Accepted Manuscript

Nucleus accumbens D2- and D1-receptor expressing medium spiny are selectively activated by morphine withdrawal and acute morphine, respectively

T Enoksson, J Bertran-Gonzalez, M.J Christie

PII: S0028-3908(12)00081-0 DOI: 10.1016/j.neuropharm.2012.02.020 Reference: NP 4568

To appear in: Neuropharmacology

Received Date: 2 January 2012 Revised Date: 23 January 2012 Accepted Date: 21 February 2012

Please cite this article as: Enoksson, T, Bertran-Gonzalez, J, Christie, M.J, D2- and D1-receptor expressing medium spiny neurons are selectively activated by morphine withdrawal and acute morphine, respectively, Neuropharmacology (2012), doi: 10.1016/j.neuropharm.2012.02.020

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Nucleus accumbens D2- and D1-receptor expressing medium spiny neurons are selectively activated by morphine withdrawal and acute morphine, respectively

Enoksson Ta,c, Bertran-Gonzalez Ja, Christie MJa,b aBrain and Mind Research Institute and bDiscipline of Pharmacology, The University of Sydney, NSW 2006, Australia. cPresent address; Department of Pharmaceutical Biosciences, Division of Pharmacology, Uppsala University, Box 591, SE-751 24 Uppsala, Sweden

Corresponding authors: MJ Christie and J Bertran-Gonzalez.

Email: [email protected] or [email protected]

Postal Address: Discipline of Pharmacology D06, University of Sydney NSW 2006 Australia

Phone: +612 9351 0899,

Fax: +612 9114 4015.

Key words: MANUSCRIPT morphine, morphine withdrawal, nucleus accumbens, D1 receptor, D2 receptor Abbreviations:

DARPP-32; and cAMP-regulated phosphoprotein 32 kDa,

D1R; dopamine D1-receptor,

D2R; dopamine D2-receptor, eGFP; enhanced green fluorescent protein,

MOR; µ-opioid receptor,

MSN; medium spiny ,

NAc; nucleusACCEPTED accumbens, TBS; Tris buffered saline,

NGS; normal goat serum,

VTA; .

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Abstract

Opioids are effective analgesic agents but serious adverse effects such as tolerance and withdrawal contribute to opioid dependence and limit their use. Opioid withdrawal involves numerous brain regions and includes suppression of dopamine release and activation of neurons in the ventral . By contrast, acute opioids increase dopamine release. Like withdrawal, acute opioids also activate neurons in the ventral striatum, suggesting that different populations of ventral striatal neurons may be activated by withdrawal and acute opioid actions. Here, immunofluorescence for the activity-related immediate-early gene, c- Fos, was examined in transgenic reporter mouse lines by confocal microscopy to study the specific populations of ventral striatal neurons activated by morphine withdrawal and acute morphine. After chronic morphine, naloxone-precipitated withdrawal strongly increased expression of c-Fos immunoreactivity, predominantly in D2-receptor (D2R) medium-sized spiny neurons (MSNs) of the nucleus accumbens (NAc) core and shell regions. By contrast, a single injection of morphine exclusively activated c-Fos immunoreactivity in D1-receptor expressing (D1R) MSNs of the core and shell of the NAc. These results reveal a striking segregation of neuronal responses occurring in the two populations of MSNs of the NAc in response to morphine withdrawal and acute morphine. MANUSCRIPT

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1. Introduction

Clinical use of opioids is limited and illicit use exacerbated by adverse effects that include development of tolerance and withdrawal during chronic use (Williams et al., 2001). Abrupt cessation of chronic opioid use produces an intense withdrawal syndrome manifested by dysphoric and aversive signs in both humans and animal models (Williams et al., 2001), which contributes to drug seeking and high rates of relapse (Koob et al., 1992). It is well established that different components of the global withdrawal response arise from distinct structures in the brain (Maldonado et al., 1992; Williams et al., 2001; Hyman et al., 2006). Among these the ventral tegmental area (VTA) and nucleus accumbens (NAc) are thought to contribute to the aversive signs of opioid withdrawal (Koob et al., 1989; Stinus et al., 1990; Gracy et al., 2001). Opioid withdrawal is associated with decreased activity of dopaminergic neurons and decreased dopamine output to the NAc (Pothos et al., 1991; Rossetti et al., 1992; Diana et al., 1995; Mazie-Robison et al., 2011), and is also associated with strong induction of the activity-related immediate early gene, c-Fos, in neurons of the NAc after precipitation of withdrawal (Hayward et al., 1990; Rasmussen et al., 1995; Georges et al., 2000; Gracy et al., 2001; Hamlin et al., 2001; Bagley et al., 2011). Conversely, acutely administered opioids increase activity of dopaminergic neurons in the ventral tegmental area (VTA) (Diana et al., 1995; Johnson and North, 1992) and increase dopamine release in the NAc (Pothos et al., 1991),MANUSCRIPT an effect that is thought to mediate reward (Hyman et al., 2006). Acute opioids also increase c-Fos expression in neurons of the NAc (Chang et al., 1988; Liu et al., 1994; Garcia et al., 1995; Bontempi and Sharp, 1997; Taracha et al., 2006).

