Abstract

The consumption of drugs of abuse provokes sensitization, the development of tolerance, dependency, and eventually addiction. It is thought that these events are partially a consequence of drug-induced alterations in the organization of neuronal circuits in specific areas of the brain. In the present study, we have used intracellular injections of lucifer yellow to examine the alterations that may occur in cortical pyramidal neurons of addiction-prone Lewis rats following 15 days of self-administration of morphine. Specifically, the effects of morphine on the structure, size and branching complexity of the basal dendrites, and spine density were determined in the basal dendritic arbors of layer III pyramidal neurons in both the prelimbic and motor cortex. We found that following morphine self-administration, there was a reduction in the size and branching complexity of the dendritic arbors of pyramidal cells in the motor cortex. In contrast, prelimbic pyramidal neurons from these morphine-treated animals had larger and longer basal dendritic arbors. Furthermore, the spine density on pyramidal neurons was higher in both cortical regions of morphine self-administered rats. These results suggest that at least part of the behavioral changes produced by repeated opiate administration may be attributed to alterations in pyramidal cell structure.

Introduction

Repeated exposure to drugs of abuse has been shown to influence neuronal plasticity in brain regions related to motivation and reward (for a review, Kauer 2004). Such drugs induce adaptations that lead to addiction through cellular mechanisms similar to those involved in learning and memory processes (for a review, Wolf 2002). Indeed, common signal transduction pathways are activated by drugs of abuse and learning mechanisms (Hyman and Malenka 2001; Nestler 2001), and both processes produce similar anatomical changes (Robinson and Kolb 1997, 1999a, 1999b; Robinson and others 2001).

Several studies have analyzed the effects of drug administration on the morphology of neurons in different areas of the rat brain (Sklair-Tavron and others 1996; Robinson and Kolb 1999a, 1999b; Robinson and others 2002). These studies have shown that contingent and noncontingent cocaine, amphetamine, or morphine administration alters morphological parameters such as spine density and dendritic branching in pyramidal cortical and medium spiny accumbens neurons (Robinson and Kolb 1999a, 1999b; Robinson and others 2001, 2002; Crombag and others 2005). However, in all these studies the brain tissue was analyzed using the Golgi method and, due to the inherent limitations of this technique including inconsistency and partial staining, it is common that only some segments of the dendrites of relatively few well-labeled neurons were analyzed. Hence, it is often difficult to interpret the morphological changes identified in Golgi impregnated brain tissue under different experimental conditions. To avoid these technical problems, we have used intracellular injection of lucifer yellow into neurons in flat-mounted cortical slices of the addiction-prone Lewis rats. This inbred rat strain consistently exhibits a preference for drugs of abuse when compared with other rat strains (for a review, see Kosten and Ambrosio 2002).

In the present study, we have focused our attention on cortical pyramidal cells as they constitute 70–90% of all neurons in the cerebral cortex (DeFelipe and Fariñas 1992). In particular, we have analyzed the size, branching structure, and total number of spines in the basal dendritic arbors of cortical pyramidal cells at the base of layer III with the aim of consistently studying the same population of pyramidal cells. Furthermore, we chose this dendritic compartment of pyramidal cells for comparative purposes, given the numerous detailed quantitative studies of pyramidal cell structure in the cerebral cortex that have been performed in the basal dendritic arbors of layer III, in a wide variety of species and experimental conditions (for a review, Elston 2002, 2003; Benavides-Piccione and others 2004). The prelimbic cortex (area Cg3 according to Zilles 1985) was selected here because it is directly related with the mesocorticolimbic dopaminergic system, one of the most important reward systems in the brain (Hall and others 1977; Di Chiara and Imperato 1988; Wise and Rompre 1989; Di Chiara 1998; Robbins and Everitt 1999; Vetulani and others 2001; Steketee 2003; Robinson and Kolb 2004). We also selected motor areas of the frontal cortex (areas Fr1 and Fr3 according to Zilles 1985) because they do not appear to be directly involved in the addictive properties of drugs of abuse. Thus, these cortical areas might be useful to examine the general effects of morphine self-administration on the structure of the cortical pyramidal neurons. In order to investigate the possible alterations to pyramidal neurons in the prelimbic and motor areas of addiction-prone Lewis rats, we have used intracellular injections of lucifer yellow to reconstruct and analyze pyramidal neurons in these areas, after 15 days of morphine self-administration. We found significant modifications in the microanatomy of pyramidal cells from both the prelimbic and motor cortex in rats that self-administered morphine when compared with animals that received saline. However, these modifications were not identical in the two areas, suggesting that morphine does not equally affect the function of different areas of the cerebral cortex.

Materials and Methods

Animals

In this study, 12 adult male Lewis rats that weighed approximately 300–350 g at the beginning of training (Harlan Ibérica, Spain) were used. All animals were experimentally naive, and they were housed individually in a temperature-controlled room (23 °C). The animals were subjected to a 12-h light–dark cycle (08:00–20:00 lights on) and given free access to Purina laboratory feed and tap water prior to the initiation of the experiments. All experiments were performed in accordance with the guidelines established by the European Union regarding the use and care of laboratory animals.

Surgery

Experimentally naive subjects were surgically prepared with an intravenous catheter placed in the jugular vein. Under ketamine and diazepam anesthesia, polyvinylchloride tubing (0.064 internal diameter) was implanted in the right jugular vein at the approximate level of the atrium. The catheter was passed subcutaneously and exited in the midscapular region. The catheter then passed through a spring tether system (Alice King, Chatham, MA) that was mounted to the skull of the rat with dental cement. All subjects were housed individually following surgery and given at least 7 days to recover.

Apparatus

Twelve operant chambers (Coulburn Instruments, Allentown, PA) were used for the saline and morphine self-administration studies. Two levers designed to register a response when a force of 3.0 g was applied were placed 14 cm apart on the front wall of the chamber. A microliter injection pump (Harvard 22) was used to deliver intravenous saline or morphine injections to the rat. Drug delivery, operant data acquisition, and storage were accomplished through IBM computers (Med Associates, St Albans, VT).

