2.2. Drug Administration

Morphine sulfate was obtained from Alcaliber S.A. (Madrid, Spain) subsequent to receiving authorization from Spanish Agency of Medicines and Medical Devices (Spanish Government). PD168,077 maleate was supplied by Tocris Bioscience (Avonmouth, UK). The specificity of PD168,077 as a D₄R agonist has been extensively proved in prior works using the highly selective antagonist L745,870 [16,25,26,27]. All drugs were dissolved in the vehicle solution consistent in 2% dimethyl sulfoxide (DMSO) and 0.9% NaCl. We have demonstrated in a previous report that this amount of DMSO had no effect on receptors function [30].

Rats received 7 or 14 days of continuous administration of vehicle, morphine (20 mg/kg/d) and/or PD168,077 (1 mg/kg/d) by an osmotic pump (Alzet^® osmotic pumps, Cupertino, CA, USA) that was subcutaneously implanted under deep anesthesia (75 mg/kg ketamine and 0.5 mg/kg medetomidine, i.p.) between the shoulder blades. Skin was sutured with sterile non absorbable suture and the surgical site was disinfected with a topical antiseptic (povidone iodine 10% solution). During the surgery and recovery, animals were kept warn using a heating pad. Animals were sacrificed the last day of the continuous administration of drugs.

2.3. Electrophysiology

2.4. Cell Reconstruction and Morphometric Analysis

After the recordings, sections were fixed with 4% paraformaldehyde, rinsed with 0.1 M phosphate-buffered saline, pH 7.4 (PBS) and incubated with streptavidin-conjugated Alexa Fluor^® 488 (Table S1) diluted 1:500 in PBS with 0.1% Triton X-100 and 0.02% sodium azide for 24 h in the dark. After washing with PBS, the sections were mounted on glass slides and coverslipped with an aqueous-based mounting medium.

Only completely Alexa Fluor^® 488 filled neurons were analyzed. Serial optical sections (0.1 μm step size) of each neuron were acquired with a laser-scanning confocal microscope (Leica, TCS NT) using a 63 × 1.3 numerical aperture oil-immersion. Images were acquired at high-resolution, with an image size of 1024 × 1024 pixels, and a voxel size of 0.27 × 0.27 × 1 μm for the x, y, and z axes, respectively. Image stacks were deconvolved to reduce signal blurring and each cell were three-dimensional reconstructed, traced, and analyzed using the image analysis system NeuronStudio (Icahn School of Medicine at Mount Sinai) [31]. For Sholl analysis, the number of intersections were counted between dendritic branches and a series of concentric spheres, starting at 10 μm from the soma and at radial increment of 1 μm. To quantify spine density, a digital zoom (6.7-fold augmentation) was applied and at least 10–15 dendritic segments (10 μm length) per branch order from each neuron were obtained. Each segment was manually inspected and appropriate corrections made using the NeuronStudio interface. Dendritic protrusions (thin, stubby, and mushroom spines) were classified according to their shapes: mushroom with large head and short neck; thin with thin head and long neck; and stubby with large head and no apparent neck.

2.5. Immunohistochemistry

Animals (n = 24) were transcardially perfused with 0.1 M PBS followed by 4% paraformaldehyde under deep sodium pentobarbital anesthesia (60 mg/kg, i.p.). The brains were rapidly removed, overnight post-fixed in the same fixative, cryoprotected in 30% sucrose in PBS (72 h) and frozen in dry ice. Rostro-caudal series of coronal sections (30 μm thick) were obtained with a freezing microtome (CM 1325, Leica, Weztlar, Germany) and stored in PBS containing 0.02% sodium azide. Free-floating sections were taken from the CPu (+1.0 to −0.30 mm from Bregma) and substantia nigra (−5.30 to −5.80 mm from Bregma) and processed for either single or double immunolabeling procedures.

Details regarding the antibodies used in the present study are listed in Table 1 (primary antibodies) and Table S1 (secondary antibodies). All primary antibodies were diluted in phosphate-buffered saline containing 0.2% Triton X-100 (PBS-TX) and 0.1% sodium azide.