The finding that both naloxone-precipitated opioid withdrawal and acute morphine administration increased c-Fos expression in NAc, despite their opposing effects on dopamine release and aversion, suggests that different populations of MSNs may be involved. Most neurons in the striatum and the NAc (∼95%) are GABAergic medium-sized spiny neurons (MSNs) (Kawaguchi, 1997), and are subdivided into two intermingled populations according to their projection targets and their selective expression of types (Gerfen et al., 1990). Striatonigral MSNs express D1-type receptors (D1Rs) and constituteACCEPTED the ‘direct-pathway’ of the , whereas striatopallidal MSNs express D2-type receptors (D2Rs) and constitute the ‘indirect-pathway’ (Alexander et al., 1986; Gerfen et al., 1990).

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Previous double in situ hybridization studies of c-fos, D1R and preproenkephalin mRNAs in rats found considerable overlap in induction of c-fos in NAc striatonigral and striatopallidal populations during opioid withdrawal (Georges et al., 2000). More recently, bacterial artificial chromosome (BAC) transgenic animals with targeted expression of eGFP in D1R (drd1a-eGFP mouse) and D2R (drd2-eGFP) expressing MSNs (Gong et al., 2003) have been used to unequivocally identify D1R (striatonigral) and D2R (striatopallidal) expressing MSNs (Valjent et al., 2009; Bertran-Gonzalez et al., 2010). This approach has identified strong functional segregation among striatonigral and striatopallidal populations in response to drugs selectively acting on D1Rs and/or D2Rs (Valjent et al., 2009). The present study therefore used these two BAC transgenic lines to determine if D1R or D2R expressing populations of NAc MSNs are differentially activated by opioid withdrawal. The effects of naloxone-precipitated opioid-withdrawal, as well as acute morphine, on the co-localization of c-Fos immunoreactivity and eGFP were examined by confocal microscopy in different territories of the NAc. A striking segregation of the neuronal populations engaged in the two opioid-related responses was observed. Naloxone-precipitated morphine withdrawal induced c-Fos expression predominantly in D2R MSNs, whereas acute morphine induced c-Fos expression exclusively in D1R MSNs.

2. Materials and Methods MANUSCRIPT 2.1 Animals

A total of 28 male adult reporter mice (Swiss-Webster) carrying drd2-eGFP or drd1a-eGFP bacterial artificial chromosome (BAC) transgenes were used in this study (Gene Expression Atlas; Gong et al., 2003). Mice were kept in flow-regulated home cages (Tecniplast Smart Flow) in stable conditions of temperature (21°C) and humidity (50%), and were maintained under a 12 h light/dark cycle with food and water ad libitum. All experiments were performed according to the ethical guidelines of the National Health and Medical Research Council of Australia and were approved by the University of Sydney Animal Care and Ethics Committee. 2.2 Drugs andACCEPTED drug administration protocols Morphine dependence and naloxone-precipitated withdrawal. Physical dependence to morphine was induced over a six-day period by three injections of a sustained-release morphine (free base) emulsion (300 mg/kg, GlaxoSmithKline Australia) on alternate days (days 1, 3 and 5) as described previously (Hacker et al., 2006; Bagley et al., 2011). Briefly,