Experimental Procedures

Self-administration behavior was studied according to procedures described previously (Ambrosio and others 1995). Briefly, prior to surgical implantation of the intravenous catheter, the animals were trained to press a lever for food reinforcement under a fixed ratio (FR) schedule of reinforcement (FR1). Initially, a single lever press on the left-hand lever resulted in delivery of a food pellet (45 mg, P.J. Noyes Company, Lancaster, NH) and turned on a stimulus light above the lever. A programed 30-s time out (TO) period in which responses had no programed consequences followed the delivery of each food pellet (FR1: TO 30 s). When the animal's behavior under the FR1 schedule of food-reinforced behavior was stable, the catheter was surgically implanted as described above. After the postoperative period, 12 littermate male Lewis rats were randomly assigned to one of the 2 groups: intravenous self-administration of 1 mg/kg/injections of morphine sulfate (n = 6) or intravenous self-administration of saline (0.9% NaCl; n = 6). The animals were allowed to self-administer morphine or saline in daily 12-h sessions between 20:00 and 8:00 h for 15 days. Immediately after the last session of saline or morphine self-administration, the animals were intracardially perfused with 4% paraformaldehyde in 0.1 mol/l phosphate buffer (PB; pH = 7.4), and the brains were removed for processing by the lucifer yellow method.

Intracellular Injections

The right and left brain hemispheres were gently flattened between 2 glass slides (Welker and Woolsey 1974), weighed and left overnight in 4% paraformaldehyde in PB. Vibratome sections (150 μm) were obtained from each hemisphere parallel to the cortical surface. The left hemisphere was used to study cell morphology in the prelimbic cortex (Cg3 area according to Zilles 1985; Fig. 1A,C), whereas the right hemisphere was used to analyze the motor cortex (primary motor; Fr1 and Fr3 according to Zilles 1985; or M1 according to Donoghue and Wise 1982; Fig. 1B,D). Cortical areas were identified based on previously published cytoarchitectonic studies (Paxinos and Watson 1982; Öngür and Price 2000). The cell injection and cell reconstruction methods used have been described in detail elsewhere (Elston and Rosa 1997; Benavides-Piccione and others 2005). Briefly, sections were prelabeled with 4,6-diamidino-2-phenylindole (D9542; Sigma, St Louis, MO), and a continuous current was used to inject cells with lucifer yellow (8% in 0.1 M Tris buffer, pH 7.4). Neurons were injected with a continuous current until the individual dendrites of each cell could be traced to an abrupt end at their distal tips, and the dendritic spines were readily visible. After injecting the neurons (we lost one of the animals from the saline self-administration group during this part of the experiment for methodological reasons), the sections were first processed with a rabbit antibody to lucifer yellow produced at the Cajal Institute (1:400 000 in stock solution: 2% bovine serum albumin [A3425; Sigma], 1% Triton X-100 [30632; BDH Chemicals, Poole, UK], and 5% sucrose in PB) and then with a biotinylated donkey antirabbit secondary antibody (1:200 in stock solution; RPN1004; Amersham Pharmacia Biotech, Little Chalfont, UK). Immunolabeled cells were visualized with a biotin–horseradish peroxidase complex (1:200 in PB; RPN1051; Amersham Pharmacia Biotech), using 3,3′-diaminobenzidine (D8001; Sigma) as the chromogen (Figs 1 and 2).

Figure 1

(A, B) Low-power photomicrographs of sections of the rat cerebral cortex parallel to the cortical surface, showing the regions where the cells were injected in the prelimbic cortex (A), medial view of the left hemisphere, and in the motor cortex (B), lateral view of the right hemisphere. These layer III neurons were injected with lucifer yellow and then processed with a light-stable diaminobenzidine reaction product. (C, D) High magnification of the boxed areas in A and B, respectively. Scale bar = 1.25 mm in A, B; 230 μm in C, D.

Figure 1

(A, B) Low-power photomicrographs of sections of the rat cerebral cortex parallel to the cortical surface, showing the regions where the cells were injected in the prelimbic cortex (A), medial view of the left hemisphere, and in the motor cortex (B), lateral view of the right hemisphere. These layer III neurons were injected with lucifer yellow and then processed with a light-stable diaminobenzidine reaction product. (C, D) High magnification of the boxed areas in A and B, respectively. Scale bar = 1.25 mm in A, B; 230 μm in C, D.

Figure 2

(A–D) Photomicrographs illustrating individual pyramidal neurons in the prelimbic (A, C) and motor (B, D) cortex in saline (A, B) and morphine self-administered animals (C, D). Note the increase in the complexity of dendritic branching of the pyramidal neuron in the prelimbic cortex of morphine self-administered (C) compared with saline-treated rats (A). (E–H) High-power photomicrographs of horizontally projecting dendrites from layer III pyramidal neurons in the prelimbic (E, G) and motor (F, H) cortex of saline (E, F) and morphine self-administered animals (G, H). Scale bar = 20 μm in A–D; 67.2 μm in G–H.

Figure 2

(A–D) Photomicrographs illustrating individual pyramidal neurons in the prelimbic (A, C) and motor (B, D) cortex in saline (A, B) and morphine self-administered animals (C, D). Note the increase in the complexity of dendritic branching of the pyramidal neuron in the prelimbic cortex of morphine self-administered (C) compared with saline-treated rats (A). (E–H) High-power photomicrographs of horizontally projecting dendrites from layer III pyramidal neurons in the prelimbic (E, G) and motor (F, H) cortex of saline (E, F) and morphine self-administered animals (G, H). Scale bar = 20 μm in A–D; 67.2 μm in G–H.

Cell Reconstruction and Morphometric Analysis

Only cells that were identified as pyramidal neurons (recognized by the labeling of the proximal portion of the apical dendrite) and whose entire basal dendritic arbor was completely filled and contained within the section were included in this analysis. These neurons were reconstructed in 3 dimensions using Neurolucida (MicroBrightField, Williston, ND; Fig. 3), and several morphological parameters that included the following features of the basal dendritic tree were measured:

  • Basal dendritic field area or the area of the dendritic field of a neuron calculated as the area enclosed by a polygon that joins the most distal points of dendritic processes (convex area).

  • Branching complexity (Sholl analysis): number of dendritic branches that intersect concentric spheres (centered on the cell body) of increasing 25 μm radii as a function of the distance from the soma.

  • Number of branches by branch order.

  • Total dendritic length (per cell) of basal dendritic arbor.

  • Dendritic length per distance, calculated as the length of dendritic segments as a function of the distance from the soma in the same concentric 25-μm radial spheres.

  • Length per branch order calculated as the length of dendrites by branch order.

  • A vertex analysis classifying the nodes by their connectivity at the vertices (connection points for the branches) and on the connectivity of the next order of vertices. This analysis compares the structure of the dendritic trees by combing the topological and spatial properties, such as nodes and branch lengths. To describe the overall structure of a dendritic arbor, the following parameters are taken into account: bifurcating nodes that have 2 terminating branches attached (Va), 1 terminating branch attached (Vb), and 0 terminating branches attached (Vc).

