Effects of morphine withdrawal on the membrane properties of medium spiny neurons in the nucleus accumbens shell

Brain Research Bulletin 90 (2013) 92–99

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Effects of morphine withdrawal on the membrane properties of medium spiny neurons in the nucleus accumbens shell夽 Xiaobo Wu, Meimei Shi, Hengli Ling, Chunling Wei, Yihui Liu, Zhiqiang Liu ∗ , Wei Ren Key Laboratory of MOE for Modern Teaching Technology, College of Life Sciences, Shaanxi Normal University, Xi’an, Shaanxi 710062, PR China

a r t i c l e

i n f o

Article history: Received 29 February 2012 Received in revised form 6 September 2012 Accepted 26 September 2012 Available online 13 October 2012 Keywords: Intrinsic excitability Spike adaptation Nucleus accumbens Medium spiny neurons Morphine withdrawal

a b s t r a c t Medium spiny neurons (MSNs) in the nucleus accumbens (NAc) undergo persistent alterations in their biological and physiological characteristics upon exposure to drugs of abuse. Previous studies demonstrated that the biochemical, morphological, and intrinsic physiological properties of MSNs are heterogeneous and provided new insights into the physiological and molecular roles of individual MSNs in addictive behaviors. However, it remains unclear whether MSNs in the NAc shell (NAcSh), an important region for mediating behavioral sensitization, are electrophysiologically heterogeneous and how such heterogeneity is relevant to neuroadaptation associated with drug addiction. Here, the membrane properties, i.e., the intrinsic excitability and spike adaptation, of MSNs in the NAcSh from saline- or morphine-treated rats were investigated in vitro by whole-cell recording. In saline-treated rats, three distinct cell types were identified by their membrane properties: type I neurons showed high levels of intrinsic excitability and rapid spike adaptation; type II neurons showed moderate levels of intrinsic excitability and relatively slow spike frequency adaptation; type III neurons showed low levels of intrinsic excitability and putative strong spike adaptation. MSNs in rats undergoing withdrawal from chronic morphine treatment (10–14 days after the last injection) also exhibited the typical firing behaviors of these three types of neurons. However, the membrane properties of the MSNs were differentially altered after withdrawal. There was an enhancement in intrinsic excitability in type II MSNs and a promotion of spike adaptation in type I MSNs. The apamin-sensitive afterhyperpolarization current (IAHP ) and the apamin-insensitive IAHP of the NAcSh MSNs were attenuated after chronic morphine withdrawal. These findings suggest that individual MSNs in the NAcSh manifest unique electrophysiological properties, which might contribute to psychostimulant-induced neuroadaptation. © 2012 Elsevier Inc. All rights reserved.

1. Introduction The nucleus accumbens (NAc) has been extensively studied as an important site that is associated with drug addiction (Nestler, 2001). More than 95% of the neurons in the NAc are GABAergic medium spiny neurons (MSNs). Repeated exposure to cocaine can induce persistent adaptive changes in the intrinsic excitability of MSNs in the NAc and these changes have been proposed to underlie addictive behaviors (Kalivas and Hu, 2006; Kourrich and Thomas, 2009; Mu et al., 2010). MSNs have heterogeneous biochemical, morphological, and intrinsic physiological properties. Different sub-populations of MSNs can be distinguished from each other by their compartmental

夽 This work was supported by grants from the National Natural Science Foundation of China (NSFC 81171264, 31040037) and the Fundamental Research Funds for the Central Universities of China (GK201101001). ∗ Corresponding author. Tel.: +86 029 85310269; fax: +86 029 85310269. E-mail address: [email protected] (Z. Liu). 0361-9230/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brainresbull.2012.09.015

distribution, afferents, sites of projection, biochemical markers, and patterns of receptor and neuropeptide expression (Gerfen, 1992; Graybiel, 2000). Cell type-specific analysis methodologies for MSNs (fluorescent reporter mice that express GFP, transgenic mice that express special receptors, and knock-in mice) have provided profound new insights into the precise molecular mechanisms of each MSN subtype and their regulation by drug addiction (Valjent et al., 2009; Lobo et al., 2010; Lobo and Nestler, 2011). For example, recent studies have shown that drugs of abuse exert the greatest influence on MSNs via enrichment of dopamine D1 receptors (D1Rs) (Self, 2010; Kim et al., 2011). MSNs in the striatum also display electrophysiological heterogeneity and can be divided into an adapting group and a non-adapting group according to their passive and active membrane properties (Venance and Glowinski, 2003). However, it remains unclear whether MSNs in the NAc shell (NAcSh), which is considered an important region for mediating the effects of drugs of abuse as well as behavioral sensitization, can also be divided into neuronal subtypes on an electrophysiological basis. Furthermore, cell type-specific electrophysiological changes in NAcSh MSNs after repeated exposure to morphine have not been explored.