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mice were transiently anesthetized with isoflurane prior to morphine injections. The morphine base was suspended in a vehicle comprising 50% saline, 40% light liquid paraffin and 10% mannide monooleate, warmed and injected s.c. at three different sites. This sustained release preparation, originally developed by Collier et al. (1972) has been extensively used to produce sustained morphine actions for several days and opioid tolerance, and dependence without the need to subject animals to invasive surgery (e.g. Hacker et al., 2006; Chieng et al., 2005; Bagley et al., 2011). On day 6, opioid withdrawal was precipitated by injecting naloxone (5 mg/kg i.p.) or its vehicle (isotonic saline). Withdrawn mice were then kept in separate cages and perfused two hours after the injection. To avoid the possibility of novelty or stress-induced induction of c-Fos in NAc, opioid withdrawal signs were not explicitly scored in separate observation chambers in the present study (Hebb et al., 2004). Nonetheless, withdrawal signs appeared comparable to those we have reported recently with the same treatment schedule (Hacker et al., 2006; Chieng et al., 2005; Bagley et al., 2011). These included jumps, wet dog shakes, paw tremor, teeth chattering, ptosis, weight loss and diarrhea (e.g. Suppl. Table 1 in Bagley et al., 2011). Control mice were subjected to the morphine dependence protocol but were perfused on day 6 without induction of naloxone-precipitated withdrawal, either without injection or following injection of vehicle (see Fig. 2A). Acute morphine protocol. A supramaximal locomotorMANUSCRIPT stimulatory (e.g. Bohn et al., 2003) but non-toxic (e.g. Sora et al., 2001) dose of morphine-HCl (Acute morphine; 50 mg/kg, GlaxoSmithKline Australia) or vehicle (Control; 0.9% (w/v) NaCl) was injected s.c. 2 h prior to processing for immunofluorescence.

2.3 Tissue processing

Mice were rapidly anaesthetized with lethabarb (300 mg/kg, i.p., Virbac) and transcardially perfused with 4% (w/v) paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.5). Brains were post-fixed overnight in the same solution and stored at 4°C. Unfrozen 30-µm thick sections were cut with a vibratome (VT1000, Leica Microsystems) and stored at -30°C in a solution containing 30% (vol/vol) ethylene glycol, 30% (vol/vol) glycerol and 0.1 M sodium phosphate buffer, until they were processed for immunofluorescence. Brain regions were identifiedACCEPTED using a mouse brain atlas (Dong, 2008), and sections close to three rostro- caudal coordinates (1.42, 1.145 and 0.945 mm from Bregma) were selected for immunofluorescence.

2.4 Immunofluorescence

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eGFP and DARPP-32 detection. Free-floating sections were rinsed three times (10 min each) in Tris-buffered saline (TBS; 0.25 M Tris and 0.5 M NaCl, pH 7.5), incubated for 5 min in TBS containing 3% H2O2 and 10% methanol (v/v), and then rinsed three times for 10 min each in TBS. After 20 min incubation in 0.2% Triton X-100 in TBS, sections were rinsed three times in TBS again, and were incubated overnight at 4°C with purified mouse monoclonal anti-DARPP-32 antibodies (1:500, BD Transduction Laboratories) in TBS. The next day, sections were then rinsed three times for 10 min in TBS and incubated for 45 minutes at room temperature with goat Cy3-coupled secondary antibodies (1:400, Invitrogen). Sections were finally rinsed for 10 min twice in TBS and twice in TB (0.25 M Tris) before mounting in Vectashield (Vector Laboratories) fluorescence medium. eGFP and c-Fos detection. Free-floating sections were rinsed three times (10 min each) in phosphate-buffered saline (PBS; 0.1M, pH 7.5) and blocked and permeabilized at room temperature for 2 h in PBS containing 10% normal goat serum (NGS) and 0.5% Triton- X100. Sections were then incubated for 48 h at 4°C with polyclonal rabbit anti-c-Fos primary antibody (1:500, Santa Cruz Biotechnology, Inc) in PBS with 2% NGS and 0.2% Triton-X100. Unbound primary antibodies were washed in PBS (3 rinses, 10 min each) prior to 90 min incubation with Cy3-conjugated goat anti-rabbit secondary antibody (1:1000, Molecular Probes) in PBS with 2% NGS and 0.2% Triton X-100 at room temperature. In sections from drd1a-eGFP mice, polyclonal chickenMANUSCRIPT anti-eGFP primary antibodies (1:500, Aves Lab, Inc) and FITC-conjugated goat anti-chicken antibodies (1:1000, Jackson ImmunoResearch Laboratories, Inc) were added to the primary and secondary antibody incubations, respectively. Sections were rinsed 3 times in PBS (10 min) before mounting in Fluoromount-G (SouthernBiotech) fluorescence medium.