  • The ratio Va/Vb > 1 suggests that the tree is nonrandom and symmetrical, whereas a ratio < 0.5 suggests that the tree is nonrandom and asymmetrical. Values ≈ 1 suggest that the growth of terminal nodes is a random process. All these values are expressed by branch order per cell.

Figure 3

Neurolucida drawings of layer III pyramidal neurons, as seen in the plane of a section parallel to the cortical surface, from saline and morphine self-administered rats in prelimbic and motor cortex. The cells illustrated have basal dendritic arbors, which approximated to the average size for each group. Scale bar = 125 μm.

Figure 3

Neurolucida drawings of layer III pyramidal neurons, as seen in the plane of a section parallel to the cortical surface, from saline and morphine self-administered rats in prelimbic and motor cortex. The cells illustrated have basal dendritic arbors, which approximated to the average size for each group. Scale bar = 125 μm.

Spine Density

The density of dendritic spines on the labeled pyramidal cells was determined by counting the number of spines in each 10 μm segment (×100 oil objective) starting at the soma and continuing to the distal tips of 20 dendrites selected at random that projected horizontally from different cells (e.g., see Eayrs and Goodhead 1959; Valverde 1967), in each cortical area and animal; no distinction was made between sessile and pedunculated types. The total number of spines found in the “average” pyramidal cell basal dendritic arbor was calculated by multiplying the average number of spines of a given portion of dendrite by the average number of branches for the corresponding region. This analysis was carried out over the entire dendritic arbor (Elston 2001).

Statistical Analysis

In the self-administration study, we analyzed the number of injections per hour across the sessions and the average number of injections per hour throughout the entire study. Because the data did not satisfy the requirements of analysis of variance (ANOVA), we used a nonparametric Mann-Whitney U-test to compare the number of injections per hour across the sessions in rats that self-administered saline and morphine. The Mann-Whitney U-test was also used to compare the total average of self-administered injections in both groups during the 15 sessions. Finally, a Wilcoxon signed-ranks test was run to determine if there were significant differences in self-administered injections between the first and last session within each group of animals. Differences were considered significant if the probability of error was less than 5%.

Statistical comparisons of morphological parameters were performed by one-way ANOVA and repeated measures ANOVA. One-way ANOVA was used for the basal dendritic area, dendritic length, dichotomous vertices, and the vertex ratio parameters. Repeated measures ANOVA was applied to the branching pattern, number of branches per order, length per distance, and density of spines. When these tests revealed significant differences in the data set, post hoc Bonferroni tests were performed. All statistical comparisons were made using SPSS statistical package (SPSS Science, Chicago, IL).

Results

Morphine Self-Administration Behavior

The number of self-administered injections per hour was registered for saline- and morphine-treated rats under a FR1 schedule of reinforcement throughout the sessions (Fig. 4A). Similarly, the average number of injections self-administered by both groups in the 15 sessions was calculated (Fig. 4B). Statistical analysis revealed significant differences in the average total number of injections self-administered in the majority of the sessions (see Fig. 4A), as well as throughout the entire study (P < 0.01). As can be seen, the rats that received morphine self-administered a significantly higher number of injections per hour across the sessions and a higher average number of injections throughout the entire study when compared with animals receiving saline. We also found that rats that received morphine administered a higher number of injections in the last session when compared with the first (P < 0.05, as revealed by the Wilcoxon signed-ranks test). Indeed, saline animals self-administered a lower number of injections per hour, and the number of injections remained similar between the different sessions. In contrast, the number of self-administered injections gradually increased as the sessions elapsed in the rats that received morphine. These results show that rats are responsive to the reinforcement properties of morphine.

Figure 4

(A) Graph showing the number of injections per hour self-administered by saline- and morphine-treated rats under a FR1 schedule of reinforcement throughout the sessions. (B) Graph showing the total average number of self-administered injections for the 15 sessions in both groups.

Figure 4

(A) Graph showing the number of injections per hour self-administered by saline- and morphine-treated rats under a FR1 schedule of reinforcement throughout the sessions. (B) Graph showing the total average number of self-administered injections for the 15 sessions in both groups.

Morphology of Layer III Pyramidal Cells

We reconstructed 150 pyramidal cells from the base of layer III of the prelimbic and motor cortex in rats that self-administered saline and morphine (Fig. 3) in order to carry out the morphometric analysis (Tables 1–4). The neurons selected displayed a clear apical dendrite, and their basal dendritic arbors were completely filled with dye and entirely contained within the section. Because no quantitative information was available regarding the possible differences in the morphometric parameters of the basal dendritic arbors of layer III pyramidal neurons in the rat motor and prelimbic cortex, we first compared the structure of the pyramidal neurons in these 2 regions.

Table 1

Summary of certain morphometric parameters

 Prelimbic saline Prelimbic morphine Motor saline Motor morphine 
Basal dendritic area (×104 μm2) (mean ± SE) 6.02 ± 0.41 7.24 ± 0.26 8.46 ± 0.26 7.05 ± 0.13 
Dendritic length (×103 μm) (mean ± SE) 2.26 ± 0.16 2.84 ± 0.12 3.35 ± 0.13 2.59 ± 0.09 
Ratio Va/Vb (mean ± SE) 1.54 ± 0.18 1.66 ± 0.2 1.35 ± 0.07 1.49 ± 0.12 
Dichotomous vertices (mean ± SE)     
    Va 7.66 ± 0.57 8.25 ± 0.31 11.55 ± 0.39 9.46 ± 0.37 
    Vb 5.83 ± 0.61 6.60 ± 0.37 9.27 ± 0.55 7.46 ± 2.58 
    Vc 3.38 ± 0.47 3.58 ± 0.27 6.96 ± 0.38 5.00 ± 0.31 
Total number of spines 2382 3360 2782 2787 
 Prelimbic saline Prelimbic morphine Motor saline Motor morphine 
Basal dendritic area (×104 μm2) (mean ± SE) 6.02 ± 0.41 7.24 ± 0.26 8.46 ± 0.26 7.05 ± 0.13 
Dendritic length (×103 μm) (mean ± SE) 2.26 ± 0.16 2.84 ± 0.12 3.35 ± 0.13 2.59 ± 0.09 
Ratio Va/Vb (mean ± SE) 1.54 ± 0.18 1.66 ± 0.2 1.35 ± 0.07 1.49 ± 0.12 
Dichotomous vertices (mean ± SE)     
    Va 7.66 ± 0.57 8.25 ± 0.31 11.55 ± 0.39 9.46 ± 0.37 
    Vb 5.83 ± 0.61 6.60 ± 0.37 9.27 ± 0.55 7.46 ± 2.58 
    Vc 3.38 ± 0.47 3.58 ± 0.27 6.96 ± 0.38 5.00 ± 0.31 
Total number of spines 2382 3360 2782 2787 

Note: SE, standard error.