X. Wu et al. / Brain Research Bulletin 90 (2013) 92–99

Here, we investigated the membrane properties of MSNs in the NAcSh using whole-cell recording on brain slice preparations from saline- or chronically morphine-treated rats. In the salinetreated rats, three cell types were identified by their distinct spiking patterns in response to depolarizing pulses: repetitive spike discharge with firing ceasing, repetitive spike discharge without firing ceasing, and non-repetitive spike firing. MSNs with different firing patterns could also be distinguished from each other by their passive and active membrane properties, including their varying levels of intrinsic excitability and spike adaptation. Chronic withdrawal from repeated morphine exposure modulated intrinsic excitability and spike adaptation in a cell type-specific manner in individual MSNs. Finally, the slow afterhyperpolarization currents (IAHP ) were attenuated by chronic morphine withdrawal. 2. Methods 2.1. Animals and morphine treatment All of the procedures were performed in accordance with the institutional guidelines for the Care and Use of Laboratory Animals as approved by the Animal Care Committee of the College of Life Sciences of Shaanxi Normal University. Sprague-Dawley rats (postnatal 25–32 days) received repeated injections (twice daily for 5 days) of saline or morphine hydrochloride (10 mg/kg, i.c., Qinghai Pharmaceutical Co., China). The rats were sacrificed 10–14 days after the last injection. Brain slices containing the NAcSh were prepared for electrophysiological recordings.

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2.2. Electrophysiology Saline- or morphine-treated rats were anesthetized with halothane and decapitated. The brains were sliced (300 ␮m) in the sagittal plane of the NAcSh. The slices were incubated in oxygenated artificial cerebrospinal fluid (ACSF) containing (in mM) 125 NaCl, 3 KCl, 2.4 CaCl2 , 1.2 MgCl2 , 26 NaHCO3 , 1.25 NaH2 PO4 , and 10 glucose at room temperature for at least 1 h before recording began. Then, a slice was placed in the recording chamber and was continuously perfused with oxygenated ACSF (1–2 ml/min) at room temperature. Picrotoxin (100 ␮M, Sigma) and CNQX (10 ␮M, Sigma) + d-APV (50 ␮M, Sigma) were present in the ACSF throughout the experiment to block GABAA receptors and glutamatergic transmission, respectively. Individual neurons were visualized with a 40× water-immersion objective under an infrared differential interference contrast microscope (Leica, Germany). MSNs were identified by their morphology and their hyperpolarized resting membrane potentials (−75 to −85 mV). Whole-cell current clamp recordings were performed with a Multiclamp 700B amplifier (Axon Instruments, Molecular Devices, USA). pClamp software (v10.0, Axon Instruments, Molecular Devices, USA) was used for data acquisition and analysis. The resistance of the patch pipettes was 4–6 M after they were filled with an intra-pipette solution containing (in mM) 120 K-Gluconate, 20 KCl, 10 HEPES, 0.2 EGTA, 4 Na2 ATP, 2 MgCl2 , and 0.3 GTP–Tris (pH 7.2–7.4, 285–295 mOsm). Uncompensated series resistance was usually less than 10 M, compensated 60–80% and was monitored repeatedly throughout the experiment. Signals were sampled at 10 kHz with an analog-to-digital converter (Digidata 1440A; Axon, Molecular Devices, USA). To make the recordings from different neurons comparable, the resting membrane potential was adjusted to −80 mV by injecting a small positive or negative current. Only electrophysiologically “stable” neurons were included in the data analysis. A current step protocol (from −200 to +400 pA, with a 25 pA increment, 800 ms duration and 10 s interpulse interval, Fig. 1B) was run to detect membrane properties. The rheobase current was defined as the minimal injected current that could evoke an action potential (AP). The input resistance (Rin ) was calculated from

Fig. 1. Firing patterns of the three types of NAcSh MSNs in saline-treated rats. (A) Sample traces of types I, II, and III MSNs in response to a 800 ms injected current step (upper, 400 pA; middle, 300 pA; bottom, 200 pA). (B) The membrane voltage changes in types I, II, and III MSNs induced by a series of hyperpolarizing and depolarizing step currents. (C) The steady state I–V plot showing the voltage changes as a function of injected current intensity. Two-way RM ANOVA; type, F(2,29) = 4.948, p = 0.014; type × injected current interaction, F(20,290) = 7.466, p < 0.001. *p < 0.05. Error bars represent SEM.

