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Addiction Biology
PRECLINICAL STUDY: FULL ARTICLE
doi:10.1111/j.1369-1600.2010.00218.x
Altered architecture and functional consequences
of the mesolimbic dopamine system in
cannabis dependence
Saturnino Spiga1, Alessandra Lintas2, Michele Migliore3 & Marco Diana2
Department of Animal Biology and Ecology, University of Cagliari, Italy1, “G.Minardi” Laboratory of Cognitive Neuroscience, Department of Drug Sciences,
University of Sassari, Italy2 and Institute of Biophysics, National Research Council, Italy3
ABSTRACT
Cannabinoid withdrawal produces a hypofunction of mesencephalic dopamine neurons that impinge upon medium
spiny neurons (MSN) of the forebrain. After chronic treatment with two structurally different cannabinoid agonists,
D9-tetrahydrocannabinol and CP55 940 (CP) rats were withdrawn spontaneously and pharmacologically with the CB1
antagonist SR141716A (SR). In these two conditions, evaluation of tyrosine hydroxylase (TH)-positive neurons
revealed significant morphometrical reductions in the ventrotegmental area but not substantia nigra pars compacta of
withdrawn rats. Similarly, confocal analysis of Golgi–Cox-stained sections of the nucleus accumbens revealed a
decrease in the shell, but not the core, of the spines’ density of withdrawn rats. Administration of the CB1 antagonist
SR to control rats, provoked structural abnormalities reminiscent of those observed in withdrawal conditions and
support the regulatory role of cannabinoids in neurogenesis, axonal growth and synaptogenesis by acting as
eu-proliferative signals through the CB1 receptors. Further, these measures were incorporated into a realistic computational model that predicts a strong reduction in the excitability of morphologically altered MSN, yielding a significant
reduction in action potential output. These pieces of evidence support the tenet that withdrawal from addictive
compounds alters functioning of the mesolimbic system and provide direct morphological evidence for functional
abnormalities associated with cannabinoid dependence at the level of dopaminergic neurons and their postsynaptic
counterpart and are coherent with recent hypothesis underscoring a hypodopaminergic state as a distinctive feature of
the ‘addicted brain’.
Keywords
Cannabinoids, dopamine, nucleus accumbens, THC, VTA, withdrawal.
Correspondence to: Marco Diana, Department of Drug Sciences, University of Sassari, Via Muroni 23/A, 07100 Sassari, Italy. E-mail: [email protected]
INTRODUCTION
Cannabis preparations have long been considered moderately harmful and mildly addictive possibly because until
recently, no clear-cut description of withdrawal syndrome is provided. Clinical reports, however, recently
described that chronic consumers of even low daily doses
of cannabis derivatives experience, upon cessation of
drug administration, overt abstinence signs (Haney et al.
1999, 2004; Budney & Hughes 2006), and these observations are paralleled by structural and functional
neuroimaging studies of cannabis use (Iversen 2003;
Quickfall & Crockford 2006). This abstinence syndrome is
of clinical significance (Cooper & Haney 2008), similar
to the onset and time course to that of other abused
substances, and diagnostic criteria for cannabis withdrawal syndrome have been proposed (Haney et al. 1999,
2004). Likewise, overt somatic signs of cannabinoid
withdrawal can be elicited in experimental models of
cannabinoid dependence by administering the competitive antagonist of cannabinoid CB1 receptor SR141716A
(SR) (Rinaldi-Carmona et al. 1994; Aceto et al. 1996;
Diana et al. 1998; Gonzalez et al. 2004) to animals
exposed to a chronic cannabinoid regimen, thereby offering the possibility to investigate neurobiological effects of
addicting chemicals in a condition that mimics human
addictive behaviour (Pulvirenti & Diana 2001; Melis,
Spiga & Diana 2005).
The morphological analysis of neurons and dendrites
(Zito & Svoboda 2002; Zito et al. 2004) has recently seen
© 2010 The Authors. Journal compilation © 2010 Society for the Study of Addiction
Addiction Biology
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Saturnino Spiga et al.
an explosion in the studies of the consequences of longterm administration of drugs (Sklair-Tavron et al. 1996;
Robinson & Kolb 1997, 2004; Spiga et al. 2003, 2005)
since these measures are suggested to reflect plasticity of
active synapses and therefore synaptic remodelling as a
consequence of experience and drug exposure (Robinson
& Kolb 2004). Addiction in this regard, as a condition
characterized by long-term drug exposure, can be conceptualized as one example of experience-dependent
plasticity, whereby experience (i.e. long-term exposure to
addictive drugs) may long-lastingly affect behavioural,
cognitive and psychological functions (Robinson & Kolb
2004; Melis et al. 2005).