Table 2

Dendritic length and branching pattern of the basal dendrites of layer III pyramidal cells, determined with concentric spheres of progressively larger radii (25 μm), and per branch order

  25 μm 50 μm 75 μm 100 μm 125 μm 150 μm 175 μm 
Sholl analysis (number of intersections; mean ± SE) Plc saline 9.4 ± 0.5 17.5 ± 0.9 17.5 ± 1.1 13.9 ± 1.6 7.44 ± 1.1 3.4 ± 0.7 0.9 ± 0.4 
Plc morphine 10.4 ± 0.3 18.7 ± 0.5 20 ± 0.8 16.1 ± 0.7 10.4 ± 0.7 5.2 ± 0.5 2.3 ± 0.3 
Mc saline 11.7 ± 0.5 23 ± 0.6 26.1 ± 0.8 23.3 ± 0.9 15.9 ± 1.1 8.1 ± 1 2.9 ± 0.5 
Mc morphine 11.7 ± 0.3 21.6 ± 0.6 22.1 ± 0.7 17.2 ± 0.9 8.1 ± 0.7 2.3 ± 0.3 0.4 ± 0.1 
Dendritic length (μm; mean ± SE) Plc saline 152.3 ± 8.2 473.8 ± 27.8 565.7 ± 33.5 483.4 ± 34.6 324.3 ± 36.2 175 ± 30.3 69.7 ± 19.3 
Plc morphine 165 ± 6.2 553.5 ± 16.8 670.1 ± 24.9 597.8 ± 24.2 426.4 ± 22.6 246.9 ± 19.1 116.3 ± 14.5 
Mc saline 160.4 ± 7.1 576.8 ± 16.8 747.3 ± 22.3 730.7 ± 27.1 582.2 ± 30.1 349.9 ± 30.4 152.4 ± 22.6 
Mc morphine 171.1 ± 5.1 567.9 ± 15.5 686.1 ± 19.1 586.7 ± 24.3 388.2 ± 26.9 151.7 ± 16.9 32.4 ± 5.6 
  Order 1 Order 2 Order 3 Order 4 Order 5 Order 6 Order 7 
Branches (mean ± SE) Plc saline 5.1 ± 0.3 8.8 ± 0.5 10.2 ± 0.6 9.7 ± 0.9 3.8 ± 0.5 1.5 ± 0.5 0.3 ± 0.2 
Plc morphine 5.1 ± 0.2 8.9 ± 0.3 11.7 ± 0.4 9.5 ± 0.6 5.4 ± 0.5 2.3 ± 0.3 0.7 ± 0.2 
Mc saline 4.6 ± 0.2 9.2 ± 0.3 15.5 ± 0.6 16.1 ± 0.9 9.9 ± 0.9 3.7 ± 0.6 0.9 ± 0.4 
Mc morphine 4.7 ± 0.1 9 ± 0.3 13.5 ± 0.5 12.5 ± 0.7 6.5 ± 0.5 2.2 ± 0.4 0.3 ± 0.1 
Length (μm; mean ± SE) Plc saline 174.3 ± 26.3 403.7 ± 37.3 542.6 ± 63.9 724.2 ± 73.8 281.3 ± 51.6 111.7 ± 35.2 19.6 ± 14.32 
Plc morphine 179.4 ± 16.8 460.3 ± 33.2 800.7 ± 44 712.5 ± 50.6 404.4 ± 42.5 179.6 ± 25.3 63.79 ± 16.5 
Mc saline 61.5 ± 4.5 275.6 ± 26.4 858.4 ± 45.9 1078 ± 81.9 746.1 ± 79.4 269 ± 47.9 70.3 ± 39.2 
Mc morphine 87.5 ± 10.2 322.6 ± 24.5 750 ± 56.3 807 ± 55.3 449.1 ± 41 153.4 ± 27.84 20.72 ± 9.2 
  25 μm 50 μm 75 μm 100 μm 125 μm 150 μm 175 μm 
Sholl analysis (number of intersections; mean ± SE) Plc saline 9.4 ± 0.5 17.5 ± 0.9 17.5 ± 1.1 13.9 ± 1.6 7.44 ± 1.1 3.4 ± 0.7 0.9 ± 0.4 
Plc morphine 10.4 ± 0.3 18.7 ± 0.5 20 ± 0.8 16.1 ± 0.7 10.4 ± 0.7 5.2 ± 0.5 2.3 ± 0.3 
Mc saline 11.7 ± 0.5 23 ± 0.6 26.1 ± 0.8 23.3 ± 0.9 15.9 ± 1.1 8.1 ± 1 2.9 ± 0.5 
Mc morphine 11.7 ± 0.3 21.6 ± 0.6 22.1 ± 0.7 17.2 ± 0.9 8.1 ± 0.7 2.3 ± 0.3 0.4 ± 0.1 
Dendritic length (μm; mean ± SE) Plc saline 152.3 ± 8.2 473.8 ± 27.8 565.7 ± 33.5 483.4 ± 34.6 324.3 ± 36.2 175 ± 30.3 69.7 ± 19.3 
Plc morphine 165 ± 6.2 553.5 ± 16.8 670.1 ± 24.9 597.8 ± 24.2 426.4 ± 22.6 246.9 ± 19.1 116.3 ± 14.5 
Mc saline 160.4 ± 7.1 576.8 ± 16.8 747.3 ± 22.3 730.7 ± 27.1 582.2 ± 30.1 349.9 ± 30.4 152.4 ± 22.6 
Mc morphine 171.1 ± 5.1 567.9 ± 15.5 686.1 ± 19.1 586.7 ± 24.3 388.2 ± 26.9 151.7 ± 16.9 32.4 ± 5.6 
  Order 1 Order 2 Order 3 Order 4 Order 5 Order 6 Order 7 
Branches (mean ± SE) Plc saline 5.1 ± 0.3 8.8 ± 0.5 10.2 ± 0.6 9.7 ± 0.9 3.8 ± 0.5 1.5 ± 0.5 0.3 ± 0.2 
Plc morphine 5.1 ± 0.2 8.9 ± 0.3 11.7 ± 0.4 9.5 ± 0.6 5.4 ± 0.5 2.3 ± 0.3 0.7 ± 0.2 
Mc saline 4.6 ± 0.2 9.2 ± 0.3 15.5 ± 0.6 16.1 ± 0.9 9.9 ± 0.9 3.7 ± 0.6 0.9 ± 0.4 
Mc morphine 4.7 ± 0.1 9 ± 0.3 13.5 ± 0.5 12.5 ± 0.7 6.5 ± 0.5 2.2 ± 0.4 0.3 ± 0.1 
Length (μm; mean ± SE) Plc saline 174.3 ± 26.3 403.7 ± 37.3 542.6 ± 63.9 724.2 ± 73.8 281.3 ± 51.6 111.7 ± 35.2 19.6 ± 14.32 
Plc morphine 179.4 ± 16.8 460.3 ± 33.2 800.7 ± 44 712.5 ± 50.6 404.4 ± 42.5 179.6 ± 25.3 63.79 ± 16.5 
Mc saline 61.5 ± 4.5 275.6 ± 26.4 858.4 ± 45.9 1078 ± 81.9 746.1 ± 79.4 269 ± 47.9 70.3 ± 39.2 
Mc morphine 87.5 ± 10.2 322.6 ± 24.5 750 ± 56.3 807 ± 55.3 449.1 ± 41 153.4 ± 27.84 20.72 ± 9.2 