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the voltage response to a 40 pA depolarizing current pulse. The fast afterhyperpolarization potential (f-AHP) was sampled after the first spike, usually elicited by the rheobase current. The AP threshold was determined by differentiating the AP waveform and by setting a rising rate of 10 mV/ms as the AP inflection point. AP amplitude was measured as the difference between the peak and threshold values. The action potential half-width was determined as the duration of the action potential at the membrane voltage halfway between the threshold and the peak of the action potential. The initial firing frequency was calculated as the 1/1st interspike interval evoked by an 800 ms pulse. The number of spikes was calculated from the sum of the spikes at each current step. In voltage-clamp experiments, the cells were held at −90 mV and tetrodotoxin (TTX; 0.5 mM) was added to the ACSF. A voltage protocol (comprising a 100 ms voltage pulse to +30 mV, followed by a 5 s voltage-step to −50 mV, Fig. 3A) was used to test IAHP as described (Kato et al., 2006). The voltage protocol was applied every 30 s. The current was multiplied by the time (in ms, measured between 5 ms after the offset of the voltage pulse to +30 mV and the time when the current returned to the baseline) to obtain the total charge (pC) of the IAHP , and the total charge was divided by the membrane capacitance to obtain the charge density (pC/pF) (Kato et al., 2006). 2.3. Statistics Double-blind analysis was used. The results were reported as the mean ± SEM. Statistical analysis was performed using Student’s t test, repeated measure (RM), or one- or two-way ANOVA followed by post hoc LSD (least significant difference) test. The statistical results are primarily presented as the F and p values of the main effect. p < 0.05 was considered statistically significant.

3. Results 3.1. Categories of NAcSh MSNs on the basis of firing behaviors Extensive data analysis was performed on 32 neurons from 11 saline-treated rats. These MSNs exhibited a hyperpolarized resting membrane potential, low input resistance (Table 1), and obvious inward rectification at hyperpolarized membrane potentials (Fig. 1B and C), as reported previously (Kita et al., 1984; Cepeda et al., 2008). Based on the firing patterns in response to injected step-currents, the NAcSh MSNs were classified into 3 groups. The groups were: repetitive spike discharge with firing ceasing, repetitive spike discharge without firing ceasing, and non-repetitive spike discharge with only one or two spikes, which we tentatively designated as type I, type II, and type III MSNs, respectively (Fig. 1A). Among the observed NAcSh MSNs, the percentages of type I, type II, and type III were 34.4%, 46.9% and 18.7%, respectively. The passive membrane properties and AP properties of each type of MSNs were summarized (Table 1). The level of intrinsic excitability in the MSNs was mainly evaluated by integrating the values of AP rheobase, AP threshold, Rin , number of spikes at each current step, and resting membrane potential (RMP), whereas the degree of spike adaptation was tested by the spike train duration, spike frequency adaptation