2.6. Western Blot

Animals (n = 16) were sacrificed by decapitation and the brains immediately removed. The substantia nigra was dissected and homogenized in a lysis buffer containing 2 mM orthovanadate and a mixture of protease inhibitors (Roche Diagnostics GmbH, Switzerland). After incubating on ice for 30 min, samples were centrifuged at 8000× g for 10 min at 4 °C. The supernatant was used for western blotting. Equal amounts of protein (50 μg) was loaded and resolved in 12 % SDS-polyacrylamide gel electrophoresis and then transferred onto PVDF membranes (Hybond-P, GE Healthcare, UK). Membranes were incubated overnight at 4 °C with a specific primary antibody (Table 1), followed by HRP-conjugated secondary antibody. Bands of the membranes were visualized using chemiluminescence detection (ECL, GE Healthcare, UK). Comparisons between the experimental groups were performed by determining bands immunoreactivity by densitometry with the image analyzing system ImageJ1.48v (NIH).

3.2. D₄R Activation Counteracts Dendritic Spine Depletion Induced by Morphine

We next analyzed the effects of the continuous treatment (14 days) with morphine and/or PD168,077 on MSNs spine density (Figure 2A). Spine density was significantly reduced in the striatal MSNs of morphine-treated animals as compared to control rats (by 25%) (Figure 2B). This spine loss was observed in distal dendrites (3rd, 4th, 5th, and 6th branch order) (Figure 2C) and it was associated with the selective reduction in stubby (by 48%) and mushroom (by 42%) spines (Figure 2D). The D₄R agonist produced an even greater decrease in the overall spine density (by 45%) (Figure 2B), but in this case it occurred throughout the entire dendritic arbor (Figure 2C). The analysis of spine morphology indicated that such PD168,077-induced lower spine density was due to a decrease in the number of thin (by 40%), stubby (by 58%) and mushroom (by 60%) spines (Figure 2D).

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Figure 2

D₄R activation restores spine density depletion induced by morphine. (A) Representative confocal images (upper panels) and reconstructions (lower panels) of dendrites showing the effect of continuous treatment (14 days) with vehicle, morphine (20 mg/kg/d), PD168,077 (1 mg/kg/d) and morphine + PD168,077 (20 mg/kg/d and 1 mg/kg/d) on spine density. (B) Overall spine density and (C) spine density as a function of dendritic branch order. (D) Spine density of the three classified spines (thin, stubby and mushroom) based on shape. (E) Histogram shows the semi-quantitative analysis of spinophilin IR in the CPu. Data represents mean ± SEM, n = 23 rats per treatment. p < 0.05 vehicle vs. morphine; * p < 0.05 vehicle vs. PD168,077; # p < 0.05 vehicle vs. morphine+PD168,077; & p < 0.05 morphine vs. PD168,077; Kruskal-Wallis test followed by Dunn’s method. Scale bar is 5 μm. Abbreviations: m, mushroom; s, stuby; t, thin.

When PD168,077 was administered together with morphine, spine density was apparently reestablished (Figure 2A–C), as it was observed for thin-shaped spines (Figure 2D). However, it should be noted that morphine + PD168,077 was unable to restore the loss of stubby spines and only partially did for mushroom spines (Figure 2D).

Since spinophilin is a protein highly enriched in dendritic spines where it plays a key role in modulating spine density and synaptic activity [33], we evaluated its possible regulation by morphine and/or PD168,077. It was showed that the continuous treatment with morphine or PD168,077 alone downregulated striatal levels of spinophilin, which was specifically counteracted by the co-treatment of both drugs (Figure 2E and Figure S1). Thus, the drugs-induced changes in spinophilin protein levels corroborated the modulation of spine density described above.

3.3. Changes in Striatal MSN Cell Bodies after Continuous Administration of Morphine and/or PD168,077

Given the specific remodeling of the MSNs dendritic arbors and dendritic spines in response to continuous treatment with morphine or/and PD168,077, we next examined whether these changes appear together with morphological alterations in the soma (Figure 3A). We observed that morphine or PD168,077 did not affect the morphology of the striatal MSNs somata, demonstrated by an absence of changes in the size (surface area and volume; Figure 3B,C) and shape (circularity; Figure 3D). However, the combined treatment with both drugs significantly increased the size of the striatal projecting neurons (surface area: by 33%; volume: by 55%) (Figure 3B,C).