Note: SE, standard error. The numbers in bold indicate the maximum value (peak) for each morphological parameter and group analyzed.

Table 3

Density of the basal dendritic spines of layer III pyramidal cells determined by counting the number of spines per 10 μm from the soma to the tip of each basal dendrite of prelimbic (Plc) and motor (Mc) areas in saline (Sa) and morphine self-administered (Mp) rats

 0–10 μm 11–20 μm 21–30 μm 31–40 μm 41–50 μm 51–60 μm 61–70 μm 71–80 μm 81–90 μm 91–100 μm 101–110 μm 11–120 μm 121–130 μm 131–140 μm 141–150 μm 
Plc Sa 0 ± 0 0.8 ± 0.4 5.3 ± 0.9 11.1 ± 1.3 14.7 ± 1.1 16.5 ± 1.3 18.9 ± 1.2 18.6 ± 1.1 17.4 ± 1.1 17.5 ± 1.3 15.2 ± 1.2 14.2 ± 1.2 13.2 ± 1.7 10.9 ± 1.6 12.1 ± 1.7 
Plc Mp 0 ± 0 1.4 ± 0.6 7.1 ± 1.6 13.3 ± 1.3 18 ± 1.14 18.8 ± 1.2 20 ± 0.9 19.6 ± 0.9 20.4 ± 1.0 21.1 ± 1.4 18.3 ± 1.6 17.1 ± 1.7 17.2 ± 1.5 14.7 ± 2.1 15.8 ± 1.3 
Mc Sa 0.05 ± 0.05 1 ± 0.4 3.7 ± 0.7 7.7 ± 0.7 10.2 ± 0.8 11.6 ± 0.7 12.5 ± 0.7 13.2 ± 0.7 11.9 ± 0.6 11.8 ± 0.4 11.3 ± 1.3 10.3 ± 1.1 8.6 ± 1.1 10.8 ± 1.1 8.8 ± 1.4 
Mc Mp 0.05 ± 0.05 2.1 ± 0.5 7.4 ± 1 12.5 ± 0.9 14.9 ± 0.8 16.1 ± 0.7 17.2 ± 0.9 17 ± 0.6 17.7 ± 0.7 15.4 ± 0.8 12.7 ± 1.0 12.8 ± 1.2 10.3 ± 1.6 12.3 ± 2.1 11.4 ± 2.2 
 0–10 μm 11–20 μm 21–30 μm 31–40 μm 41–50 μm 51–60 μm 61–70 μm 71–80 μm 81–90 μm 91–100 μm 101–110 μm 11–120 μm 121–130 μm 131–140 μm 141–150 μm 
Plc Sa 0 ± 0 0.8 ± 0.4 5.3 ± 0.9 11.1 ± 1.3 14.7 ± 1.1 16.5 ± 1.3 18.9 ± 1.2 18.6 ± 1.1 17.4 ± 1.1 17.5 ± 1.3 15.2 ± 1.2 14.2 ± 1.2 13.2 ± 1.7 10.9 ± 1.6 12.1 ± 1.7 
Plc Mp 0 ± 0 1.4 ± 0.6 7.1 ± 1.6 13.3 ± 1.3 18 ± 1.14 18.8 ± 1.2 20 ± 0.9 19.6 ± 0.9 20.4 ± 1.0 21.1 ± 1.4 18.3 ± 1.6 17.1 ± 1.7 17.2 ± 1.5 14.7 ± 2.1 15.8 ± 1.3 
Mc Sa 0.05 ± 0.05 1 ± 0.4 3.7 ± 0.7 7.7 ± 0.7 10.2 ± 0.8 11.6 ± 0.7 12.5 ± 0.7 13.2 ± 0.7 11.9 ± 0.6 11.8 ± 0.4 11.3 ± 1.3 10.3 ± 1.1 8.6 ± 1.1 10.8 ± 1.1 8.8 ± 1.4 
Mc Mp 0.05 ± 0.05 2.1 ± 0.5 7.4 ± 1 12.5 ± 0.9 14.9 ± 0.8 16.1 ± 0.7 17.2 ± 0.9 17 ± 0.6 17.7 ± 0.7 15.4 ± 0.8 12.7 ± 1.0 12.8 ± 1.2 10.3 ± 1.6 12.3 ± 2.1 11.4 ± 2.2 

Note: The numbers in bold indicate the maximum value (peak) for each morphological parameter and group analyzed.