(SFA) ratio, and initial firing frequency. We also compared AP properties (amplitude, half-width, speed of rise and decay, f-AHP peak amplitude) of these three types of MSNs (Table 1). 3.2. Type I MSNs Typical firing responses of type I MSNs in response to depolarizing current injections are shown in Fig. 1A. Although type I neurons were able to fire repetitively, they exhibited a spike ceasing phenomenon in the process of depolarization at most of the injected current steps, especially at the high current steps (250–400 pA). The current–voltage (I–V) plot of type I MSNs showed obvious inward rectification at hyperpolarized membrane potentials (Fig. 1B and C). Type I MSNs had high input resistance (Rin ), a low AP threshold, and a low rheobase current that made it easy for them to discharge (Table 1). The number of spikes remained relatively stable with increased depolarizing currents (4–8 spikes at each current step) (Fig. 2B). The SFA ratio was utilized to estimate spike adaptation. The SFA ratio was calculated for each cell using the formula adaptation ratio = Finitl /Ffinal , where Finitl is the initial spike frequency (1/1st interspike interval) and Ffinal is the average frequency calculated from the last three interspike intervals at each step (Venance and Glowinski, 2003; Vandecasteele et al., 2011). Type I MSNs displayed fast spike frequency adaptation (Fig. 2C). The spike train duration was short due to spike ceasing. This result showed that the spike train duration of these neurons decreased (Fig. 2D), whereas the initial firing frequency increased (Fig. 2E) as the injected depolarizing currents increased. Other passive membrane and single AP characteristics, such as the resting membrane potential (RMP), AP half-width, AP amplitude, and fast-afterhyperpolarization (f-AHP) amplitude are shown in Table 1. Thus, type I neurons are characterized by high levels of intrinsic excitability and exhibit rapid spike adaptation to terminate repetitive spike discharge during depolarizing current injections. 3.3. Type II MSNs These neurons were able to fire repetitively without spike ceasing throughout the tested current-step injections (Fig. 1A). Compared to type I neurons, type II neurons displayed a lower Rin (p < 0.01), a higher AP threshold (p < 0.05), a higher rheobase current (p < 0.01) (Table 1), a faster inward rectification at hyperpolarized membrane potentials (p < 0.05) (Fig. 1B and C), and a larger spike number (p < 0.05) (Fig. 2B). Unlike type I MSNs, type II neurons exhibited a rapid enhancement in spike number with an increase in depolarizing current (4–8 spikes for each step at low

Table 1 Passive and active membrane properties of NAcSh MSN subtypes in rats undergoing chronic withdrawal after repeated morphine exposure or in saline-treated rats. Type I

Passive membrane properties Rin (M) RMP (mV) Active membrane properties Rheobase (pA) AP threshold (mV) AP amplitude (mV) AP half-width (ms) Rise (dV/dt) (mV/ms) Decay (dV/dt) (mV/ms) f-AHP peak amplitude (mV)

Type II

Type III

Saline (n = 11)

Morphine (n = 12)

Saline (n = 15)

Morphine (n = 20)

Saline (n = 6)

Morphine (n = 5)

234.3 ± 25.5 −80.3 ± 0.7

299.2 ± 35.3 −80.6 ± 0.5

174.8 ± 11.1## −81.7 ± 0.4

175.1 ± 8.9 −81.7 ± 0.5

126.4 ± 5.4## −81.2 ± 0.6

131.2 ± 7.8 −81.2 ± 0.5

145.5 −35.4 75.5 1.6 129.4 37.7 9.0

± ± ± ± ± ± ±

9.4 0.9 2.3 0.1 7.8 3.9 0.7

122.9 −34.8 75.8 1.6 126.2 36.8 9.5

± ± ± ± ± ± ±

11.3 1.5 1.5 0.04 7.5 1.5 1.2

190.0 −31.5 72.8 1.7 126.7 37.1 9.8

± ± ± ± ± ± ±

8.4## 0.9# 2.1 0.1 9.6 1.5 0.6

161.3 −32.4 76.04 1.5 145.7 42.3 10.4

± ± ± ± ± ± ±

9.5* 0.6* 1.9 0.1* 5.4 1.4* 0.5

341.7 −30.9 75.7 1.5 131.5 41.7 9.4

± ± ± ± ± ± ±

15.4## 1.0# 1.0 0.1 4.7 4.1 0.3

325.0 −28.9 74.7 1.5 125.5 40.4 9.1

± ± ± ± ± ± ±

25.0 1.2 3.2 0.1 14.1 3.0 0.5

Values represent the mean ± SEM for the number of neurons indicated. Rin , input resistance; RMP, resting membrane potential; AP, action potential; f-AHP, fast afterhyperpolarization. # p < 0.05 represents significant differences in the three types of MSNs from saline-treated rats, one-way ANOVA test. ## p < 0.01 represents significant differences in the three types of MSNs from saline-treated rats, one-way ANOVA test. * p < 0.05 represents significant differences when compared to the saline group within types, Student’s t test.

