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. Author manuscript; available in PMC: 2014 Oct 3.
Published in final edited form as: Neuron. 2013 Oct 2;80(1):159–170. doi: 10.1016/j.neuron.2013.07.019

Repeated cocaine weakens GABAB-Girk signaling in Layer 5/6 pyramidal neurons in the prelimbic cortex

Matthew Hearing 1, Lydia Kotecki 1, Ezequiel Marron Fernandez de Velasco 1, Ana Fajardo-Serrano 2, Rafael Luján 2, Kevin Wickman 1,†
PMCID: PMC3793643  NIHMSID: NIHMS509806  PMID: 24094109

Summary

Repeated cocaine exposure triggers adaptations in Layer 5/6 glutamatergic neurons in the medial prefrontal cortex (mPFC) that promote behavioral sensitization and drug-seeking behavior. While suppression of metabotropic inhibitory signaling has been implicated in these behaviors, underlying mechanisms are unknown. Here, we show that Girk/KIR3 channels mediate most of the GABAB receptor (GABABR)-dependent inhibition of Layer 5/6 pyramidal neurons in the mPFC and that repeated cocaine suppresses this pathway. This adaptation was selective for GABABR-dependent Girk signaling in Layer 5/6 pyramidal neurons of the prelimbic cortex (PrLC) and involved a D1/5 dopamine receptor- and phosphorylation-dependent internalization of GABABR and Girk channels. Persistent suppression of Girk signaling in Layer 5/6 of the dorsal mPFC enhanced cocaine-induced locomotor activity and occluded behavioral sensitization. Thus, the cocaine-induced suppression of GABABR-Girk signaling in Layer 5/6 pyramidal neurons of the prelimbic cortex appears to represent an early adaptation critical for promoting addiction-related behavior.

Introduction

Drug addiction is marked by subtle yet pervasive disruptions in cognitive function that facilitate its development and contribute to a high rate of relapse in addicts, even after sustained periods of abstinence (Garavan and Hester, 2007). The prefrontal cortex (PFC) is a critical substrate for many higher-order cognitive functions, including decision-making, inhibitory behavior, and processing of reward-related information (Rushworth et al., 2011). Prolonged exposure to drugs like cocaine promotes structural, physiological, and functional abnormalities within the PFC that compromise these functions. For example, activity in areas of the PFC of cocaine addicts is decreased during withdrawal but increased during intoxication and exposure to drug-associated cues, the latter of which is associated with feelings of craving (Goldstein and Volkow, 2011). Lesions of analogous brain regions in the rodent (the medial PFC, or mPFC) facilitate the acquisition of cocaine self-administration (Weissenborn et al., 1997), while reversal of cocaine-induced prefrontal hypoactivity prevents the development of compulsive drug-seeking behavior (Chen et al., 2013).

Glutamatergic pyramidal neurons in the deepest layers of the mPFC (Layers 5 and 6) are the primary output neurons of the mPFC and represent a major source of excitatory input to the ventral tegmental area (VTA) and nucleus accumbens (NAc). Collectively, these structures comprise the ‘reward circuit’ that mediates response to natural rewards and drugs of abuse (Koob and Volkow, 2010). Frontal corticostriatal glutamate projections play a critical role in modulating neurotransmission in the mesocorticolimbic system (Sesack et al., 1989; Taber and Fibiger, 1995). Moreover, adaptations in glutamate transmission are produced by repeated exposure to cocaine that contributes to the expression of addiction-related behavior. For example, cocaine-induced augmentation of glutamatergic output to the VTA and NAc is critical for the development and expression of behavioral sensitization and reinstatement of drug-seeking behavior (Steketee and Kalivas, 2011).

Repeated cocaine exposure enhances the responsiveness of mPFC pyramidal neurons to cocaine and cocaine-related stimuli (Sun and Rebec, 2006). This phenomenon, and the related cocaine-induced augmentation of glutamatergic output from the mPFC to the VTA and NAc, has been linked to adaptations in multiple ion conductances in Layer 5/6 mPFC pyramidal neurons (Dong et al., 2005; Nasif et al., 2005a; Nasif et al., 2005b; Lu et al., 2010). Repeated cocaine also weakens signaling mediated by inhibitory G protein-coupled receptors, including the D2 dopamine receptor (D2R) and GABAB receptor (GABABR), pathways that normally temper the excitability of mPFC pyramidal neurons (Hearing et al., 2012). Indeed, decreased D2R- and GABABR-dependent modulation of glutamatergic output from the mPFC to the VTA and NAc has been proposed to contribute to the development of cocaine sensitization (Beyer and Steketee, 2002; Jayaram and Steketee, 2004).

The mechanisms underlying the cocaine-induced suppression of metabotropic inhibitory signaling in the mPFC are unresolved. Reductions in D2R availability and expression have been reported in the PFC of human cocaine addicts and in rats following cocaine self-administration (Volkow et al., 1993; Briand et al., 2008). In contrast, no change in expression level of GABABR or inhibitory (Gi/o) G proteins (Striplin and Kalivas, 1992; Li et al., 2002), or functional coupling between GABABR and G proteins (Kushner and Unterwald, 2001), has been observed in the mPFC following repeated cocaine exposure. Here, we sought insight into the impact of cocaine on metabotropic inhibitory signaling in Layer 5/6 mPFC pyramidal neurons, focusing on the postsynaptic GABABR signaling pathway.

Results

GABABR-dependent inhibition of Layer 5/6 pyramidal neurons of the prelimbic cortex

Sub-regions of the mPFC demonstrate distinct patterns of connectivity and regulate different behavioral functions (Dalley et al., 2004; Vertes, 2004; Marquis et al., 2007). Increased excitatory output from the dorsal mPFC, which includes the interface between the cingulate (cg) and prelimbic (PrLC) cortices, is crucial for the expression of sensitization and reinstatement of drug-seeking behavior (Tzschentke and Schmidt, 2000; McFarland and Kalivas, 2001; Steketee and Kalivas, 2011; Chen et al., 2013). Thus, we began by evaluating GABABR-dependent signaling in Layer 5/6 pyramidal neurons in the PrLC (Fig. 1A).

Figure 1. GABABR-dependent signaling and excitability in Layer 5/6 PrLC pyramidal neurons.

Figure 1

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(A) Depiction of a coronal section containing the mPFC. The shaded region denotes the area of the prelimbic cortex (PrLC) targeted for analysis. Given the lack of definitive anatomic borders, a small number of neurons in the ventral cingulate (cg) and dorsal infralimbic (ILC) sub-regions of the mPFC was likely included in the analysis.

(B) Outward current (upper trace) evoked by baclofen (200 μM) in a wild-type Layer 5/6 PrLC pyramidal neuron (Vhold = −60 mV). The baclofen-induced current (IBaclofen) was associated with a decrease in input resistance (RIN, lower trace) and was reversed by the GABABR antagonist CGP54626 (CGP, 2 μM). Scale bars: 50 pA/5 min.