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Figure 3

Co-treatment with the D4R agonist and morphine increases the size of the striatal MSNs. (A) Representative XY, XZ and YZ confocal images of MSNs soma showing the effect of continuous treatment (14 days) with vehicle, morphine (20 mg/kg/d), PD168,077 (1 mg/kg/d) and morphine + PD168,077 (20 mg/kg/d and 1 mg/kg/d). (B,C) Histograms represent the effect of drugs treatment on surface area (μm2) and volume (μm3) of the striatal MSNs. (D) Graph depicts the circularity (index for soma shape) of MSNs evaluated in the three axis (XY, XZ and XY). Data represents mean ± SEM, n = 23 neurons on average per treatment. One-way ANOVA followed by Tukey test, # p < 0.05 vehicle vs. morphine + PD168,077. Scale bar is 30 μm.

3.4. D₄R Activation during Continuous Treatment with Morphine Changes the Passive Properties and Excitability of Striatal MSNs

Since the continuous treatment with morphine and/or PD168,077 regulated dendritic arborization, dendritic spines density and soma size of striatal MSNs, we hypothesized that their electrical activity might be also altered. We used whole-cell patch-clamp recordings to evaluate the intrinsic properties and excitability of striatal MSNs from rats treated for 14 days with vehicle, morphine, PD168,077 or morphine + PD168,077 (Figure 4A). The combined treatment with morphine + PD168,077 yielded significant differences in several passive and active MSNs membrane properties: (i) increased membrane capacitance (Figure 4B); (ii) reduced membrane resistance (Figure 4C); (iii) increased rheobase (Figure 4F); and (iv) depolarized membrane potential (Figure 4G). However, the action potential threshold (Figure 4D) and the first action potential latency (Figure 4E) were not altered by any of the drug treatments.

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Figure 4

The combined treatment of the D4R agonist PD168,077 with morphine induces changes in passive properties and in excitability of striatal MSNs. (A) Representative examples of the voltage responses of identified MSNs of rats treated for 14 days with saline (black), morphine (red; 20 mg/kg/d), PD 168,077 (green; 1 mg/kg/d), and morphine + PD 168,077 (blue, 20 mg/kg/d and 1 mg/kg/d) to two second-long current injection of +300, +200, and −200 pA (top to bottom), respectively. All traces were obtained from neurons that were recorded using a pipette solution complemented with biocytin, and included in the post-recording morphological analysis. (B–G) Population graphs depicting significant differences in membrane capacitance (B), membrane resistance (RM, C), action potential threshold (APth, D), first action potential latency (E), rheobase (F), and membrane potential (Vm, G) only in the morphine + PD168,077 group. (H) Graph showing voltage deflections in response to injection of negative currents (−50 pA steps) in the four experimental groups. (I) Graph showing driven activity in response to injection of positive currents (+50 pA steps) in the four experimental groups. Note that current injection evokes significantly milder responses in the morphine + PD showed group. The data only include values obtained from the neurons that were reconstructed and thus included in the post-recording morphological analysis. In B–G, data were compared using Kruskal-Wallis test followed by a Dunn’s post hoc test. In H,I, data were compared using mixed model ANOVA followed by Dunnet’s post hoc test * p < 0.05.

In addition to the changes in the passive properties, excitability was also reduced in the morphine + PD168,077 group compared with vehicle, morphine or PD168,077. The combined treatment group showed significantly smaller voltage-deflection (Figure 4H) and slower firing frequency (Figure 4I) in response to negative or positive current injection (50 pA steps), respectively.

3.5. Regulation of GIRK1 and GIRK2 Expression in the Striatal MSNs and Nigral Dopamine Neurons by Morphine and/or PD168,077 Treatments

Changes in the intrinsic and firing properties of striatal MSNs induced by the co-treatment with morphine + PD168,077 suggest a putative modulation of the heteromeric G protein-gated inward rectifier K⁺ channels (GIRK), since these channels are the predominant channel open at the resting membrane potential [34] and it have been largely involved in the control of neuron excitability [35]. This hypothesis is supported by our previous results demonstrating a deregulation in the gene expression pattern of several GIRK subunits after the acute treatment with morphine and/or PD168,077 [36].

GIRK1 immunoreactivity (IR) was found in the striatal MSNs displaying a punctate labeling in the cytoplasm (Figure 5A) and in most of the striatal interneurons (PV, SS, and ChAT) showing an intense reticulum-like distribution in the soma and primary dendrites (Figure 5A,B). GIRK2 IR was no detected in the CPu (Figure 5C). Both GIRK1 IR (Figure 5D) and GIRK2 IR (Figure 5E) were present in almost all dopaminergic cells of the SNc identified with an anti-TH antibody. GIRK1 IR showed a reticulum-like distribution in the cell bodies (Figure 5D) whereas GIRK2 was demonstrated in both the perikarya and dendritic branches, even in those that extend down into the substantia nigra pars reticulata (SNr) (Figure 5E).