Table 4

Statistical comparisons between the morphological characteristics from layer III pyramidal cells in the motor and prelimbic cortex of saline and morphine rats

 Prelimbic saline–morphine Motor saline–morphine Saline motor–prelimbic Morphine motor–prelimbic 
Branching pattern (ANOVA F3,144 = 13.043, P < 0.0001) — — 
Basal dendritic area (ANOVA F3,147 = 10.91, P < 0.0001) — 
No. branches per order (ANOVA F3,144 = 15.88, P < 0.0001) — — 
Dendritic length (ANOVA F3,147 = 8.37, P < 0.0001) — 
Length per distance (ANOVA F3,144 = 8.65, P < 0.0001) — 
Length per order (ANOVA F3,144 = 8.20, P < 0.0001) — — 
Dichotomous vertices     
    Va (ANOVA F3,147 = 15.52, P < 0.0001) — — 
    Vb (ANOVA F3,147 = 7.59, P < 0.0001) — — — 
    Vc (ANOVA F3,147 = 20.25, P < 0.0001) — 
Vertex ratio (Va/Vb) (ANOVA F3,147 = 0.47, P = 0.69) — — — — 
Density of spine (ANOVA F3,74 = 24.99, P < 0.0001) 
 Prelimbic saline–morphine Motor saline–morphine Saline motor–prelimbic Morphine motor–prelimbic 
Branching pattern (ANOVA F3,144 = 13.043, P < 0.0001) — — 
Basal dendritic area (ANOVA F3,147 = 10.91, P < 0.0001) — 
No. branches per order (ANOVA F3,144 = 15.88, P < 0.0001) — — 
Dendritic length (ANOVA F3,147 = 8.37, P < 0.0001) — 
Length per distance (ANOVA F3,144 = 8.65, P < 0.0001) — 
Length per order (ANOVA F3,144 = 8.20, P < 0.0001) — — 
Dichotomous vertices     
    Va (ANOVA F3,147 = 15.52, P < 0.0001) — — 
    Vb (ANOVA F3,147 = 7.59, P < 0.0001) — — — 
    Vc (ANOVA F3,147 = 20.25, P < 0.0001) — 
Vertex ratio (Va/Vb) (ANOVA F3,147 = 0.47, P = 0.69) — — — — 
Density of spine (ANOVA F3,74 = 24.99, P < 0.0001) 
*

Post hoc Bonferroni analysis, P < 0.05.

Animals That Self-Administered Saline: A Comparison between the Prelimbic and Motor Areas

In animals that self-administered saline, the basal dendritic arbors of pyramidal cells in the prelimbic cortex had less dendritic branches than those in the motor areas. For example, the peak branching complexity of pyramidal cells in the prelimbic cortex (17.5 ± 1.1 mean ± standard error of the mean) was significantly lower than that in the motor cortex (26.1 ± 0.8, repeated measures ANOVA revealed the difference to be significant F3,144 = 13.043, P < 0.0001; Table 4). This difference could not be attributed to the area covered by the basal dendritic field because this did not significantly differ between the prelimbic and motor cortex. However, in the prelimbic cortex the dendritic arbor had shorter dendritic segments and a lower number of intersections per order, as well as a lower number of Va, Vb, and Vc dichotomous vertices (Fig. 5 and Tables 1–4).

Figure 5

Graphs representing the different morphological parameters analyzed in this study in saline prelimbic and motor cortex. (A) Basal dendritic field area. (B) Sholl analysis. (C) Number of branches per order. (D) Total basal dendritic length. (E) Dendritic length per distance. (F) Length per branch order. (G) Vertex analysis. (H) The ratio Va/Vb. (I) Total number of spines. (J) Density of spines. (G) The number of Va type vertices in white, Vb in gray, and Vc in black. (AJ) (except G) Morphological parameters of pyramidal neurons from the prelimbic (black) and the motor cortex (gray).

Figure 5

Graphs representing the different morphological parameters analyzed in this study in saline prelimbic and motor cortex. (A) Basal dendritic field area. (B) Sholl analysis. (C) Number of branches per order. (D) Total basal dendritic length. (E) Dendritic length per distance. (F) Length per branch order. (G) Vertex analysis. (H) The ratio Va/Vb. (I) Total number of spines. (J) Density of spines. (G) The number of Va type vertices in white, Vb in gray, and Vc in black. (AJ) (except G) Morphological parameters of pyramidal neurons from the prelimbic (black) and the motor cortex (gray).

The density of dendritic spines also differed significantly between pyramidal cells in the prelimbic and motor cortex (repeated measures ANOVA F3,74 = 24.99, P < 0.0001). The spine density was significantly higher in the prelimbic cortex (peak spine density 18.9 ± 1.2) than in the motor cortex (13.2 ± 0.7; Fig. 5 and Tables 3 and 4). When the data from the Sholl analysis was combined with that of the spine densities, we could estimate the total “average” number of spines per pyramidal neuron. We found that the total number of spines per pyramidal cell in the prelimbic cortex (2382) was approximately 17% lower than those in the motor cortex (2782), despite the fact that the density of spines on pyramidal neurons was higher in the prelimbic cortex than in the motor cortex. Indeed, in the motor cortex this difference in the spine density was compensated for by the lengthening of the pyramidal neuron dendrites.

Morphine Self-Administering Animals

Self-administration of morphine significantly lengthened the dendrites of pyramidal neurons in the prelimbic cortex (2.84 ± 0.12 × 103 μm) when compared with those in saline-treated animals (2.26 ± 0.16 × 103 μm, ANOVA F3,147 = 8.37, P < 0.0001). Moreover, pyramidal cells in the prelimbic cortex displayed a higher spine density than that observed in animals that received saline (repeated measures ANOVA F3,74 = 24.99, P < 0.0001). Therefore, the total number of spines increased by 42% in the prelimbic cortex of morphine self-administered rats (3360 in morphine self-administered rats and 2382 in those that received saline; Fig. 6 and Tables 1–4).

Figure 6

Graphs representing the different morphological parameters analyzed in this study in the prelimbic cortex. (A) Basal dendritic field area. (B) Sholl analysis. (C) Number of branches per order. (D) Total basal dendritic length. (E) Dendritic length per distance. (F) Length per branch order. (G) Vertex analysis. (H) The ratio Va/Vb. (I) Total number of spines. (J) Density of spines. (G) The number of Va type vertices in white, Vb in gray, and Vc in black. (AJ) (except G) Morphological parameters of pyramidal neurons from saline (black) and morphine (gray) self-administered rats.

Figure 6

Graphs representing the different morphological parameters analyzed in this study in the prelimbic cortex. (A) Basal dendritic field area. (B) Sholl analysis. (C) Number of branches per order. (D) Total basal dendritic length. (E) Dendritic length per distance. (F) Length per branch order. (G) Vertex analysis. (H) The ratio Va/Vb. (I) Total number of spines. (J) Density of spines. (G) The number of Va type vertices in white, Vb in gray, and Vc in black. (AJ) (except G) Morphological parameters of pyramidal neurons from saline (black) and morphine (gray) self-administered rats.