(C) Concentration-response curve for IBaclofen in Layer 5/6 PrLC pyramidal neurons. Responses to lower concentrations were normalized to the current induced by 200 μM baclofen (Norm. Response %). The EC50 and Hill Slope were 2.1±0.1 μM and 0.54±0.02, respectively (n=6).

(D) IBaclofen measured in a wild-type Layer 5/6 PrLC pyramidal neuron in the presence of external Ba2+ (0.3 mM, blue), and in a neuron from a Girk2–/– mouse (gray). Scale bars: 50 pA/5 min.

(E) IBaclofen in Layer 5/6 PrLC pyramidal neurons from wild-type mice in the presence of external Ba2+ (0.3 or 1 mM, blue), with (+) or without (−) 8Br-cAMP (10 mM) in the pipette solution, as compared to control (ACSF, black). A significant effect of group was observed (F(4,43)=147.8, P<0.001; n=4-16/group). ***P<0.001 vs. ACSF; #,##P<0.05 and 0.01, respectively; ^^P<0.01 vs. 0.3 mM Ba2+ (no 8Br-cAMP).

(F) IBaclofen in Layer 5/6 PrLC pyramidal neurons from wild-type (wt), Girk1–/–, Girk2–/–, Girk2/3–/– and Girk3–/– mice. A significant effect of genotype was observed (F(4,54)=36.2, P<0.001; n=9-16/group). ***P<0.001 vs. wt; ##P<0.01 vs. Girk3–/–.

(G) Spiking elicited by current injection (140 pA/1 s) in Layer 5/6 PrLC pyramidal neurons from wild-type (top) and Girk2–/– (bottom) mice. Scale: 20 mV, 200 ms.

(H) Current required to evoke an action potential (AP, Threshold) in neurons from Girk2–/– mice, as compared to wild-type (wt) (t(23)=4.1, P<0.01; n=10-14/group). **P<0.01 vs. wt.

(I) Current-spike plots for wild-type and Girk2–/– Layer 5/6 PrLC pyramidal neurons (F(10,223)=2.4, P<0.01; n=9-12/group). *P<0.05 vs. wild-type (within current injection group).

All data are presented in this and remaining figures as the mean ± SEM. See Table S1 for electrophysiological properties of Layer 5/6 PrLC pyramidal neurons from wild-type and Girk–/– mice, and Figure S1 for Girk1-4 gene expression analysis in the mPFC and analysis of sEPSC data from wild-type and Girk2–/– mice.

Bath application of a saturating dose of the GABABR agonist baclofen (200 μM) evoked a robust outward current (IBaclofen) that showed little desensitization after 10 min of continuous drug application, but was reversed by the GABABR antagonist CGP54626 (Fig. 1B). IBaclofen was dose-dependent, with an EC50 of 2.1±0.1 μM (Fig. 1C). IBaclofen was correlated with a decrease in input resistance and it reversed polarity at −88±2 mV (n=11), consistent with the activation of a K+ channel. Indeed, IBaclofen was suppressed by low concentrations of external Ba2+ (0.3-1 mM) (Fig. 1D,E), pointing to the involvement of an inwardly-rectifying K+ channel.

GABABRs activate G protein-gated inwardly-rectifying K+ (Girk/KIR3) channels in many neurons, including Layer 2/3 mPFC pyramidal cells (Wang et al., 2010). To test whether Girk channels mediate the GABABR-dependent inhibition of Layer 5/6 PrLC pyramidal neurons, we next compared IBaclofen in slices from wild-type and Girk–/– mice. While Girk ablation had no effect on many parameters of Layer 5/6 PrLC pyramidal neurons (Table S1), IBaclofen was ~60% smaller in neurons from Girk–/– mice, with the smallest currents seen in slices from Girk1–/– and Girk2–/– mice (Fig. 1D,F). These findings were consistent with RT-PCR data from micropunches containing the mPFC, which showed expression of Girk1, Girk2, and Girk3 in this brain region (Fig. S1A).

Girk1 cannot form functional homomeric Girk channels due to lack of an ER export signal (Ma et al., 2002). Thus, to better understand the residual IBaclofen measured in Girk–/– mice, we evaluated Layer 5/6 PrLC pyramidal neurons in slices from Girk2/3–/– mice. IBaclofen in neurons from Girk2/3–/– mice was comparable to that measured in slices from Girk1–/– and Girk2–/– mice (Fig. 1F), arguing that compensatory up-regulation of Girk3 is not responsible for the residual current. We next asked whether up-regulation of Girk4 could explain the residual current measured in slices from Girk1–/– and Girk2–/– mice. While sparse Girk4 expression was detected in the deepest layers of the most posterior aspect of the mPFC (Fig. S1B), these rare neurons were located outside the region targeted for analysis. Moreover, no difference in Girk4 mRNA levels were observed in mPFC micropunches from wild-type and Girk2/3–/– mice (Fig. S1C). Thus, the residual IBaclofen seen in Girk–/– mice appears to be Girk-independent.

The amplitude of the fraction of IBaclofen resistant to Ba2+ (0.3 mM) was not significantly different in neurons from wild-type (70±5 pA, n=10) and Girk2–/– (53±13 pA, n=6) mice, nor was it different from the residual current in neurons from Girk2–/– mice measured in the absence of Ba2+ (79±8 pA; F(2,25)=1.98, P=0.16, n=12). These findings argue that constitutive Girk ablation did not cause significant alterations in the Girk-independent component of IBaclofen in Layer 5/6 PrLC pyramidal neurons. Importantly, the Girk-independent component of IBaclofen was significantly lower when 8Br-cAMP was included in the pipette solution (Fig. 1E), arguing that this component of IBaclofen is mediated by a cAMP-dependent current.

Girk channels and the excitability of Layer 5/6 PrLC pyramidal neurons

Having established that Girk channels mediate the majority of the direct GABABR-dependent inhibition of Layer 5/6 PrLC pyramidal neurons, we next investigated the contribution of Girk channels to the excitability of these neurons. Neurons from Girk2–/– mice exhibited a reduced threshold for spiking induced by current injection and an overall leftward shift in the current-spike relationship (Fig. 1G-I). Previous work has shown that Girk2 ablation can enhance excitatory neurotransmission in neurons within the VTA and NAc (Arora et al., 2010). To address the possibility that Girk ablation may enhance excitatory neurotransmission in Layer 5/6 PrLC pyramidal neurons, we measured spontaneous EPSCs (sEPSCs) in slices from wild-type and Girk2–/– mice (Fig. S1D,E). sEPSCs were readily observed in slices from wild-type mice with amplitudes and frequencies consistent with previous report (Wang and Zheng, 2001). sEPSC frequency, but not amplitude, was elevated significantly in slices from Girk2–/– mice. Collectively, these data suggest that loss of Girk-dependent signaling leads to enhanced excitability of Layer 5/6 PrLC pyramidal neurons.