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Figure 5

Effects of treatment with morphine and/or PD168,077 on GIRK1 and GIRK2 expression in the CPu and SNc. (A) Representative photomicrographs of coronal section of the CPu showing GIRK1 immunoreactivity (IR). The majority of the GIRK1 IR cells are MSNs (left panel in A) displaying a punctate labeling pattern, although few of them are larger cells depicting a reticular-like staining (right panels in A). (B) Photomicrographs illustrating by dual labeled immunohistochemistry with anti-GIRK1 (green) and anti-ChAT, anti-PV, anti-SS, and anti-CR (red) the expression of GIRK1 in striatal interneurons. (C) Representative photomicrograph showing the absence of GIRK2 expression in the CPu. (D,E) Photomicrographs illustrating GIRK1 (D) and GIRK2 (E) expression in the SN and their dual expression with tyrosine hydroxylase (TH). (F–H) Graphs represent the semi-quantitative analysis of GIRK1 IR in the CPu (F) and GIRK1 IR (G) and GIRK2 IR (H) in the SNc after the continuous (7 days) treatment with morphine (20 mg/kg/d) and/or PD168,077 (1 mg/kg/d). Next to the graphs, representative photomicrographs of GIRK1 and GIRK2 IR in the CPu and SNc are shown. Data represents mean ± SEM, n = 6 per treatment per treatment. Kruskal–Wallis test followed by Dunn’s method, p < 0.05 vehicle vs. morphine; * p < 0.05 vehicle vs. PD168,077; # p < 0.05 vehicle vs. morphine + PD168,077; & p < 0.05 morphine vs. morphine + PD168,077. Scale bars are: A,C: 30 μm; B: 10 μm; D,E (left panels): 200 μm; D,E (right panels): 10 μm; F,G,H: 10 μm. Abbreviations: CPu, caudate putamen; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata.

The continuous treatment (7 days) with morphine or PD168,077 did not alter GIRK1 IR in the striatal MSNs. However, the combined administration of both drugs produced a significant increase in GIRK1 IR (by 23%) (Figure 5F). No changes in GIRK1 IR were detected in ChAT interneurons (Figure S2A), which were identified by their morphological characteristics.

In the SNc, GIRK1 IR and GIRK2 IR were significantly downregulated by morphine (GIRK1: by 10%: GIRK2: by 32%) and PD168,077 (GIRK1: by 15%; GIRK2: by 38%) (Figure 5G,H). However, the co-administration of morphine with PD168,077 completely (for GIRK1; Figure 5G) or partially (for GIRK2; Figure 5H) prevented these effects.

We also observed that the expression of GIRK1 and/or GIRK2 was regulated even after a single dose of morphine and/or PD168,007 in the CPu and SNc, although with a different pattern (Figure S2B).

4.1. Morphine-Induced Structural Plasticity in Striatal MSNs

There is strong evidence that morphine elicits structural and functional plasticity mostly in brain regions associated with reward, learning, or incentive motivation, such as the NAc, VTA, PFC, or hippocampus [5,15,42]. These neuroplastic changes persist for long after the discontinuation of morphine treatment. In fact, a recent research has found that morphine-induced alterations in proteins related to synaptic plasticity and cytoskeletal organization persist even six months after cessation of drug administration [43]. However, little was known about whether morphine also produced structural plasticity in the CPu, despite this region is critically involved in drug-related habits formation and consolidation of addiction [1,8,44].

Results of the present study clearly demonstrated that a continuous treatment with morphine triggers a retraction of the dendritic arbor and a significant loss of dendritic spines in the dorsal striatal MSNs. This observation is consistent with previous reports describing a similar pattern of long-term morphine-induced structural plasticity in several regions belonging to the reward circuit [2,5,12,45]. However, more recent studies have highlighted some contradictory data depicting an opposite effect, especially in the NAc, the orbital prefrontal cortex and the dentate gyrus of the hippocampus [13,14,15,46]. These differences could be attributed to several factors, such as the paradigm of drug administration, the animal sacrifice time point, the brain region analyzed, or even the cell type studied.