2.5 Fluorescence imaging and quantification

Double-labeled images were obtained with a sequential laser scanning confocal microscope (Olympus Fluoview FV300, BX61WI microscope) using a 60X objective (UPFL 60X oil). In the c-Fos study, 10 image stacks of 235.47 µm2 (step size: 2 µm) were sequentially taken for green FITC/eGFP fluorescence (channel 01, Ar laser intensity usually 20.0%; PMT: 700v; offset: 3%) and red CY3 fluorescence (channel 02, HeNe green laser intensity usuallyACCEPTED 20.0%; PMT: 720v; offset 3%). For each of the 3 rostro-caudal sections analyzed, image stacks were acquired bilaterally per region, so a total of 12 stacks were obtained per individual (see Fig. 1). The total number c-Fos expressing neurons was first assessed by identifying all immunoreactive cells in each stack (channel 02). Stacks were then overlapped with the corresponding eGFP series of images (channel 01). The number

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of c-Fos and eGFP co-localized neurons was then re-quantified, and the number of non co- localized neurons was obtained as the total number minus the number of co-localized neurons. Final numbers are the sum of counted neurons per hemisphere. Total values from each hemisphere were finally averaged per individual.

2.6 Statistical analyses

Data are expressed as mean values ± SEM (n = 3-4 animals per group), and were analyzed using two-way ANOVA. Post hoc comparisons between groups were made using Bonferroni multiple comparison test. In all cases, significance threshold was set at p < 0.05. Statistical analysis was performed with PRISM 5.0 software (GraphPad, Inc).

3. Results

3.1 drd2-eGFP BAC transgenic mice provide clear identification of the two MSN populations in the NAc core and shell

BAC transgenic mice have been established as a very good tool for the identification of the two main MSN populations in the striatum (Valjent et al., 2009; Bertran-Gonzalez et al., 2010). drd2-eGFP mice are particularly useful because accurate distinction of D2R neurons is possible without any method of signal amplification, MANUSCRIPT due to their high intensity of fluorescence (Matamales et al., 2009). Confocal microscopy on drd2-eGFP mice combined with DARPP-32 immunostaining, a reliable marker of both populations of MSNs (Ouimet et al., 1984; Matamales et al., 2009), was used to systematically identify D2R neurons in regions unambiguously comprised within the NAc core and the NAc shell at three different rostro-caudal levels (Fig. 1A). Fluorescence in these mice was confined to the striatum and (Fig. 1B), and very clearly identified D2R neurons in the selected NAc core and NAc shell regions at a higher magnification (Fig. 1B, insets 1 and 2). Double staining with DARPP-32, revealed that approximately half of the neurons expressed eGFP in both selected regions (Fig. 1C and D), confirming the accuracy of drd2-eGFP mice for the identification of not only D2R neurons (those expressing fluorescence) but also D1R neurons (thoseACCEPTED lacking fluorescence) (Matamales et al., 2009). 3.2 Naloxone-precipitated opioid withdrawal induces c-Fos expression very selectively in D2R MSNs of drd2-eGFP mice

Induction of c-Fos expression has long been related to neuronal activation (Sheng and Greenberg, 1990; Hughes and Dragunow, 1995), and has been shown to occur in striatal 7

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neurons in response to multiple pharmacological treatments, especially those engaging D1Rs and D2Rs (Robertson et al., 1991; Young et al., 1991; Berretta et al., 1992; Moratalla et al., 1996). In the NAc, acute morphine injections induce strong c-Fos expression in striatal neurons (Chang et al., 1988; Garcia et al., 1995; Taracha et al., 2006), and this activation is dependent upon D1, NMDA and µ-opioid (MOR) receptors (Liu et al., 1994; Bontempi and Sharp, 1997). Naloxone-precipitated morphine withdrawal induces high levels of c-Fos expression in striatal neurons, an effect that is particularly strong in the NAc but less prominent in the dorsal striatum (Hayward et al., 1990; Rasmussen et al., 1995; Georges et al., 2000; Gracy et al., 2001).

The treatment schedule used here has consistently been reported to induce intense naloxone-precipitated withdrawal behavior (see Methods, e.g. Bagley et al., 2011). The effect of naloxone-precipitated withdrawal on c-Fos expression was first examined in the NAc of drd2-eGFP mice (Fig. 2). c-Fos expression was greatly enhanced in NAc core and shell 2 hours after injection of naloxone compared to controls, and almost all immunoreactivity was confined to D2R+ neurons (Fig. 2B-D). No differences in basal c-Fos expression were observed between uninjected and vehicle challenged mice (Fig. 2C, D). In a separate experiment (Bagley et al., 2011), we also observed low levels of basal c-Fos expression in NAc and no significant differences between three control groups (all n = 6), including chronic vehicle/saline challenge (18 ±MANUSCRIPT 3 c-Fos positive neurons per series in NAc shell), chronic vehicle/naloxone challenge (20 ± 3) and chronic morphine/saline challenge (13 ±2).