Cocaine effects on Layer 5/6 PrLC pyramidal neurons

We next asked whether repeated cocaine exposure altered GABABR-dependent signaling and/or excitability of Layer 5/6 PrLC pyramidal neurons (Fig. 2). Mice were subjected to a 5-d cocaine dosing regimen that induced behavioral sensitization (Fig. 2A,B). IBaclofen measured 1-2 d after the last injection was ~50% smaller in Layer 5/6 PrLC pyramidal neurons from cocaine-treated mice as compared to saline controls (Fig. 2C,D). The cocaine-induced suppression of GABABR-dependent signaling correlated with a significant depolarizing shift in resting membrane potential (saline: −68±2 mV, n=9; cocaine: −62±1 mV, n=11; t(18)=−2.9, P<0.01), and enhanced excitability of Layer 5/6 PrLC pyramidal neurons (Fig. 2E,F). In contrast, a single cocaine injection had no effect on IBaclofen (Fig. 2D) or excitability (not shown) of these neurons. The effects of cocaine on GABABR signaling and excitability of Layer 5/6 PrLC pyramidal neurons were surprisingly durable, persisting for up to 6 weeks following the last cocaine injection (Fig. 2G-I). In contrast, cocaine-induced effects on sEPSCs frequencies and amplitude were time-dependent, with enhanced frequencies observed during early withdrawal and enhanced amplitudes observed after longer withdrawal periods (Fig. S2A-D).

Figure 2. Cocaine-induced adaptations in Layer 5/6 PrLC pyramidal neurons.

Figure 2

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(A) Left, The repeated cocaine treatment study included two acclimation days (H, sal), 5 days of saline or cocaine (15 mg/kg i.p.) injection, and 3 time-frames (1-2 d, 10-12 d, or 28-40 d after the final injection) for electrophysiological analysis. Right, Depiction of a coronal slice containing the mPFC. PrLC, prelimbic cortex; ILC, infralimbic cortex; cg, cingulate cortex.

(B) Distance traveled (m) following saline (sal) injection on the 2nd acclimation day, and after the 1st and 5th injections, by saline- and cocaine-treated mice (F(2,109)=37.1, P<0.001; n=9-10/group). ***P<0.001 vs. saline-treated mice (within day); ##P<0.01.

(C) IBaclofen in Layer 5/6 PrLC pyramidal neurons from mice given repeated saline (black) or cocaine (gray), measured 1-2 d after the last injection. Scale bars: 50 pA/5 min.

(D) IBaclofen in Layer 5/6 PrLC pyramidal neurons measured 1-2 d after a single injection of cocaine (coc) or saline (sal) or after completing the repeated dosing regimen. While a single cocaine injection was without effect on IBaclofen (t(14)= −1.3, P=0.23; n=8/group), repeated cocaine treatment suppressed IBaclofen by ~50% (t(18)=4.6, P<0.001). ***P<0.001 vs. sal.

(E) AP threshold in Layer 5/6 PrLC pyramidal neurons, measured 1-2 d after the last injection of saline or cocaine. Repeated cocaine suppressed the AP threshold by ~35% (t(24)=2.4, P<0.05; n=12-14/group). A single cocaine injection was without effect on AP threshold (t(14)=1.5, P=0.17; n=5-10/group; not shown)

(F) Current-spike plots for Layer 5/6 PrLC pyramidal neurons from repeated saline- and cocaine-treated mice, measured 1-2 d following the final injection (F(8,224)=5.3; P<0.05; n=12-14/group). *,**P<0.05 and 0.01, respectively, vs. sal (within current injection group). Current-spike data were not significantly different in animals treated with a single cocaine or saline injection (F(8,134)=1.9; P=0.07; n=5-10/group; not shown).

(G) IBaclofen in Layer 5/6 PrLC pyramidal neurons in saline- and cocaine-treated mice, measured 10-12 d (t(30)=4.6, P<0.001; n=14-18/group) or 28-40 d (t(29)= 3.2, P=0.004; n=11-20/group) following the final injection. **,***P<0.01 and 0.001, respectively, vs. sal.

(H) AP threshold in Layer 5/6 PrLC pyramidal neurons from repeated saline- and cocaine-treated mice, measured 10-12 d (t(34)= 4.8, P<0.001; n=15-21/group) or 28-40 d (t(38)= 4.8, P=<0.001; n=17-23/group) after the last injection. ***P<0.001 vs. sal.

(I) Current-spike plots for Layer 5/6 PrLC pyramidal neurons from saline- and cocaine-treated mice, measured 10-12 d (F(8,311)=1.1; P<0.001; n=15-17/group) or 28-40 d (F(11,421)=4.9; P<0.001; n=17-23/group) following the final injection **,*** P<0.01 and P<0.001, respectively, vs. sal (within current injection group).

See Figure S2 for sEPSC analysis in Layer 5/6 PrLC neurons from saline- and cocaine-treated mice.

Anatomic and molecular specificity of the cocaine-induced adaptation in PrLC neurons

To examine the anatomic specificity of the cocaine-induced adaptations in GABABR-dependent signaling and excitability of Layer 5/6 PrLC pyramidal neurons, we measured IBaclofen in Layer 5/6 pyramidal neurons of the infralimbic (ILC) cortex, Layer 2/3 pyramidal neurons in the PrLC, and Layer 5/6 pyramidal neurons in primary and secondary motor cortices (M1/M2). Importantly, IBaclofen and excitability measures were unaltered by repeated cocaine exposure in these cortical pyramidal neurons (Fig. S3A-C).

α2-adrenergic receptors (α2-AR) and D2/3 dopamine receptors (D2/3R) are expressed in PFC pyramidal neurons (Vincent et al., 1993; Aoki et al., 1998). Thus, we asked whether repeated cocaine suppressed the direct inhibitory effects of these receptor systems on Layer 5/6 PrLC pyramidal neurons. Bath application of the α2-AR and D2/3R agonists guanfacine (20 μM) and quinpirole (20 μM), respectively, evoked small (<50 pA) and transient outward currents in 60% of Layer 5/6 PrLC pyramidal neurons (not shown); no significant differences in current amplitudes were seen between slices from cocaine- and saline-treated animals (Fig. S3D-E).