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Fig. 2. Chronic withdrawal from repeated morphine exposure specifically increased intrinsic excitability in type II MSNs and spike adaptation in type I MSNs. Sample traces of type I MSNs (A1) and type II MSNs (A2) induced by a 325 pA depolarizing current in the saline (Sal)- and morphine (Mor)-treated groups. (B) The spike numbers of type I and type II MSNs in the NAcSh in saline (Sal)- and morphine (Mor)-treated rats. Two-way RM ANOVA. Saline type I vs. type II: F(1,24) = 8.365, p = 0.009; type × injected current interaction, F(10,240) = 41.869, p < 0.001. Saline type I vs. morphine type I: F(1,21) = 0.665, p = 0.424; treatment × injected current interaction, F(10,210) = 0.930, p = 0.507. Saline type II vs. morphine type II: F(1,33) = 5.523, p = 0.025; treatment × injected current interaction, F(10,330) = 0.194, p = 0.997. (C) SFA ratio of type I and type II MSNs from saline (Sal)and morphine (Mor)-treated rats. Saline type I vs. type II: F(1,24) = 26.871, p < 0.001; type × injected current interaction, F(4,96) = 4.222, p = 0.004. Saline type I vs. morphine type I: F(1,21) = 4.98, p = 0.037; treatment × injected current interaction, F(4,84) = 3.247, p = 0.016. Saline type II vs. morphine type II: F(1,33) = 2.432, p = 0.128; treatment × injected current interaction, F(4,132) = 0.588, p = 0.672. (D) The spike train duration of type I and type II MSNs from saline (Sal)- and morphine (Mor)-treated rats. Saline type I vs. type II: F(1,24) = 28.961, p < 0.001; type × injected current interaction, F(6,144) = 16.442, p < 0.001. Saline type I vs. morphine type I: F(1,21) = 4.543, p = 0.045; treatment × injected current interaction, F(6,126) = 0.298, p = 0.937. Saline type II vs. morphine type II: F(1,33) = 5.384, p = 0.027; treatment × injected current interaction, F(6,198) = 3.093, p = 0.007. (E) The initial firing frequency of type I and type II MSNs from saline (Sal)- and morphine-treated (Mor) rats. Saline type I vs. type II: F(1,24) = 9.295, p < 0.001; type × injected current interaction, F(10,240) = 0.954, p = 0.485. Saline type I vs. morphine type I: F(1,21) = 5.175, p = 0.034; treatment × injected current interaction, F(10,210) = 0.263, p = 0.988. Saline type II vs. morphine type II: F(1,33) = 3.055, p = 0.090; treatment × injected current interaction, F(10,330) = 1.648, p = 0.092. Error bars represent SEM.