During withdrawal, c-Fos immunoreactivity was profoundly elevated (319 ± 32.1 neurons, n = 4 vs. 51.5 ± 20.7, n = 4, p < 0.001) throughout the rostro-caudal extent of the NAc (see Fig. 1A for rostro-caudal levels sampled). A large, significant increase of c-Fos expression occurred in D2R+ (~9-fold) versus D2R- neurons in both the NAc core and shell, whereas a smaller increase (~3 fold) was observed in D2R- neurons (Fig. 2C and D), which was significant (P < 0.05) only in the NAc core. The increase of c-Fos expression in D2R+ neurons was significantly greater than D2R- neurons in both NAc core and shell (Fig. 2C and D).

3.3 Naloxone-precipitated opioid withdrawal induces c-Fos expression selectively in (presumed)ACCEPTED D2R MSNs of drd1a-eGFP mice

Recent studies have reported several behavioral and physiological alterations in drd2-eGFP mice (Kramer et al., 2011) that could potentially influence opioid withdrawal effects on NAc neurons. It was also not possible to rule out whether or not the trend for withdrawal-induced 8

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c-Fos in D2R- neurons could include the small populations of NAc non-MSNs in drd2-eGFP mice (Kawaguchi, 1997). The next experiment therefore sought to confirm the distribution of naloxone-induced c-Fos expression found in D2R MSNs by using drd1a-eGFP BAC transgenic mice (Fig. 3), another reporter line that specifically tags D1R expressing population of striatal MSNs with no major physiological alterations. After application of the same naloxone-precipitated withdrawal procedure to drd1a-eGFP mice (see Fig. 2A), the pattern of c-Fos immunoreactivity distribution in the NAc largely matched that found in drd2- eGFP mice. As found in the drd2-eGFP mice, withdrawal-induced increased c-Fos was largely confined to presumed D2R expressing (D1R-) neurons in drd1a-eGFP mice (Fig. 3A). As in the case of drd2-eGFP mice, rostro-caudal quantification of c-Fos-positive neurons in the NAc core of drd1a-eGFP mice also showed a large increase of c-Fos neurons (266.8 ± 12, n = 4 vs. 20.8 ± 1.6, n = 3, p < 0.0001), suggesting that overall c-Fos induction in drd2-eGFP mice was not aberrant. Overall induction of c-Fos was also similar to that we have previously reported in other mice using the same treatment protocol (Bagley et al., 2011).

As found in the drd2-eGFP mice (see above), a large increase of immunoreactivity (~22 fold) was found in presumed D2R+ (observed as D1R-) neurons of the NAc core, and a

MANUSCRIPT+ much lower and non-significant increase (∼3 fold) in D1R neurons after naloxone-

precipitated withdrawal (Fig. 3B). In the NAc shell, naloxone-precipitated withdrawal induced a marked increase of c-Fos immunoreactivity in D1R- neurons (~27 fold), although a smaller (~7 fold), significant increase of c-Fos immunoreactivity also occurred in D1R+ neurons (Fig. 3C). Taking the findings for drd2-eGFP and drd1a-eGFP mice together, these results clearly demonstrate that the strong neuronal activation occurring in the NAc following naloxone-precipitated withdrawal predominantly involves D2R striatopallidal MSNs of the indirect pathway, with a lesser involvement of D1R MSNs.

3.4 Acute morphineACCEPTED treatment induces a selective increase of c-Fos expression in D1R population of striatal MSNs

The sub-populations of NAc MSNs in which c-Fos is induced in response to acute morphine have not been studied directly. The distribution of c-Fos induction in response to a single