In contrast, pyramidal neurons from the frontal cortex of animals that self-administered morphine displayed less complex branching of their dendritic arbors (Fig. 7 and Tables 1–4). In these animals, the dendrites were shorter (2.59 ± 0.09 × 103 μm) than in rats that self-administered saline (3.35 ± 0.13 × 103 μm), and they not only had less branches (peak branching = 22.1 ± 0.7 when compared with 26.1 ± 0.8 in self-administered saline animals; ANOVA F3,144 = 13.043, P < 0.0001) but also fewer branches per order and less vertices (ANOVA F3,147 = 15.52, P < 0.0001 for Va; ANOVA F3,147 = 7.59, P < 0.0001 for Vb; ANOVA F3,147 = 20.25, P < 0.0001 for Vc). However, motor pyramidal neurons did display a higher spine density in animals that received morphine than in self-administered saline animals (peak spine density of 17.2 ± 0.9 and 13.2 ± 0.7, respectively; Tables 3 and 4). As a result, the total number of spines per neuron did not vary in the motor cortex following morphine self-administration (2782 in saline and 2787 in morphine self-administered rats; Table 1, Fig. 7).

Figure 7

Graphs representing the different morphological parameters analyzed in this study in the motor cortex. (A) Basal dendritic field area. (B) Sholl analysis. (C) Number of branches per order. (D) Total basal dendritic length. (E) Dendritic length per distance. (F) Length per branch order. (G) Vertex analysis. (H) The ratio Va/Vb. (I) Total number of spines. (J) Density of spines. (G) The number of Va type vertices in white, Vb in gray, and Vc in black. (A–J) (except G) Morphological parameters of pyramidal neurons from saline (black) and morphine (gray) self-administered rats.

Figure 7

Graphs representing the different morphological parameters analyzed in this study in the motor cortex. (A) Basal dendritic field area. (B) Sholl analysis. (C) Number of branches per order. (D) Total basal dendritic length. (E) Dendritic length per distance. (F) Length per branch order. (G) Vertex analysis. (H) The ratio Va/Vb. (I) Total number of spines. (J) Density of spines. (G) The number of Va type vertices in white, Vb in gray, and Vc in black. (A–J) (except G) Morphological parameters of pyramidal neurons from saline (black) and morphine (gray) self-administered rats.

Finally, when the dendritic arbors of the prelimbic and motor cortex were compared in rats that self-administered morphine, the only parameters that were significantly different were the number of Vc dichotomous vertices (ANOVA F3,147 = 20.25, P < 0.0001) and the spine density (F3,74 = 24.99, P < 0.0001, Table 4). All the other parameters considered did not appear to differ from one area to the other. Therefore, most of the morphological differences observed between the motor and prelimbic cortex in saline self-administering animals disappeared in rats that self-administered morphine (Table 4 and Figs 6 and 7).

Discussion

We can draw two principal conclusions from the data generated in this study. First, there are significant differences between the prelimbic and motor cortex in terms of the structure of the basal dendritic arbor and the density of spines in layer III pyramidal cells in self-administered saline animals. Second, self-administration of morphine affects the structure and spine density of the basal dendritic arbor of these pyramidal neurons in both the prelimbic and motor cortex, but morphine self-administration affects these two cortical regions in different ways.

Comparison between the Basal Dendritic Arbors of Pyramidal Cells in the Prelimbic and Motor Cortex in Rats That Self-Administered Saline

In the present study, we found that the basal dendritic arbors of pyramidal cells in the prelimbic cortex are smaller and less branched in rats that self-administered saline than in those of the motor areas. In addition, pyramidal cells in the prelimbic cortex displayed a significantly higher density of spines than in the motor cortex. However, the average number of spines per pyramidal neuron in the prelimbic cortex was approximately 17% lower than in the motor cortex. This was probably due to the fact that the basal dendrites are longer in the motor cortex than in the prelimbic cortex, compensating for the lower density of spines displayed by motor pyramidal neurons. Nevertheless, our results should be interpreted with caution because cells from one cortical area were sampled in one hemisphere, and their structure was compared with those from another cortical area in the contralateral hemisphere. Therefore, we cannot rule out the possibility that the differences found between the prelimbic and motor cortex were at least in part due to interhemispheric differences. These morphological differences may have important functional implications. For example, a large dendritic arbor implies that a wider region of the cortex must be sampled, whereas a more complex branching pattern may determine the degree to which the integrations of inputs are compartmentalized within their arbors (Koch and others 1982). Greater potential for compartmentalization results in a significant increase in the representational power and a greater capacity for learning and memory (Poirazi and Mel 2001; reviewed in Elston 2003). Because each dendritic spine receives at least one excitatory glutamatergic synapse (Colonnier 1968; Jones and Powell 1969; Peters and Kaiserman-Abramof 1969; reviewed in DeFelipe and Fariñas 1992), the 17% lower number of spines per prelimbic pyramidal neuron suggests that these pyramidal neurons integrate less excitatory information than those in the motor cortex. In addition, the higher density of spines observed in prelimbic pyramidal neurons implies that the spines are not separated as much and therefore, there is a different input pattern between dendritic branches. In theory, this may have important functional consequences (Poirazi and Mel 2001; Stepanyant and others 2002).

Effects of Morphine Self-Administration

Morphine self-administration produced different effects on the pyramidal neurons present in the motor and prelimbic areas examined. The basal dendritic arbors of pyramidal neurons in the prelimbic cortex were larger and longer, whereas in the motor cortex there was a decrease in size, length, and dendritic branching complexity. The spine density increased in both areas. Nevertheless, a 42% increase in the total number of spines in the prelimbic cortex was observed, whereas the total number of spines did not vary in the motor cortex (2782 in saline vs. 2787 in morphine self-administered rats). Moreover, the absence of a systematic correlation between the changes in structure of basal dendritic arbor and spine density indicates that these variables are regulated independently.