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depolarization, 14–18 at high depolarization) (Fig. 2B). Type II neurons also had a lesser degree of spike frequency adaptation than type I neurons (p < 0.001) (Fig. 2C). For the absence of spike ceasing in the process of membrane depolarization, type II neurons had a longer spike train duration than type I neurons (p < 0.001) (Fig. 2D). The initial firing frequency of type II neurons in response to a series of depolarizing current steps was significantly lower than that of type I neurons (p < 0.001) (Fig. 2E). There were no significant differences in the RMP, AP half-width, AP amplitude, speed of AP rise and decay, and f-AHP amplitude between type I and type II neurons (Table 1). These results suggest that type II neurons have lower levels of intrinsic excitability and spike frequency adaptation than type I neurons. 3.4. Type III MSNs Type III neurons were encountered less frequently. It was very difficult for this type of neuron to fire in response to low depolarizing current pulses and only a few incidental spikes could be induced by very high depolarizing currents (300–400 pA) (Fig. 1A). Compared to type I and type II neurons, type III neurons displayed the lowest Rin (one-way ANOVA, F(2,29) = 7.207, p < 0.01), the highest AP threshold (one-way ANOVA, F(2,29) = 5.758, p < 0.01), and the largest rheobase current (one-way ANOVA, F(2,29) = 70.803, p < 0.001) (Table 1). Type III neurons had a similar inward rectification as type II neurons (p > 0.05) (Fig. 1B and C). There were no significant differences in other aspects of AP properties between type III neurons and type I or II neurons (Table 1). Thus, type III neurons have the lowest levels of intrinsic excitability and the putative strongest adaptation among all of the observed NAcSh MSNs. 3.5. Effects of morphine treatment on the firing behaviors of NAcSh MSN subtypes To assess the possible effects of chronic withdrawal from repeated morphine exposure (twice daily for 5 days) on the firing behavior of NAcSh MSNs, we measured their passive membrane properties and active membrane properties after 10–14 days of withdrawal from morphine exposure. The MSNs of morphinetreated rats can also be classified into 3 types, as in saline-treated rats (Table 1; Fig. 2A). Type I, type II, and type III neurons represented approximately 32.4%, 54.1%, and 13.5% of the total observed neurons (37 cells from 10 rats), respectively. Similar to saline-treated rats, the type I neurons of the morphine-treated rats displayed a stable spike number throughout depolarizing steps (p > 0.05) (Fig. 2B). However, in morphinetreated type I neurons, the SFA ratio (p < 0.05) (Fig. 2C) and the initial firing frequency (p < 0.05) (Fig. 2E) were significantly increased. Accordingly, the spike train duration was decreased (p < 0.05) (Fig. 2D), which means that the repetitive spike discharge ceased early. However, the rheobase, Rin , AP threshold, and single AP dynamic properties (AP amplitude, AP half-width, RMP, etc.) of type I neurons showed no significant changes (Table 1). These results suggest that after chronic withdrawal from repeated morphine exposure the spike adaptation but not the intrinsic excitability of type I NAcSh MSNs increased. In contrast, the intrinsic excitability of type II neurons was significantly changed in rats that underwent chronic morphine withdrawal. Compared to saline-treated rats, the type II neurons of morphine-treated rats exhibited a decreased rheobase current (p < 0.05), a reduced AP threshold (p < 0.05) (Table 1), an increased number of spikes (p < 0.05) (Fig. 2B), and an increased spike train duration (p < 0.05) (Fig. 2D). Type II neurons of morphine-treated rats also showed shortened AP half-widths (p < 0.05) and increased speed of AP decay time (p < 0.05) (Table 1). However, there were no significant differences in the SFA ratio (p > 0.05) (Fig. 2C) and the

initial firing frequency (p > 0.05) (Fig. 2E) between type II neurons from morphine- and saline-treated rats. Thus, after chronic withdrawal from repeated morphine exposure, the intrinsic excitability and AP properties but not the spike adaptations of type II NAcSh MSNs are changed. The type III neurons still fired a few spikes in response to a depolarizing current during chronic morphine withdrawal. There were no significant differences in the membrane and AP properties between saline- and morphine-treated type III neurons (Table 1). 3.6. Effects of morphine treatment on the slow IAHP in NAcSh MSNs IAHP plays a fundamentally important role in the regulation of neuronal firing properties (Bennett et al., 2000; Savic´ et al., 2001). Given that there was no significant change in the f-AHP between the saline- and morphine-treated rats (Table 1), we tested the two slow components of IAHP , the apamin-sensitive current and the apamininsensitive current. A depolarizing step (100 ms duration) elicited a slow current with a greater than 1.5 s decay time, as shown in Fig. 3A, indicating that the slow IAHP component was present in NAcSh MSNs. The application of 100 nM apamin attenuated the total slow IAHP by 43–51% in 12 recorded cells. The apamin-sensitive current component was calculated by digital subtraction of the apamin-insensitive current from the total slow IAHP of the same cell (Fig. 3B). Compared to the saline group, NAcSh MSNs of morphinetreated rats showed significant decreases in the charge densities of the total IAHP (p < 0.01), the apamin-sensitive current (p < 0.05) and the apamin-insensitive current (p < 0.05) (Fig. 3C). 4. Discussion In the present study, we first identified three functional subtypes of MSNs in the NAcSh based on their distinct firing patterns in response to depolarizing current pulses. They were: repetitive spike discharge with firing ceasing (type I), repetitive spike discharge without firing ceasing (type II), and non-repetitive spike discharge (type III). We then demonstrated that these cell types could be distinguished from each other by their membrane properties. Type I neurons exhibited the highest levels of intrinsic excitability and rapid spike frequency adaptation, type II neurons had moderate levels of intrinsic excitability and relatively slow spike adaptation, and type III neurons had the lowest levels of intrinsic excitability and the putative strongest spike adaptation. Finally, we found that after chronic withdrawal from repeated morphine exposure the intrinsic excitability of type II but not type I MSNs and the spike adaptation of type I but not type II MSNs were enhanced. Additionally, both the apamin-sensitive IAHP and the apamin-insensitive IAHP of MSNs decreased after morphine withdrawal. As a ventral extension of the striatum, the NAc has been proposed to be involved in a number of functions, including motivation, attention, and reward. It is also the target brain region of several classes of psychoactive drugs (Nielsen and Scheel-Krüger, 1986; Swerdlow and Koob, 1987). The NAc has been functionally subdivided into two parts: the core, which is involved in the emotional perception of stimuli and the initiation of learning, and the shell, which mediates the expression of learned behaviors in response to motivationally relevant stimuli (Ito et al., 2004). NAc MSNs are commonly divided into two major subtypes on the basis of their expression of D1Rs (D1R-MSNs) or dopamine D2 receptors (D2R-MSNs) (Le Moine and Bloch, 1995). Glutamatergic input regulates the activity of these two types of MSNs, whereas dopaminergic input modulates their functional responses via stimulation of the distinct dopamine receptor subtypes, positively modulating excitatory glutamatergic input through D1R signaling and negatively