Increased glutamatergic transmission arising from the mPFC to the VTA and NAc is a hallmark of cocaine-induced sensitization (Steketee and Kalivas, 2011). To determine if the cocaine-induced suppression of GABABR-dependent signaling and enhanced excitability occurred in Layer 5/6 PrLC pyramidal neurons projecting to the VTA and/or NAc, we used a retrograde labeling approach. Infusions made into the ventral midbrain targeted the VTA, but also reached areas such as the parabrachial pigmented nucleus, paranigral nucleus, and interfasicular nucleus (Fig. 3A,B). Consistent with past studies (Gabbott et al., 2005), Retrobeads™ injected into the ventral midbrain primarily labeled populations of neurons in Layers 5/6 of the PrLC and ILC, with some additional labeling in Layer 2/3 and the cingulate region (Fig. 3B). Infusions made into the NAc targeted primarily the core sub-region of the accumbens, as it receives input primarily from the PrLC (Brog et al., 1993) (Fig. 3C,D). While injections into the NAc labeled some Layer 2/3 neurons, most of labeled neurons were found in Layers 5/6 of the PrLC and ILC (Fig. 3D).

Figure 3. Cocaine adaptations in Layer 5/6 PrLC pyramidal neurons projecting to the ventral midbrain and NAc.

Figure 3

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(A) Retrobead™ injection sites in the ventral midbrain (VM); the respective tracer areas of the individual experiments in each target region are marked with different colors.

(B) Representative Retrobead™ injection site in the ventral midbrain (VM) in a 50 μm coronal section counterstained with green Nissl (left panel). While injections centered on the VTA, neighboring areas including the parabrachial pigmented nucleus (PBP), paranigral nucleus (PN), and interfasicular nucleus (IF) were unavoidably targeted. Corresponding labeling in the PrLC (right panel; scale, 250 μm), with numbers showing approximate locations of cortical layers. fr, fasciculus retroflexus; SN, substantia nigra; MM, medial mammilary nucleus.

(C) Retrobead™ injection sites in the NAc; the respective tracer areas of the individual experiments in each target region are marked with different colors.

(D) Retrobead™ injection site in the NAc in a 50 μm coronal section counterstained with green Nissl (left panel). While injections were centered on the NAc core, some labeling of the NAc shell was evident. Corresponding labeling in the PrLC (right panel; scale, 250 μm), with numbers showing approximate locations of cortical layers. ac, anterior commisure; CPu, caudate-putamen; fmi, forceps minor of the corpus callosum; LV, lateral ventricle.

(E) RetroBead™-labeled Layer 5/6 mPFC pyramidal neurons in an acutely-isolated brain slice, as visualized via infrared microscopy (left panel) and epifluorescence (scale bar, 25 μm). The dashed circle highlights the cell targeted for evaluation (note the pipette visible in the left panel).

(F) Summary of IBaclofen in Layer 5/6 PrLC pyramidal neurons, measured 1-2 d following the last injection. IBaclofen did not differ in RetroBead™-positive (Rb+) and negative (Rb-) Layer 5/6 PrLC pyramidal neurons from saline-treated mice (left, t(6)=−1.0, P=0.4; n=3-5/group). Cocaine treatment suppressed IBaclofen in Layer 5/6 PrLC pyramidal neurons that project to the ventral midbrain (VM, t(20)=3.9, P<0.001; n=10-12/group) and NAc (t(9)=5.6, P<0.001; n=5-6/group). ***P<0.001 vs. sal.

(G) AP threshold in Layer 5/6 PrLC pyramidal neurons projecting to the VM (t(20)=2.6, P<0.05; n=11/group) and NAc (t(13)=2.3, P<0.05; n=6-9/group) in saline- and cocaine-treated mice *P<0.05 vs. sal.

(H) Current-spike plots for Layer 5/6 PrLC pyramidal neurons projecting to the VM in saline- and cocaine-treated mice (F(13,257)=4.6, P<0.001; n=9-10/group). **P<0.01 vs. sal (within current injection group).

(I) Current-spike plots for Layer 5/6 PrLC pyramidal neurons projecting to the NAc in saline- and cocaine-treated mice (F(13,209)=2.8; P<0.05; n=6-9/group). **P<0.01 vs. sal (within current injection group).

See Figure S3 for data relating to the anatomic and molecular specificity of the cocaine-induced suppression of IBaclofen.

While Retrobead™ uptake did not alter IBaclofen (Fig. 3F) or excitability of Layer 5/6 PrLC pyramidal cells (not shown), IBaclofen was notably larger in the retrograde labeling experiments, a distinction attributable to the older age of the mice required for these studies (Fig. 3F). More importantly, repeated cocaine significantly reduced IBaclofen in, and augmented the excitability of, Layer 5/6 PrLC pyramidal neurons that project to the medial portions (including the VTA) of the ventral midbrain (Fig. 3F-H) and NAc (Fig. 3F,G,I).

Mechanisms underlying the cocaine-induced adaptation in PrLC pyramidal neurons

Systemic cocaine administration increases DA levels in the PFC, exerting both direct and indirect effects on the output of Layer 5/6 mPFC pyramidal neurons through activation of D1- and D2-like receptors in both pyramidal and non-pyramidal cells (Seamans and Yang, 2004; Hyman et al., 2006). To test whether DA receptor activation mediates the repeated cocaine-induced adaptations in Layer 5/6 PrLC pyramidal neurons, we pre-treated mice with the D2/3R antagonist sulpiride or the D1/5R antagonist SCH23390 prior to each cocaine injection (Fig. 4). Sulpiride pre-treatment had no effect on the cocaine-induced suppression of IBaclofen or on the excitability of Layer 5/6 PrLC pyramidal neurons. In contrast, SCH23390 pre-treatment prevented both the cocaine-induced suppression of IBaclofen and enhanced excitability, indicating that D1/5R activation is required for the repeated cocaine-induced suppression of IBaclofen and increased excitability of Layer 5/6 PrLC pyramidal neurons.

Figure 4. Dopamine signaling and the cocaine-induced adaptations in Layer 5/6 PrLC pyramidal neurons.

Figure 4

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(A) IBaclofen in Layer 5/6 PrLC pyramidal neurons in mice given repeated saline (sal) or cocaine (coc) injections (noted above the histogram), with daily pre-treatments (noted below the histogram) of either saline (black), SCH23390 (0.2 mg/kg i.p, blue) or sulpiride (50 mg/kg i.p., red). IBaclofen was measured 1-2 d after the final drug treatment (F(3,31)=6.4, P<0.01; n=5-10/group). **P<0.01 vs. sal/sal.

(B) AP threshold in Layer 5/6 PrLC pyramidal neurons from sal/sal, sal/coc, SCH/coc, and sulp/coc-treated mice (F(3,42)=5.8, P<0.01; n=6-14/group). **P<0.01 vs. sal/sal; #P<0.05 vs. SCH/coc.

(C) Current-spike plots for Layer 5/6 PrLC pyramidal cells from sal/sal, sal/coc, SCH/coc, and sulp/coc-treated mice (F(24,347)=2.6, P<0.001). **P<0.01 sal/coc and sulp/coc vs. sal/sal; #P<0.05 sal/coc and sulp/coc vs. SCH/coc.