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injection of morphine (50 mg/kg) was examined in drd2-eGFP mice (Fig. 4). Two hours after injection, morphine-treated mice displayed an increase in c-Fos immunoreactive neurons in both the core and shell of the NAc compared to vehicle controls (Fig. 4A), confirming the effects of this drug in ventral striatal regions (Garcia et al., 1995; Bontempi and Sharp, 1997; Taracha et al., 2006). Although significant, the overall induction of c-Fos by acute morphine was substantially less than that observed during naloxone-precipitated withdrawal (77.9 ± 9.08, n = 4 vs. 35.33 ± 2.4, n = 3, p < 0.05), particularly in the NAc core (Fig. 4B, C). Interestingly, co-localization with eGFP revealed that the morphine-induced increase in c- Fos immunoreactive neurons occurred exclusively in neurons that did not express eGFP fluorescence (Fig, 4A). The quantification study revealed that, whereas both eGFP-positive (D2R+) and eGFP-negative (D2R-) neurons expressed equivalent basal levels of c-Fos immunoreactive neurons, acute morphine treatment increased c-Fos expression selectively in D2R- neurons, whereas D2R+ c-Fos levels remained unchanged (Fig. 4B, C). As previously reported (Taracha et al., 2006), the extent of neuronal activation was found to be higher in the NAc shell, although the distribution of the activated cells was restricted to D2R- neurons in both NAc core and shell (Fig. 4B, C). These results suggest the exclusive activation of the D1R striatonigral system of the NAc in response to a single injection of morphine. 4. Discussion MANUSCRIPT The aim of the present study was to unambiguously identify the sub-populations of MSNs activated by naloxone-precipitated opioid-withdrawal, as well as acute morphine, in the NAc since both opioid-related conditions generate strong cellular responses in ventral striatal neurons despite their very different neurobehavioral effects. To this end, we took advantage of the recently generated BAC transgenic reporter mouse lines (Gong et al., 2003), which have been shown to be useful for sensitive and selective identification of the two different populations of projection MSNs (Bertran-Gonzalez et al., 2008; Matamales et al., 2009).

Our results confirm previous studies that eGFP labeling in these lines permits reliable visualization of virtually all of the D1R and D2R classes of MSNs (Bertran-Gonzalez et al., 2008, Matamales et al., 2009). As shown in Fig. 1, drd2-eGFP mice showed that about one half of the ACCEPTEDstriatal projection MSNs, which were reliably identified through DARPP-32 immunodetection, expressed a clear eGFP signal in the core and shell regions of the NAc, as expected. This is fully in agreement with previous reports validating in these mice that DARPP-32 immunostaining labels all striatal neurons (MSNs) except for local (Bertran-Gonzalez et al., 2008, Matamales et al., 2009). These studies established that

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approximately a half of DARPP-32 positive (medium spiny) striatal neurons are eGFP positive in each of drd2-eGFP and drd1a-eGFP mice and, importantly, that all DARPP-32 positive neurons are eGFP positive when the two lines are crossed (Matamales et al., 2009). Using these mice we found a striking segregation between the MSNs engaged by withdrawal (predominantly D2R MSNs) and acute morphine (exclusively D1R MSNs), respectively.

The mechanisms responsible for selective engagement of D2R neurons during opioid withdrawal are not certain. Although opioid withdrawal directly modulates synaptic activity in NAc neurons in vitro (e.g. Chieng and Williams, 1998), most studies suggest that the effects of morphine withdrawal in the NAc are a consequence of alterations of dopamine neuron firing in the VTA (Williams et al., 2001) and are not due to a local regulation of MOR (Maldonado et al., 1992). In contrast to the effects of acute morphine, morphine withdrawal induces a profound depression of dopamine neuron activity in the VTA (Diana et al., 1995), both through enhanced GABAergic (Madhavan et al., 2010) and decreased glutamatergic transmission onto these neurons (Manzoni and Williams, 1999). Opioid withdrawal also enhances c-Fos expression in GABAergic VTA neurons (Bagley et al., 2011). An intriguing question from these studies is how an abrupt and virtually complete cessation of dopamine neuron firing induced by morphine withdrawal (Diana et al., 1995) can lead to such a strong neuronal activation in ventral striatal regions (HaywardMANUSCRIPT et al., 1990; Rasmussen et al., 1995; Georges et al., 2000; Gracy et al., 2001). It is tempting to speculate that nearly complete elimination of dopamine release in the NAc during withdrawal removes the sustained stimulation of high affinity, inhibitory D2Rs (Richfield et al., 1989). This would be expected to trigger the strongly segregated neuronal responses in neurons containing

D2Rs observed in the present study because the sustained, Gαi-mediated inhibition of cAMP signaling by D2Rs would be released, as has been shown to occur in response to D2R blockade in the dorsal striatum (Bertran-Gonzalez et al., 2009).