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Fig. 3. Effects of morphine treatment on the IAHP of NAcSh MSNs. (A) The voltage protocol and a sample trace of IAHP in NAcSh MSNs. The total IAHP charge was measured between 5 ms after the offset of the voltage pulse to +30 mV and the time when the current returned to the baseline. (B) Sample traces showing that 100 nM apamin attenuated the current (upper panel); the residual current was subtracted from the control current to obtain the apamin-sensitive IAHP (lower panel). The traces shown here were the averages of 5 traces. (C) Summarized data of charge densities (pC/pF) for apamin-sensitive and apamin-insensitive currents in saline- (n = 12) and morphine- (n = 6) treated rats. *p < 0.05, **p < 0.01, Student’s t test. Error bars represent SEM.

modulating glutamatergic input through D2R signaling (Lobo and Nestler, 2011; Surmeier et al., 2007; Gerfen and Surmeier, 2011). These opposing dopamine-dependent modulations result in the exhibition of higher intrinsic excitability in D2R-MSNs than in D1RMSNs (Gertler et al., 2008). Our study demonstrated that individual MSNs in the NAcSh can also be divided into subtypes by their membrane properties. Interestingly, the levels of intrinsic excitability and adaptation of individual MSNs in the NAcSh are graded among the different subtypes. The graded levels of intrinsic excitability and spike adaptation of the NAcSh MSN subtypes may reflect individual differences in MSNs in their combinations and/or density of ion channels. A variety of inward Na+ and Ca2+ currents and outward K+ currents are involved in shaping neuronal APs and firing patterns (Hille, 2001; Bhattacharjee and Kaczmarek, 2005). BK-type Ca2+ -dependent K+ channels, SK-type Ca2+ -dependent K+ channels, A-type K currents, M-currents, voltage-activated Ca2+ channels, Na/K pump currents, and persistent Na+ currents (INaP ) are believed to participate in the modulation of neuronal excitability (Peng and Wu, 2007; Miles et al., 2005; Lin et al., 2010; Darbon et al., 2003; D’Ascenzo et al., 2009), whereas sustained IK , K+ channels sensitive to intracellular Na+ , P-type Ca2+ current, slow IAHP and fast IAHP , and slow inactivation of the fast Na+ conductance are likely to be the key currents underlying spike adaptation (Bhattacharjee and Kaczmarek, 2005; Peng and Wu, 2007; Miles et al., 2005; Ovsepian and Friel, 2008;

Sanchez et al., 2011; Wang, 1998; Teagarden et al., 2008). In this study, type II and type III MSNs showed significantly stronger inward rectification at hyperpolarized membrane potentials than type I MSNs, a result of inwardly rectifying potassium channels (Kirs) (Nisenbaum and Wilson, 1995). The IAHP is another fundamental parameter that influences neuronal firing pattern. Studies on hippocampal pyramidal neurons indicate that the apaminsensitive IAHP regulates the number of APs per stimulus while the slower apamin-insensitive IAHP underlies spike-frequency adaptation (Stocker et al., 1999). We found morphine withdrawal attenuated the slow IAHP . These results imply that intrinsic differences in the Kirs and IAHP properties of the MSN subtypes cause the graded levels of intrinsic excitability and spike adaptation (Stocker et al., 1999; Shen et al., 2007; Kreitzer and Malenka, 2007). In addition to the ionic conductance of the somatic membrane, firing patterns are also partially determined by the coupling conductance between the dendritic and somatic compartments and the ratio of dendritic to somatic areas (Venance and Glowinski, 2003). D1R-MSNs have more primary dendrites than D2R-MSNs, and experimentally grounded simulations have suggested that differences in MSN dendritic areas are a major contributor to the dichotomy of the electrophysiological properties (Gertler et al., 2008). The asymmetric modulations of the Kirs channels in the D1R- and D2R-MSNs by dendritic M1 muscarinic receptor signaling may also contribute to differences in the electrophysiological