While repeated non-contingent cocaine administration weakens the GABABR-dependent modulation of excitatory output from the mPFC (Jayaram and Steketee, 2005), no changes in the functional coupling between GABABR and G proteins or in the levels of GABABR or G protein have been reported (Nestler et al., 1990; Striplin and Kalivas, 1992; Kushner and Unterwald, 2001; Li et al., 2002). To determine whether the cocaine-induced adaptation in GABABR-dependent signaling is linked to an adaptation in downstream effector(s), we measured IBaclofen in the presence of 0.3 mM Ba2+ in Layer 5/6 PrLC pyramidal neurons from saline- and cocaine-treated wild-type mice. The Ba2+-insensitive component of IBaclofen was not significantly different between these groups (Fig. 5A), indicating that the cocaine-induced suppression of GABABR-dependent signaling is mediated by a selective suppression of the Girk-dependent component of IBaclofen. Nevertheless, no differences in Girk2 or GABABR1 subunit mRNA levels (Fig. 5B), or Girk2 or GABABR1 total protein levels (Fig. 5C), were observed in mPFC micropunches from cocaine- and saline-treated mice.

Figure 5. Subcellular trafficking and the cocaine-induced suppression of IBaclofen.

Figure 5

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(A) IBaclofen measured in the presence of 0.3 mM extracellular Ba2+ in Layer 5/6 PrLC pyramidal neurons from repeated saline- and cocaine-treated wild-type mice (t(8)=0.75, P=0.48).

(B) Girk2 and GABABR1 (BR1) mRNA levels in mPFC tissue from repeated saline- (black) and cocaine (gray)-treated mice (n=4/group), measured by qRT-PCR 1 d after the final injection (Girk2: t(6)= 0.16, P=0.9; GABABR1: t(6)=0.91, P=0.4).

(C) Top: Representative sub-sections of immunoblots showing Girk2 (left) and GABABR1 (BR1) (right) protein levels in tissue punches of the mPFC from repeated saline and cocaine-treated mice, 1 d after the final injection. Bottom: Quantification of Girk2 (t(6)= −0.91, P=0.4) and GABABR1 (t(6)= −0.13, P=0.8) protein levels, with normalization to the levels of β-actin (n=4/group).

(D) Plasma membrane-associated immunogold particle density for Girk2 and GABABR1 (BR1) in Layer 5/6 PrLC pyramidal neuron spines (left, Girk2: t(118)=4.4, P<0.001; GABABR1: t(118)=4.5, P<0.001; n=60/group) and dendrites (right, Girk2: t(118)=4.6, P<0.001; GABABR1: t=4.4, P<0.001; n=60/group) from saline- and cocaine-treated mice (n=6/group). ***P<0.001 coc vs. sal.

(E) Distribution of Girk2 immunogold particles at the plasma membrane (PM) and intracellular (intra) sites, expressed as a percentage of total particles analyzed in spines and dendrites of Layer 5/6 PrLC pyramidal neurons from saline- and cocaine-treated mice (n=420-430 total particles/group). The redistribution of GABABR1 was comparable to that seen for Girk2 (not shown). **P<0.01 intra-sal vs. PM-sal; ##P<0.01 intra-coc vs. PM-coc.

See Figure S4 for immunoelectron micrographs from which data in panels D & E were extracted.

To test whether the cocaine-induced suppression of IBaclofen reflected an internalization of Girk channels, we used quantitative immunoelectron microscopy to compare the subcellular distributions of Girk2 and GABABR1 in Layer 5/6 PrLC pyramidal neurons of cocaine- and saline-treated mice. Consistent with the quantitative immunoblotting data, there was no obvious difference in total Girk2 or GABABR1 immunolabeling in Layer 5/6 PrLC pyramidal neurons from cocaine-treated and saline-treated mice. In samples from saline-treated controls, Girk2 and GABABR1 immunoparticles were found mainly in the extrasynaptic plasma membrane of dendritic shafts and spines, with ~35% of immunolabeling observed at intracellular sites (Fig. 5D,E; Fig. S4). In samples from cocaine-treated mice, the density of Girk2 and GABABR1 immunolabeling in dendritic and spine membranes was significantly reduced, with a corresponding increase in intracellular labeling (Fig. 5D,E). Thus, repeated cocaine treatment triggered a redistribution of Girk2 and GABABR1 from the dendritic membrane to intracellular sites in Layer 5/6 PrLC pyramidal neurons.

Surface trafficking of GABABR and Girk channels is regulated by phosphorylation-dependent mechanisms. For example, phosphorylation of Ser-9 on Girk2 promotes internalization of Girk2-containing channels, while dephosphorylation of Ser-783 on GABABR2 is associated with reduced surface expression of the GABABR (Chung et al., 2009; Terunuma et al., 2010; Padgett et al., 2012). Using phospho-specific antibodies, we next evaluated whether the phosphorylation of Girk2(Ser-9) or GABABR2(Ser-783) was altered by repeated cocaine in mPFC tissue punches from saline- and cocaine-treated mice. While no differences in the level of phosphorylated Girk2(Ser-9) were detected between groups, repeated cocaine treatment correlated with a significant reduction in the phosphorylation of GABABR2(Ser-783) (Fig. 6A).

Figure 6. Reversal of cocaine-induced adaptations in Layer 5/6 PrLC pyramidal neurons.

Figure 6

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(A) Top: Representative immunoblots examining levels of phosphorylated Girk2(Ser-9) (pSer9, left) and GABABR2(Ser-783) (pSer783, right) in mPFC micropunches from repeated saline and cocaine-treated mice, measured 1-2 d after the final injection. Bottom: Quantification of Girk2(pSer-9) (t(6)= −0.85, P=0.43; n=4/group) and GABABR2(pSer-783) (t(10)= 3.9, P=0.003; n=6/group) with normalization to the total level of Girk2 and GABABR2 (BR2) respectively. **P<0.01 vs. sal.

(B) Representative IBaclofen traces in Layer 5/6 PrLC pyramidal neurons from repeated saline (red) and cocaine-treated mice (blue), measured with 100 nM okadaic acid (OA) in the pipette solution, next to a control recording in a neuron from a repeated cocaine-treated mouse measured in the absence of OA (gray). Scale bars: 50 pA/5 min.

(C) IBaclofen measured in Layer 5/6 PrLC pyramidal neurons from saline- and cocaine-treated mice, in the absence or presence of OA (F(3,21)=10.22, P<0.001). **P<0.01 vs. sal (no OA); ##P<0.01 vs. coc/OA.

(D) AP threshold in Layer 5/6 PrLC pyramidal neurons from saline and cocaine-treated mice, measured with and without OA in the pipette solution (F(3,25)=7.2, P<0.002; n=6-8/group). **P<0.01 vs. vs. sal (no OA); ###P<0.001 vs. coc/OA.