If the mechanism of selective c-Fos induction in D2R versus D1R MSNs did depend indirectly on dopamine tone, this might also explain the selective induction of c-Fos in D1R+ after acute administration of morphine. It is generally accepted that acute effects of systemic morphine on striatal neurons are the consequence of a strong regulation exerted by GABAergicACCEPTED interneurons of the VTA on dopamine neurons through a MOR-dependent mechanism (Di Chiara and North, 1992; Johnson and North, 1992). Accordingly, systemically administered morphine stimulates MORs strongly expressed in midbrain

GABAergic synapses, which engages Gαi-mediated intracellular signaling events that decrease the GABAergic tone exerted on dopamine neurons of the VTA (Di Chiara and 11

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North, 1992; Johnson and North, 1992), presumably arising from local GABAergic interneurons (Johnson and North, 1992; Chieng et al., 2011) and GABAergic rostromedial tegmental nucleus neurons (Jalabert et al., 2011; Matsui and Williams, 2011). This in turn disinhibits dopamine neurons, inducing release of dopamine in target regions such as the NAc. Importantly, enhanced dopamine release in the NAc caused by other stimuli produces strong c-Fos induction in MSNs, an effect that has been shown to be dependent upon D1- and NMDA-receptors (Liu et al., 1994; Bontempi and Sharp, 1997). Since c-Fos expression (as well as a multitude of other signaling events; Valjent et al., 2009) is mediated by D1Rs in response to drugs increasing dopamine tone, only neurons expressing these receptors are expected to be excited after acute morphine administration, as was observed after administration of psychostimulants (Bertran-Gonzalez et al., 2008).

It is difficult to reconcile our findings with the double in situ hybridization study of Georges et al. (2000) in morphine-withdrawn rats. In their study, naltrexone-precipitated withdrawal induced c-fos mRNA expression with a trend for selective expression in D1R mRNA- positive and pre-pro-enkephalin A mRNA-negative (striatonigral) neurons of the NAc (Georges et al., 2000). Interestingly, however, they reported in the same animals a preferential distribution of c-fos mRNA in striatopallidal (presumably D2R positive) neurons of dorsal striatal regions. Besides possible differences due to species, the method of MSN phenotyping (in situ hybridization vs. targeted expressionMANUSCRIPT of eGFP) and/or neuronal activity detection (mRNA vs. protein), the different degrees of D1R and D2R co-expression reported in different striatal territories may explain the contrasting results (Bertran-Gonzalez et al., 2008; Perreault et al., 2010). It is important to consider recent estimates of D1R and D2R co-expressing neurons performed in BAC transgenic mice, in which only 5% of the MSNs are estimated to co-express these receptors in the dorsal striatum and the NAc core, whereas up to 17% of the MSNs seem to do so in the NAc shell (Bertran-Gonzalez et al., 2008; Matamales et al., 2009).

Importantly, co-expression D1R and D2R in ~17% of MSNs in NAc shell could also reconcile the differences observed here in drd1a- and drd2-eGFP mice following morphine withdrawal. A similar induction of c-Fos in D2R expressing neurons was found in the NAc core and shell of both drd2-eGFP (D2R+) and drd1a-eGFP mice (D1R-) after precipitation of withdrawal.ACCEPTED However, withdrawn drd1a-eGFP mice also showed a small, significant increase in c-Fos expression in D1R+ neurons of the shell but not the core (see Figs. 2 and 3). According to the functional model discussed above, the effect induced by morphine withdrawal in the NAc would be directly mediated by D2Rs, and should therefore be exclusively dependent on the distribution of D2Rs but not D1Rs. Thus, a more accurate 12

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localization of the effect is expected in the drd2-eGFP line, which labels all the neurons that express D2Rs, independently of their co-expression with D1Rs. By contrast, since the levels of D1R and D2R co-expression occur in about 17% of the neurons in BAC-transgenic mice (Bertran-Gonzalez et al., 2008; Matamales et al., 2009), the expected proportion of D1R+ neurons in drd1a-eGFP mice showing the effect should approach this number, which likely corresponds to D1R and D2R co-expressing neurons. New transgenic lines accurately labeling both populations with different fluorophores in the same individuals already exist, and should help to resolve this question (Shuen et al., 2008; Ade et al., 2011).

5. Conclusions

In conclusion, the present study has identified clearly segregated cellular activation D2R and D1R expressing MSNs of the NAc induced by opioid withdrawal and acute morphine, respectively. This characteristic pattern of neuronal responses in the NAc is crucial for the understanding of the addictive and aversive properties of opioids during acute administration and withdrawal, respectively. Both factors contribute to the limitations of opioids for the therapeutic treatment of pain disorders.

