.x. [DOI] [PubMed] [Google Scholar]
  58. Neisewander JL, Acosta JI. Stimulation of 5-HT2C receptors attenuates cue and cocaine-primed reinstatement of cocaine-seeking behavior in rats. Behavioural pharmacology. 2007;18:791–800. doi: 10.1097/FBP.0b013e3282f1c94b. [DOI] [PubMed] [Google Scholar]
  59. Nelson CL, Milovanovic M, Wetter JB, Ford KA, Wolf ME. Behavioral sensitization to amphetamine is not accompanied by changes in glutamate receptor surface expression in the rat nucleus accumbens. Journal of neurochemistry. 2009;109:35–51. doi: 10.1111/j.1471-4159.2009.05911.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. North RA, Uchimura N. 5-Hydroxytryptamine acts at 5-HT2 receptors to decrease potassium conductance in rat nucleus accumbens neurones. The Journal of physiology. 1989;417:1–12. doi: 10.1113/jphysiol.1989.sp017786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Paladini CA, Fiorillo CD, Morikawa H, Williams JT. Amphetamine selectively blocks inhibitory glutamate transmission in dopamine neurons. Nature neuroscience. 2001;4:275–281. doi: 10.1038/85124. [DOI] [PubMed] [Google Scholar]
  62. Paladini CA, Williams JT. Noradrenergic inhibition of midbrain dopamine neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2004;24:4568–4575. doi: 10.1523/JNEUROSCI.5735-03.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Reichel CM, Ramsey LA, Schwendt M, McGinty JF, See RE. Methamphetamine-induced changes in the object recognition memory circuit. Neuropharmacology. 2012;62:1119–1126. doi: 10.1016/j.neuropharm.2011.11.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Analytical biochemistry. 1985;150:76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  65. Speake T, Kibble JD, Brown PD. Kv1.1 and Kv1.3 channels contribute to the delayed-rectifying K+ conductance in rat choroid plexus epithelial cells. American journal of physiology Cell physiology. 2004;286:C611–620. doi: 10.1152/ajpcell.00292.2003. [DOI] [PubMed] [Google Scholar]
  66. Stevens DR, McCarley RW, Greene RW. Serotonin1 and serotonin2 receptors hyperpolarize and depolarize separate populations of medial pontine reticular formation neurons in vitro. Neuroscience. 1992;47:545–553. doi: 10.1016/0306-4522(92)90164-w. [DOI] [PubMed] [Google Scholar]
  67. Tang XC, McFarland K, Cagle S, Kalivas PW. Cocaine-induced reinstatement requires endogenous stimulation of mu-opioid receptors in the ventral pallidum. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2005;25:4512–4520. doi: 10.1523/JNEUROSCI.0685-05.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Valenti O, Conn PJ, Marino MJ. Distinct physiological roles of the Gq-coupled metabotropic glutamate receptors Co-expressed in the same neuronal populations. Journal of cellular physiology. 2002;191:125–137. doi: 10.1002/jcp.10081. [DOI] [PubMed] [Google Scholar]
  69. Weber M, Schmitt A, Wischmeyer E, Doring F. Excitability of pontine startle processing neurones is regulated by the two-pore-domain K+ channel TASK-3 coupled to 5-HT2C receptors. The European journal of neuroscience. 2008;28:931–940. doi: 10.1111/j.1460-9568.2008.06400.x. [DOI] [PubMed] [Google Scholar]
  70. Werry TD, Gregory KJ, Sexton PM, Christopoulos A. Characterization of serotonin 5-HT2C receptor signaling to extracellular signal-regulated kinases 1 and 2. Journal of neurochemistry. 2005;93:1603–1615. doi: 10.1111/j.1471-4159.2005.03161.x. [DOI] [PubMed] [Google Scholar]
  71. Willins DL, Meltzer HY. Serotonin 5-HT2C agonists selectively inhibit morphine-induced dopamine efflux in the nucleus accumbens. Brain research. 1998;781:291–299. doi: 10.1016/s0006-8993(97)01267-5. [DOI] [PubMed] [Google Scholar]
  72. Wilson CJ, Groves PM. Fine structure and synaptic connections of the common spiny neuron of the rat neostriatum: a study employing intracellular inject of horseradish peroxidase. The Journal of comparative neurology. 1980;194:599–615. doi: 10.1002/cne.901940308. [DOI] [PubMed] [Google Scholar]
  73. Xiang Z, Wang L, Kitai ST. Modulation of spontaneous firing in rat subthalamic neurons by 5-HT receptor subtypes. Journal of neurophysiology. 2005;93:1145–1157. doi: 10.1152/jn.00561.2004. [DOI] [PubMed] [Google Scholar]
  74. Zahm DS. The ventral striatopallidal parts of the basal ganglia in the rat--II. Compartmentation of ventral pallidal efferents. Neuroscience. 1989;30:33–50. doi: 10.1016/0306-4522(89)90351-5. [DOI] [PubMed] [Google Scholar]
  75. Zahm DS, Brog JS. On the significance of subterritories in the “accumbens” part of the rat ventral striatum. Neuroscience. 1992;50:751–767. doi: 10.1016/0306-4522(92)90202-d. [DOI] [PubMed] [Google Scholar]
  76. Zahm DS, Heimer L. Ventral striatopallidal parts of the basal ganglia in the rat: I. Neurochemical compartmentation as reflected by the distributions of neurotensin and substance P immunoreactivity. The Journal of comparative neurology. 1988;272:516–535. doi: 10.1002/cne.902720406. [DOI] [PubMed] [Google Scholar]
  77. Zahm DS, Heimer L. Two transpallidal pathways originating in the rat nucleus accumbens. The Journal of comparative neurology. 1990;302:437–446. doi: 10.1002/cne.903020302. [DOI] [PubMed] [Google Scholar]
  78. Zhang XF, Cooper DC, White FJ. Repeated cocaine treatment decreases whole-cell calcium current in rat nucleus accumbens neurons. The Journal of pharmacology and experimental therapeutics. 2002;301:1119–1125. doi: 10.1124/jpet.301.3.1119. [DOI] [PubMed] [Google Scholar]
  79. Zhang XF, Hu XT, White FJ. Whole-cell plasticity in cocaine withdrawal: reduced sodium currents in nucleus accumbens neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience. 1998;18:488–498. doi: 10.1523/JNEUROSCI.18-01-00488.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Supplementary Table 1. Input resistance

NIHMS628546-supplement-1.docx (146KB, docx)
2
NIHMS628546-supplement-2.docx (146.8KB, docx)

RESOURCES