(E) Current-spike plots for Layer 5/6 PrLC pyramidal neurons from saline and cocaine-treated mice, measured with and without OA in the pipette solution (F(24,224)=4.7, P<0.001; n=6-8/group). *,**,***P<0.05, P<0.01, P<0.001, respectively, vs. sal and coc/OA.

As phosphatase PP2A can dephosphorylate GABABR2(pSer-783) (Terunuma et al., 2010), we next evaluated the effect of acute intracellular application of okadaic acid (OA; 100 nM) on IBaclofen and excitability in Layer 5/6 PrLC pyramidal neurons (Fig. 6B-E). In saline-injected mice, there was no significant effect of OA on IBaclofen or excitability, suggesting that tonic phosphatase activity does not regulate GABABR-dependent signaling or excitability in these neurons. In cocaine-treated animals, however, OA normalized IBaclofen and excitability in Layer 5/6 PrLC pyramidal neurons, suggesting that the redistribution of GABABR and Girk2 from the surface membrane to intracellular sites is linked to the dephosphorylation of GABABR2.

Girk signaling in the mPFC and behavioral sensitization

Enhanced glutamatergic output from the prelimbic sub-region of the mPFC contributes to the development of cocaine-induced behavioral sensitization (Steketee and Kalivas, 2011). Data presented above suggest that the suppression of Girk signaling in Layer 5/6 PrLC pyramidal neurons contributes, at least in part, to the cocaine-induced increase in excitability of these neurons. To test whether the cocaine-induced suppression of Girk signaling in the mPFC contributes to the development of behavioral sensitization, we used a viral RNAi strategy to persistently suppress Girk signaling in Layer 5/6 of the dorsal aspect of the mPFC. We infused bilaterally a mixture of lentiviruses harboring shRNAs for Girk1 and Girk2 (Girk1/2-LV), or a GFP-non-target control, into deep cortical layers of the dorsal mPFC of wild-type mice (Fig. 7A; Fig. S5). Girk2 protein levels were significantly lower in mPFC micropunches from Girk1/2-LV mice as compared to the control group (Fig. 7B). Mice given intra-mPFC Girk1/2-LV displayed normal motor activity during habituation (not shown) and in response to saline injection, but showed elevated responses to the first 3 cocaine injections (Fig. 7C). Importantly, cocaine-induced activity levels for Girk1/2-LV-treated mice did not differ between the 1st and 5th injections, and were indistinguishable from those exhibited by control animals after their 5th cocaine injection. In contrast, control animals displayed a progressive increase in cocaine-induced motor activity over the 5 injections. Collectively, these data suggest that the persistent suppression of Girk signaling in Layer 5/6 of the dorsal mPFC can pre-sensitize mice to the motor-stimulatory effect of cocaine.

Figure 7. Suppression of Girk signaling in the mPFC and behavioral sensitization.

Figure 7

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(A) Coronal brain sections (50 μm) showing eGFP labeling in the dorsal mPFC, 28 d after co-injection of two lentiviruses expressing Girk1- and Girk2-specific shRNAs (Girk-LV; scale, 500 μm). The inset shows virus-infected cells in the mPFC (scale, 25 μm). fmi, forceps minor of the corpus callosum.

(B) Top: Representative sub-sections of immunoblots showing Girk2 (left) and Girk1 (right) protein levels in tissue punches of the mPFC from repeated saline and cocaine-treated mice, 1 d after the final injection. Bottom: Quantification of Girk2 (t(17)= 3.1, P=0.008), with normalization to the levels of β-actin (n=9-10/group).

(C) Distance traveled (m) following saline (sal) and 5 daily cocaine injections (15 mg/kg, i.p.) in mice receiving intra-mPFC infusions of Girk1/2 lentivirus (Girk-LV) or a non-target GFP shRNA (eGFP-control; F(5,83)=2.6, P<0.05; n=7-9/group) *,**P<0.05 and 0.01, respectively, vs. eGFP-control; ##P<0.01, injection 1 vs. 5 (eGFP-control).

See Figure S5 for time-dependent expression of eGFP in virus-infused mice.

Discussion

Repeated cocaine exposure increases the excitability of Layer 5/6 mPFC pyramidal neurons, making them more responsive to cocaine and cocaine-associated cues (Sun and Rebec, 2006). One consequence of these adaptations is enhanced glutamatergic output to the VTA and NAc, which is critical for the development and expression of addictive behaviors such as sensitization (Jayaram and Steketee, 2004; Steketee and Kalivas, 2011). GABABR-dependent signaling plays a fundamental role in regulating mPFC glutamatergic output to limbic structures (Jayaram and Steketee, 2004; Harte and O'Connor, 2005), and repeated cocaine exposure reduces GABABR-dependent inhibitory influences in the mPFC (Jayaram and Steketee, 2004; Steketee and Beyer, 2005). Here, we show that repeated cocaine suppresses GABABR-Girk signaling in Layer 5/6 PrLC pyramidal neurons in a durable and specific manner, contributing to the enhanced excitability of neurons implicated in the development of addiction-related behavior.

Pharmacologic and genetic approaches revealed that most (~70%) of the direct inhibitory effect of GABABR activation in Layer 5/6 PrLC pyramidal neurons is attributable to Girk channel activation. Importantly, repeated cocaine selectively suppressed this dominant (Girk-dependent) component of IBaclofen. The Girk-independent component of the GABABR-induced current in was suppressed by 8Br-cAMP, arguing that this conductance is modulated via the Gi/o-dependent inhibition of adenylyl cyclase activity and subsequent decline in intracellular cAMP. In light of the 8Br-cAMP sensitivity and recent studies (Deng et al., 2009; Sandoz et al., 2012), the non-Girk component of IBaclofen may be mediated by a two-pore domain Trek K+ channel. Modulation of HCN channels may also play a role, however, as they are found in Layer 5/6 pyramidal neurons (Kase and Imoto, 2012).

DA input from the VTA influences the response of the mPFC to reward (Wolf and Roth, 1987). We found that D1/5R antagonism prevented the cocaine-induced suppression of GABABR-dependent signaling and increased excitability of Layer 5/6 PrLC pyramidal neurons. Repeated cocaine exposure has been shown to up-regulate cAMP signaling mPFC pyramidal neurons (Dong et al., 2005) and reduce D2R-dependent inhibition in Layer 5/6 mPFC pyramidal neurons (Nogueira et al., 2006), the latter of which would further contribute to enhanced cAMP signaling. Thus, the cocaine-induced, D1/5R-dependent suppression of GABABR-Girk signaling in Layer 5/6 PrLC pyramidal neurons seen here may reflect enhanced activation of cAMP-dependent signaling. Indeed, Szulczyk and colleagues demonstrated that a Girk-like channel in mPFC pyramidal neurons was inhibited both by D1R activation and cAMP (Witkowski et al., 2008; Witkowski et al., 2012).