Different drugs of abuse have been seen to induce plastic changes in different regions of the rat brain related with reward, motivation, and learning (Robinson and Kolb 1999a, 1999b; Robinson and others 2001, 2002; reviewed in Robinson and Kolb 2004). Thus, morphine self-administration decreases spine density and branching in the basal and apical dendritic arbors of pyramidal neurons in layer V of the rat prelimbic cortex. These observations contrast with the results found here regarding the structure of the basal dendritic arbor of layer III pyramidal neurons in the prelimbic cortex and regarding the spine density of pyramidal neurons in both prelimbic and motor cortex. Over and above the variations that might be introduced by the different methods employed, there may also be other reasons that explain the differences observed and in particular, there are 2 factors that deserve further consideration. First, one must take into account the time at which the animals were sacrificed. Whereas we have sacrificed the rats immediately after the last session of morphine self-administration, the analysis of layer V pyramidal cells was carried out on animals sacrificed 1 month after the last dose of morphine administered. Consequently, the changes observed in those studies may be attributed to the long-lasting effects of morphine or to a readaptation to an environment without morphine, whereas any differences observed here are more readily attributable to the chronic effects of morphine. Second, it is possible that layer V pyramidal neurons are affected in a different manner to layer III pyramidal neurons or that the different rat strains analyzed (Sprague-Dawley rats vs. Lewis rats) may also have influenced the effects of morphine observed. In conclusion, it is difficult if not impossible to draw comparisons between studies that have examined different brain regions and neuronal populations, or distinct parts of dendrites (i.e., apical vs. basal).

It has been suggested that the prelimbic cortex is directly involved in the learning processes mediated by the proposed dopaminergic reward circuit, which in turn is modified by drugs of abuse including morphine (Emson and Koob 1978; Bassareo and others 1996; Simonato 1996; Schultz 1997; McBride and others 1999; Kelley 2004). In the present study, morphine self-administration might have induced morphological changes in prelimbic pyramidal neurons through the participation of the dopaminergic system. Indeed, it has been shown that chronic morphine administration augments dopamine release in various regions of this reward system, which results in plastic changes (Maldonado and others 1996; Nestler and others 1996; Abel and Lattal 2001; Kandel 2001; Morris and others 2003, reviewed in Kelley 2004; Nestler 2004). Furthermore, chronic morphine treatment induces molecular changes that lead to a decrease in the levels of neurofilament proteins. These proteins are major components of the neuronal cytoskeleton and play a fundamental role in determining neuronal morphology, as well as affecting the dynamics and function of other cytoskeletal elements (Hoffman and Lasek 1975; Tytell and others 1981; Hoffman and others 1984; Hall and others 1991; Beitner-Johnson and others 1992; reviewed in Julien and Mushynski 1998). Moreover, in the rat prefrontal cortex and particularly in the anterior cingulate cortex, the strongest dopamine innervation occurs in the superficial layers, establishing synapses with distal dendrites of both local circuit neurons and with the dendritic spines and shafts of pyramidal cells (Emson and Koob 1978; Lewis and others 1979; Oades and Halliday 1987; Van Eden and others 1987; Carr and others 1999; Carr and Sesack 2000).

Other neurotransmitter systems should also be taken into account when considering the possible functional consequences of the changes induced by morphine self-administration in the morphology of layer III pyramidal neurons in the prelimbic cortex. For example, glutamatergic projections from the anterior cingulate cortex to the medial caudate putamen have been implicated in motor and cognitive functions, many of which are potently modulated by activating N-methyl-D-aspartate (NMDA) receptors (Calabresi and others 1997; Wang and Pickel 2000). In addition, there are indications that NMDA synaptic responses in the prefrontal cortex are regulated by D1 receptors (Gurden and others 2000). Hence, the coactivation of both glutamatergic and dopaminergic systems might participate in the long-term changes associated with plasticity and conditioned learning (Kelley and others 1997; Adriani and others 1998). Similarly, morphine self-administration might have induced morphological changes in the prelimbic and motor cortical neurons through modifications in the opioid receptors, which are widely distributed in the rat cerebral cortex (Mansour and others 1987; Tempel and Zukin 1987). In this respect, we have found that morphine self-administration increased mu-opioid receptor binding in several cortical areas, including the frontal, cingulate, somatosensorial, piriform, and insular cortices in Lewis rats (Cardoso and others 2003). Therefore, as well as the possibility that the morphological changes observed in the present study may be due to the influence of morphine on the dopaminergic system, the effects of morphine on both glutamatergic and opioids systems should not be disregarded.

With respect to the pyramidal neurons in the motor cortex, this cortical region is not directly involved in the reward system affected by drugs of abuse. However, opioid receptors are widely distributed in the rat cerebral cortex (Mansour and others 1987; Tempel and Zukin 1987), and it is possible that the morphological changes produced by morphine in the motor cortex are due to a general effect in the cerebral cortex mediated by these receptors. Indeed, the increase in mu-opioid receptor binding found in the motor cortex in morphine self-administered rats (Cardoso and others 2003) might affect the pyramidal neurons of this region. In addition, it is possible that there are hemispheric differences that might affect morphine self-administration. For example, it has been reported that dopamine release in the medial prefrontal cortex is asymmetric and that this affects the self-administration of morphine in rats (Glick and others 1992). However, it remains to be determined whether there are hemispheric differences in the effects of morphine on pyramidal neurons, and thus the effects of morphine self-administration on pyramidal neurons located in other cortical regions and hemispheres should also be analyzed. In this way, it would be possible to determine to what extent the morphological changes are induced by morphine and whether the effects on the structure of the dendritic arbor and spine density in the different cortical areas follow a similar pattern or if they are area and/or hemispheric specific.

Finally, it is thought that morphological plasticity serves to balance the flow of neuronal activity with the efficiency of interneuronal communication under normal conditions. However, this is not clear in the case of the plasticity induced by drugs. For example, the plastic changes induced by morphine are not necessarily due to a direct effect because it is possible that at least some of these changes are a consequence of associative learning (Robinson and Kolb 2004). Furthermore, morphological plasticity is especially evident in dendritic arbors but other more subtle changes, such as modifications in the morphology of individual spines, might pass unnoticed (e.g., Robinson and others 2002). Therefore, further studies will be necessary to examine drug-induced morphological changes in more detail, including those of the apical and basal dendritic trees in all cortical layers. Such studies will help to distinguish between the direct effects of the drugs and other factors that may influence these changes.

Morphine sulfate was kindly provided by the Dirección General de Estupefacientes (Spain). This work was supported by the Ministerio de Educación y Ciencia (grants Biología Fundamental 2003-02745 and Genómica y Proteómica 2003-20651-C06-06 to JDeF) and the Fondo de Inversiones Sanitarias 01-05-01; the Plan Nacional sobre Drogas (PNSD) 2000–2003; the Comunidad Autónoma de Madrid 08.8/0010.1/2003, and Salud y Farmacia 2004-08148 grants to EA. IBY thanks the Ministerio de Educación y Ciencia (AP2001-0671) for their support. The authors wish to thank Rosa Ferrado and Luis Carrillo for their technical assistance. Conflict of Interest: None declared.

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