Acknowledgements: Supported by the National MANUSCRIPT Health and Medical Research Council of Australia (project grant 512390 and fellowship 511914 to MJC). Assistance of Dr B Chieng with chronic morphine administration is gratefully acknowledged. Prof BW Balleine is gratefully acknowledged for providing the transgenic mice.

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References

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Figure Legends

Fig. 1. Distribution of eGFP fluorescence in the NAc core and shell of drd2-eGFP BAC transgenic mice. (A) Three rostro-caudal coronal sections containing the NAc were selected per animal. Red squares indicate the sample regions bilaterally taken from the NAc core (c) and NAc shell (s). Coordinates are mm from Bregma (Diagrams modified from Dong, 2008). (B) Low-magnification micrograph showing the distribution of drd2-eGFP signal in all striatal regions at +1.245 mm from Bregma. Insets 1 and 2 show a higher magnification confocal image of the selected regions of the NAc core (1) and NAc shell (2) used in further analyses of this study. (C, D) Confocal images showing double staining of the MSN marker DARPP- 32 (red) and drd2-driven eGFP (green) in the NAc core (C) and shell (D). Note that drd2- driven fluorescence is present in about half of the MSNs.

Fig. 2. Naloxone-precipitated morphine withdrawal induces c-Fos expression almost exclusively in D2R-eGFP neurons of the NAc. (A) Over a six-day period, drd2-eGFP mice received 300 mg/kg s.c. morphine injections (M, days 1,3,5) and were challenged with no injection (control, C), vehicle injection (V, i.p.) or 5 mg/kg naloxone (NLX, i.p.) on day 6. (B) Single confocal micrographs showing drd2-driven eGFP fluorescence (green) combined with c-Fos immunofluorescence (red) in the NAc core of morphine-treated drd2-eGFP mice perfused 2 hours after final treatment. (C, D) QuantificationMANUSCRIPT of c-Fos immunoreactive neurons among eGFP-positive (D2R+) and eGFP-negative (D2R-) neurons in the NAc core (B) and NAc shell (C) of drd2-eGFP mice treated as in A. Data (mean ± SEM; n = 3-4 per group) were analyzed using two-way ANOVA with Bonferroni posthoc comparisons. *p < 0.05; ***p < 0.001; n.s., non significant.

Fig. 3. Naloxone-precipitated morphine withdrawal induces a matching pattern of c-Fos expression in drd1a-eGFP mice. drd1a-eGFP mice were treated as in Fig. 2A. (A) Single confocal micrographs showing drd1a-driven eGFP fluorescence (green) combined with c- Fos immunofluorescence (red) in the NAc core of morphine-treated drd1a-eGFP mice perfused 2 hours after naloxone (NLX 5 mg/kg) or not (Control). (B, C) Quantification of c- Fos immunoreactiveACCEPTED neurons among eGFP-negative (D1R-) and eGFP-positive (D1R+) neurons in the NAc core (B) and NAc shell (C) of drd1a-eGFP mice treated as in A. Data (mean ± SEM; n = 3-4 per group) were analyzed using two-way ANOVA with Bonferroni posthoc comparisons. ***p < 0.001; n.s., non significant.

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Fig. 4. Acute morphine treatment induces c-Fos expression exclusively in non D2R-eGFP neurons of the NAc. (A) Single confocal micrographs showing drd2-driven eGFP fluorescence (green) combined with c-Fos immunofluorescence (red) in the NAc core of drd2-eGFP reporter mice 2 hours after a single injection of vehicle or morphine (50 mg/kg, s.c.). (B, C) Quantification of c-Fos immunoreactive neurons among eGFP-positive (D2R+) and eGFP-negative (D2R-) neurons in the NAc core (B) and NAc shell (C) of drd2-eGFP mice treated as in A. Data (mean ± SEM; n = 3-4 per group) were analyzed using two-way ANOVA with Bonferroni posthoc comparisons. *p < 0.05; ***p < 0.001; n.s., non significant.

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D2R and D1R-GFP transgenics can identify activated (c-Fos) nucleus accumbens neurons

Both opioid withdrawal and acute morphine induce c-Fos in nucleus accumbens

Morphine withdrawal selectively induces c-Fos in D2R neurons in nucleus accumbens

Acute morphine selectively induces c-Fos in D1R neurons in nucleus accumbens

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