While D1/5R activation in PrLC pyramidal neurons may trigger the cocaine-induced suppression of GABABR-Girk signaling, D1/5Rs were blocked globally in our study. Importantly, activation of D5R signaling in the VTA was linked to the increase in excitatory transmission seen in VTA DA neurons triggered by cocaine (Argilli et al., 2008). This form of plasticity occurs following a single exposure to cocaine and facilitates a shift in the threshold for induction of plasticity in downstream targets of the VTA, including the mPFC (Mameli et al., 2009). Thus, if D1/5R antagonist pre-treatment prevented excitatory adaptations in VTA DA neurons in our study, it may also have blocked initiation of downstream adaptations in the mPFC, including the suppression of GABABR-Girk signaling, that normally occur following repeated cocaine exposure.

Local GABA interneurons and GABA-containing projection neurons arising from the VTA are major sources of GABA in the mPFC (Kawaguchi, 1993; Retaux et al., 1993; Carr and Sesack, 2000). Recently, Slesinger and colleagues demonstrated that acute cocaine and methamphetamine exposure depressed GABABR-Girk signaling in VTA GABA neurons in a D1/5R-dependent manner (Padgett et al., 2012). Repeated cocaine exposure also suppressed the influence of D2R on GABA interneurons of the mPFC, leaving an unopposed D1R-mediated enhancement of local GABA signaling (Kroener and Lavin, 2010). Together, these observations provide reasonable explanations for the elevated extracellular GABA levels seen in the mPFC of cocaine-sensitized rats (Jayaram and Steketee, 2005, 2006), and suggest the intriguing possibility that the cocaine-induced suppression of GABABR-dependent signaling in Layer 5/6 PrLC pyramidal neurons reflects a desensitization of GABABR-Girk signaling triggered by elevated GABA transmission.

Activation of the PrLC and its projections to the NAc core is necessary for the development and expression of sensitization, as well as initiating and reinstating cocaine-seeking behavior in self-administering rats (Pierce et al., 1998; Tzschentke and Schmidt, 2000; Kalivas and O'Brien, 2008). Activation of the ILC and its projections to the shell region of the NAc is responsible for inhibiting drug-seeking during extinction training (Peters et al., 2008). Akin to this functional dichotomy, we demonstrate that while Layer 5/6 pyramidal neurons in PrLC and ILC sub-regions of the mPFC showed robust GABABR-Girk responses, repeated cocaine exposure suppressed GABABR-Girk signaling selectively in the PrLC. Moreover, persistent suppression of Girk signaling, primarily targeting the dorsal aspect of the mPFC, was sufficient to potentiate acute cocaine-induced motor activity and prevent the augmentation of activity seen with repeated cocaine dosing. These data support the contention that the cocaine-induced suppression of Girk signaling contributes to increased excitatory output from Layer 5/6 PrLC pyramidal neurons that are important for key aspects of addiction-related behavior, including sensitization.

We reported previously that excitatory neurotransmission was elevated in VTA DA neurons from Girk1–/– and Girk2–/– mice, observations that correlated with elevated levels of synaptic AMPA receptors (Arora et al., 2010). Similar adaptations were observed in NAc medium spiny neurons (Arora et al., 2010). As Girk1 is not expressed in VTA DA neurons, and neither Girk1 nor Girk2 are abundantly expressed in the NAc (Karschin et al., 1996), these adaptations are likely secondary to the loss of Girk signaling in an afferent glutamatergic neuron population. Our work here shows that Girk channels containing Girk1 and Girk2 temper the excitability of Layer 5/6 PrLC pyramidal neurons, a major source of glutamate to the VTA and NAc. Thus, Girk signaling in Layer 5/6 PrLC pyramidal neurons may represent a barrier – lowered by cocaine – to persistent adaptations in excitatory neurotransmission in the VTA and/or NAc that promotes addiction-related behaviors associated with drug-seeking and relapse.

Experimental Procedures

Animals

Animal use was approved by the Institutional Animal Care and Use Committee at the University of Minnesota. Girk–/– mice have been used extensively to characterize Girk contributions to metabotropic inhibitory signaling (e.g., (Koyrakh et al., 2005)). The Tg(Kcnj5-EGFP)49Gsat line was generated as part of the GENSAT project, and bred on-site. Male C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) for all studies involving cocaine.

Drugs

Cocaine hydrochloride, baclofen, sulpiride, SCH23390, guanfacine, quinpirole and barium chloride were purchased from Sigma (St. Louis, MO). 8-Bromoadenosine 3',5'-cyclic monophosphate (8Br-cAMP) and CGP54626 were purchased from Tocris (Ellisville, MO). Okadaic acid was purchased from MP Biomedicals (Santa Ana, CA).

Motor activity

Activity studies were performed in open-field activity chambers as described (Arora et al., 2011). Animals were acclimated to testing room, handling, and chambers for 3 d prior to repeated cocaine or saline treatment. Subjects received 5 injections of cocaine (15 mg/kg i.p.) or saline over a 5-7 d period. For DA receptor antagonist experiments, mice were pre-treated with saline, SCH23390, or sulpiride 30 min prior to each saline or cocaine injection.

Electrophysiology

Coronal slices (300 μm) containing the mPFC were prepared from wild-type and Girk–/– mice (4-6 weeks), and repeated saline- or cocaine-treated C57BL/6J mice (5-10 weeks), in an ice-cold solution containing (in mM): 1.9 KCl, 1.2 Na2HPO4, 33 NaHCO3, 6 MgCl2, 0.5 CaCl2, 10 glucose, 0.4 ascorbic acid and 200 sucrose, bubbled with 95% O2/5% CO2. Slices were transferred to pre-warmed (32-35°C) ACSF (in mM): 125 NaCl, 2.5 KCl, 1.25 Na2HPO4, 25 NaHCO3, 4 MgCl2, 1 CaCl2, 10 glucose, 0.4 ascorbic acid (pH 7.3-7.4), and gradually acclimated to room temperature over the course of ≥1 h. Slices were transferred to a recording chamber and superfused with oxygenated ACSF (in mM): 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.3 MgCl2, 2.0 CaCl2, 10 glucose, 0.4 ascorbic acid (pH 7.3-7.4) at a flow rate of 2-2.5 mL/min. Bath and chamber temperatures were maintained at 29-30°C. Borosilicate (3-5 MΩ) electrodes were filled with (in mM): 140 K-gluconate, 2 MgCl2, 1.1 EGTA, 5 HEPES, 2 Na2-ATP, 0.3 Na-GTP, and 5 phosphocreatine, pH 7.4. The predicted EK for these conditions was −105 mV. In some experiments, okadaic acid (100 nM) was added to the pipette. Currents, resistances, and potentials were measured using a Multiclamp 700A amplifier and pCLAMP v.9 software (Molecular Devices; Foster City, CA) and stored on hard disk. All measured and command potentials factored in a junction potential of −15 mV predicted using JPCalc software (Molecular Devices).