Interestingly, we observed that the retraction of dendrites specifically occurred by a decrease in the size (length and volume) of the proximal dendrites, while the spines loss was attributable to a selective downregulation of mushroom and stubby spines on distal dendrites. The concurrence of these two forms of structural plasticity could have a major impact on MSNs activity. Firstly, because signals propagation effectiveness throughout dendrites is highly dependent on their morphology [47,48,49,50]. Secondly, because dendritic spines remodeling is tightly linked to synaptic strength, and therefore to the synaptic plasticity phenomena [51]. Thus, the selective loss of mushroom spines after prolonged exposure to morphine could reflects the induction of long-term depression (LTD) processes, as previously demonstrated in excitatory inputs to the dorsal striatum [52,53]. It should be noted that these two structural changes took place in specific subregions of the dendritic arbor, revealing a dichotomy between proximal and distal dendrites. Regarding this, the existence of a topographic organization of the striatal MSNs inputs has been largely suggested although not fully unraveled. Proximal dendrites seem to mainly receive intrinsic shaft connections from striatal interneurons and collateral projections from MSNs, whereas distal dendrites preferentially support extrinsic projections from the cortex, thalamus, or midbrain [54,55]. In this perspective, it is likely that morphine, by promoting morphological changes and therefore affecting synaptic inputs, has a significant impact on the function of the MSNs of the dorsal striatum and ultimately on their output.

Our results show that, although morphine can induce an overall structural plasticity in the MSNs of the CPu, it is unable to modify their passive membrane properties and intrinsic excitability. Interestingly, previous studies have demonstrated that repeated morphine exposure specifically alters the activity of the NAc MSNs which expressed the dopamine D2 receptors (D₂R) [56,57]. Thus, as in our analysis we did not identify striatal MSNs subpopulations—e.g., direct- and indirect-pathway projection neurons—it remains to be investigated in future works whether morphine specifically mediates changes in one of them. On special interest will also be to reveal the specific action of morphine on the striatonigral projection neurons located in the striosomal compartment since this neural population express MOR and provide direct inputs on nigral dopamine neurons [37,38,39,40,58] and we have observed a morphine-induced increase in MOR expression in the striosome-dendron bouquets.

4.2. D₄R Activation Alter Dendritic Arbor Complexity and Spine Density of the Striatal MSNs

Dopamine action on striatal MSNs through its interaction with dopamine receptors has a major role in both structural and synaptic plasticity occurring in these neurons [59]. In fact, the existence of opposite processes controlling synaptic plasticity induced by dopamine in form of LTP or LTD are postulated to be directed by D₁R or D₂R in the direct- and indirect-pathway MSNs, respectively [60,61,62]. These data support the classical model for the independence of the two main striatal pathways outputs [63]. However, this model is currently under review since recent data are quite conflictive. As an example, a recent report has described that D₁R is essential for the maintenance of spine plasticity in the direct-pathway MSNs but it also affects indirect-pathway neurons [64]. This could be explained on the base of intrinsic connectivity between direct- and indirect-pathways MSNs or the cross-talk of these neurons via interneurons [62]. In addition, several works confirm that a certain proportion of MSNs co-express D₁R and D₂R [65,66].

The role of other receptor subtypes different to D₁R and D₂R has been largely undervalued in the context of the CPu despite their expression in both direct- and indirect-pathways MSNs. Thus, the existence of D₄R and D₅R in the CPu [28,67] opens up the possibility to discover new dopamine signaling mechanisms underlying striatal plasticity. According to our results, we suggest a new role for the D₄R in mediating the regulation of MSNs morphology by dopamine. Here, we show that prolonged D₄R stimulation by itself expands the MSNs dendritic arbors, as the activation of these receptors increase both the number of distal dendritic branches and also of their length. This dendritic reshaping could be related to the ability of D₄R to modulate the expression of the neurotrophic factor BDNF [36] and the transcription factors CREB and ΔFosB [25], which, in turn, regulates the transcription of numerous genes encoding for cytoskeleton regulatory proteins [5]. Paradoxically, D₄R stimulation also induces a downregulation of all types of dendritic spines (i.e., thin, stubby and mushroom), which occurs across the entire dendritic arbor. However, despite the morphological alterations, the passive membrane properties and excitability remain unchanged. This effect differs from that previously described for D₂R, since overexpression of this receptor increases the excitability of the MSNs and decreases the complexity and length of their dendritic arbors [68].