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properties of D1- and D2-MSNs (Shen et al., 2007). Our previous study had shown that selective activation of GluN2Aor GluN2B-containing NMDARs enhanced spike adaptation or intrinsic excitability, respectively, suggesting that GluN2A- and GluN2B-containing NMDARs play different roles in mediating the intrinsic firing properties of neurons (Shi et al., 2011). The activation of postsynaptic D2Rs induces endocytic suppression of GluN2B-containing NMDA receptors via an increase in glycogen synthase kinase-3␤ activity in the rat prefrontal cortex, therefore attenuating NMDAR currents (Li et al., 2009). Conversely, D1R activation modulates NMDAR currents through direct protein–protein interactions between GluN2A and D1R in cultured hippocampal neurons and in HEK-293 cells (Lee et al., 2002). These direct and indirect interactions between dopamine receptor subunits and NMDAR subunits represent a biochemical cross-talk pathway between dopaminergic and glutamatergic transmission. It is possible that the NAc MSNs utilize this pathway to modulate their synaptic plasticity and firing properties in response to drug addiction. Both synaptic input and intrinsic neuronal excitability are essential for the determination of functional neuronal output. It has been widely documented that dynamic changes in both the synaptic strength and the intrinsic excitability of MSNs in the NAc contribute to development and/or relapse of drug addiction (Kourrich and Thomas, 2009; Mu et al., 2010; Thomas et al., 2001; Brebner et al., 2005; Kourrich et al., 2007). An important view is that reduced basal excitability, combined with enhanced excitation driven by drug-associated stimuli, contributes to drug addiction (Kalivas and Hu, 2006). Consistent with this idea, a phenomenon of homeostatic synapse-driven membrane plasticity (hSMP) has been recently identified in NAc MSNs; increased strength of excitatory synapses and decreased intrinsic excitability were observed during withdrawal from repeated cocaine or amphetamine exposure (Kourrich and Thomas, 2009; Mu et al., 2010). Additionally, cocaine exposure specifically decreased the intrinsic excitability of D1R-MSNs but had no significant effect on the intrinsic excitability of D2R-MSNs (Kim et al., 2011). Interestingly, our present results showed that chronic withdrawal from repeated morphine exposure induced a trichotomous modification in the membrane properties of NAcSh MSNs in a cell type-specific manner: an enhancement of intrinsic excitability in type II neurons, a promotion of spike adaptation in type I neurons, and no significant effect on type III neurons. It appears that the enhancement of intrinsic excitability and the promotion of spike adaptation in the different subtypes of MSNs partially neutralize these effects at the functional output level of the whole NAcSh. This compensation helps maintain balance in the reward-associated circuitry and might serve as another homeostatic mechanism of psychostimulant-induced neuroadaptation. Although the cellular mechanisms underlying cell type-specific modulation during chronic morphine withdrawal were not addressed in this study, our findings provide strong evidence that individual NAc MSNs differentially contribute to psychostimulantinduced neuroadaptation by changing their intrinsic membrane properties. It is also possible that the morphine withdrawalinduced modulations of the membrane properties of the MSN subtypes can be ascribed to their unique somatic primary ion channels, their distinct projection pathways (direct-pathway (striatonigral MSNs project to the substantia nigra pars reticulata) vs. indirect-pathway (striatopallidal MSNs project to the globus pallidus)), their dendritic dichotomy, and the specific interactions between NMDARs and dopamine receptors, as mentioned above. In addition, we could not exclude the possibility that firing pattern transition is involved in the effects of morphine exposure. Further studies on the distinct intracellular or synaptic mechanisms of the three types of MSNs and their dynamic

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