Pyramidal neurons in Layer 5/6 were identified based on a pyramidal-shaped soma, a long apical dendrite extending toward superficial cortical layers, a resting membrane potential ≤ −60 mV, lack of spontaneous activity, and an apparent capacitance of ≥100 pF (Connors and Gutnick, 1990; Yang et al., 1996; Kawaguchi and Kubota, 1997). For rheobase assessments, cells were held in current-clamp mode and given 1-s current pulses beginning at −60 pA and progressing to 200 pA in 20 pA increments. Agonist-induced changes in holding current were measured at a holding potential (Vhold) of −60 mV; holding current, input resistance, and series resistance values were monitored during experiments by tracking responses to periodic (0.2 Hz) voltage steps (−5 mV, 800 ms). Only experiments with stable (<20% variation) and low series resistances (<20 MΩ) were analyzed.

Retrograde labeling

Male C57BL/6J mice (6-8 wk) were anesthetized with ketamine (100 mg/kg i.p)/xylazine (10 mg/kg i.p) and placed in a stereotaxic device (David Kopf Instruments; Tujunga, CA). A 5-μL Hamilton syringe was placed into the VTA (from bregma: +3.08 mm AP, ±0.5 mm ML; from ventral skull surface: −3.7 mm DV) or the NAc (from bregma: +1.1 mm AP, ±1.0 mm ML; from ventral skull surface: −3.8 mm DV), and remained in place for 2 min prior to bilateral infusion (0.3-0.4 μL, 0.1 μL/min) of Retrobeads™ (LumaFlour Inc.; Naples, FL) using an UltraMicroPump with SYS-Micro4 controller (World Precision Instruments; Sarasota, FL). After a 10-min delay to reduce solution backflow along the infusion track, the syringe was removed. Repeated cocaine treatment began after a suitable period to allow for retrograde transport of RetroBeads™ from the VTA (18-21 d) or NAc (10-15 d) to the mPFC. Retrobead™ injection sites were determined by serial sectioning and staining with green Nissl (NeuroTrace 500/525, Molecular Probes; Eugene, OR). Mice with improper targeting or significant bead contamination outside target areas were excluded from analysis.

qRT-PCR

mPFC punches (2-mm diameter, 2-mm thick) were taken from male C57BL/6J mice (5-7 wk), 1 d after the final cocaine or saline injection. Quantitative analysis of GABABR1 and Girk2 mRNA levels was performed as described (Arora et al., 2011).

Immunoblotting

mPFC punches (2-mm diameter, 2-mm thick) were sonicated in 1% SDS lysis buffer containing a Halt phosphatase and protease inhibitor cocktail (Thermo Fisher Scientific; Rockford, IL), heated at 85°C for 10 min, and centrifuged at 4°C for 20 min at 16,000×g. Protein samples (40 μg) were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked in 5% milk/Tris-buffered saline (TBS), incubated overnight at 4°C with antibodies against Girk2 (1:200; Alomone Labs; Jerusalem, Israel), Girk2(pSer-9) (1:200; (Chung et al., 2009)), GABABR1 (1:500; (Kulik et al., 2003), GABABR2 (1:10; NeuroMab; Davis, CA), GABABR2(pSer-783) (1:200; PhosphoSolutions; Aurora, CO), or β-actin (1:10,000; Abcam; Cambridge, MA), diluted in 5% milk/TBS/0.1% Tween-20 or 1% milk/TBS/0.1% Tween-20. Membranes were washed with TBS/0.1% Tween-20 or 1% milk/TBS/0.1% Tween-20 and incubated with donkey anti-mouse (926-32212; 1:1000-5000; LI-COR Biosciences; Lincoln, NE) or anti-rabbit (926-68072; 1:5000; LI-COR) secondary antibodies. Blots were developed using the Odyssey infrared imaging system (LI-COR) and an integrated density of each band was measured using Image J software (NIH; Bethesda, MD).

Quantitative immunoelectron microscopy

The subcellular distribution of Girk2 and GABABR1 was measured using pre-embedding immunoelectron microscopy and quantitative analysis approaches, as described (Arora et al., 2011).

Lentiviral suppression of Girk signaling

pLKO.1-puro-CMV-eGFP-based shRNA constructs specific for Girk1 (TRCN0000069733, Sigma) and Girk2 (TRCN0000069814) were identified based on their ability to suppress target expression in a heterologous system (not shown). These constructs, and a non-target control, were used to generate high-titer (>3×109 TU/ml) lentiviral stocks. Male C57BL/6J mice (5-7 wks) were anesthetized and secured in a stereotaxic device. Girk1 and Girk2 knockdown lentiviral stocks were mixed, loaded into a 5-μL Hamilton syringe and lowered into the mPFC. Two infusions (0.4 μL/infusion site; 0.2 μL/min) were made to ensure sufficient coverage in the mPFC (from bregma: +1.9 mm AP, ±0.3 mm ML; from ventral skull surface: −1.2/−1.5 mm DV). Following each infusion, the syringe was left in place for 5 min to reduce backflow. Mice began the repeated cocaine regimen 21-24 d after viral infusion. Motor activity data were analyzed if bilateral eGFP expression was detected in Layer 5/6 of the dorsal portion of mPFC.

Data analysis

Statistical analysis was performed using SigmaPlot (Systat Software, Inc; San Jose, CA). Motor activity data were analyzed using 1-way, 2-way, and 2-way repeated measures (RM) ANOVA, as appropriate. sEPSCs were analyzed using Minianalysis software using a 20 pA detection threshold (Synaptosoft; Decatur, GA). IBaclofen and current injection data were analyzed with Student's t-test or 1-way ANOVA, while current-spike relationships were analyzed with 2-way RM ANOVA. Immunoelectron microscopy data were analyzed using a Mann-Whitney Rank Sum Test. Student Newman-Keuls post hoc test was used for pair-wise comparisons as appropriate. The threshold for statistical significance was P<0.05

Supplementary Material

01

Highlights.

  • Girk channels mediate GABABR-induced inhibition of Layer 5/6 mPFC pyramidal neurons

  • Cocaine suppresses GABABR-Girk signaling in Layer 5/6 mPFC pyramidal neurons

  • The cocaine-induced adaptation is selective and trafficking-dependent

  • Persistent suppression of Girk signaling in mPFC pre-sensitizes mice to cocaine

Acknowledgements

This work was supported by NIH grants to KW (MH061933, DA011806, DA029343), MH (DA007097), LK (DA007097), and the Spanish Ministry of Science and Innovation BFU2012-38348 and CONSOLIDER-Ingenio CSD2008-0000 (RL). The authors thank Dr. Hee Jung Chung for the Girk2(pSer-9) antibody, and Kelsey Mirkovic, Daniele Young, Matt Novitch, and Edward Kim for excellent technical support.

Footnotes

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