Cannabis withdrawal produces a marked reduction of
electrophysiological activity in nucleus accumbens
(NAcc)-projecting dopamine (DA) containing neurons of
the rat midbrain (Diana et al. 1998) and a reduction of
DA outflow in the NAcc shell (Tanda, Loddo & Di Chiara
1999). This functional evidence suggests that cannabinoid withdrawal may structurally alter cellular elements of the mesolimbic system as it was recently shown
for opiate dependence (Spiga et al. 2003, 2005; Diana,
Spiga & Acquas 2006). Accordingly, a chronic tetrahydrocannabinol (THC) treatment has been shown to
increase the length of the dendrites as well as the
number of dendritic branches in the shell of the NAcc
and in the medial prefrontal cortex but not in other
brain areas (Kolb et al. 2006). However, because morphological evaluations were made long after drug discontinuation (30 days), it is impossible to ascertain from
these observations the relationship between structural
changes and onset of withdrawal.
Thus, in the present study, we investigated the morphological alterations affecting neurons of the rat ventrotegmental area (VTA), substantia nigra pars compacta
(SNc) and their postsynaptic counterparts in the NAcc
subregions. TH-positive neurons and Golgi–Cox-stained
MSN were evaluated after chronic cannabinoid treatment and withdrawal in order to obtain further insights
into the morphological features of the mesolimbic DA
system and its involvement in cannabis dependence,
whereas the role of endogenous cannabinoids was investigated through administration of the CB1 receptor
antagonist SR. To further investigate on the functional
relevance of the morphological changes after withdrawal, we used a biophysical model of MSNs to estimate
the alteration in the spiking activity produced by cannabis dependence.
MATERIALS AND METHODS
Subjects, drugs and cannabinoid treatment
Male Sprague-Dawley albino rats (n = 48; Charles River,
Como, Italy), weighing 200–225 g at the beginning of
treatment were used. The rats were kept on a 12-hour
light/12-hour dark cycle with food and water available ad
libitum. Experimental protocols were approved by the
Ethical Committee (EC) of the University of Sassari and
performed in strict accordance with the EC regulations
for the use of experimental animals (CEE N°86/609), and
recommended guidelines for the care and use of experimental animals approved by the Society for Neuroscience. The rats were administered twice daily (8 am and
8 pm). Time cycle changed at 8 am and 8 pm. Drug injections took place one in the light and one in the dark for
6.5 days with either D9-THC (Sigma, Milan, Italy) or CP
55 940 (Sigma-Aldrich, Milan, Italy) emulsified in 1%
Tween 80 then diluted in a saline solution and administered i.p. in a volume of 3 ml/kg.
The animals were assigned to the following groups:
chronic saline (1% Tween) (CTRL) (n = 6); chronic CP
(0.4 mg/kg) (CP-Chr) (n = 6); chronic D9-THC (15 mg/
kg) (THC-Chr) (n = 6); 24 hours spontaneous withdrawal
from chronic CP (CP-Sw) (n = 6); 24 hours spontaneous
withdrawal from chronic THC (THC-Sw) (n = 6); SR
(5 mg/kg) precipitated withdrawal from chronic CP
(0.4 mg/kg/administration) (CP-SR) (n = 6); SR (5 mg/
kg) precipitated withdrawal from chronic D9-THC
(15 mg/kg/administration) (THC-SR) (n = 6); and SR
(5 mg/kg) in CTRL (SR) (n = 6) (Diana et al. 1998). On
the morning of day 7, the rats received the first daily
administration (vehicle, CP or THC) and 1 hour before
sacrifice were tested for signs of spontaneous and
SR-precipitated withdrawal (not shown, see Diana et al.
1998).
Histology
The animals were anesthetized with urethane (1.3 g/kg
i.p.) before transcardiac perfusion with 100 ml of ice-cold
saline solution immediately followed by 400 ml of icecold 4% paraformaldehyde. The brains were divided into
two parts at approximately -2 mm from bregma.
VTA-SNc TH-immunolabelling
The posterior (VTA- and SNc-containing) part of the
brains (Fig. 1) was postfixed for 24 hours in the 4%
paraformaldehyde solution and cryoprocteted in 30%
sucrose in phosphate-buffered saline (PBS). Coronal sections between -5.80 mm and -6.30 mm for VTA-SN
(25 mm thick) from bregma according to Paxinos &
Watson (1998) (Fig. 1) were obtained with a cryostat
(Micron Cryo-Star HM 560, Walldorf, Germany). The
sections for TH-immunolabelling were washed for
3 ¥ 5 minutes in PBS, immersed for 30 minutes in 10%
normal goat serum (NGS) in 0.1 M PBS added with 0.5%
Triton X-100 (PBS-TX) and incubated for two hours
with a mouse monoclonal anti-TH antibody (1:500;
© 2010 The Authors. Journal compilation © 2010 Society for the Study of Addiction
Addiction Biology
Mesolimbic dopamine system in cannabis dependence
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Chemicon, Temecula, CA, USA) in PBS-TX. The sections
were then washed (3 ¥ 5 minutes) in PBS-TX and incubated with a biotinylated antimouse IgG (1:300, Vector
Laboratories, Burlingame, CA, USA) in PBS-TX and 1%
NGS for 30 minutes, rinsed (3 ¥ 5 minutes) in PBS-TX
and incubated with avidin-TRITC (1:200, SigmaAldrich) in PBS-TX and 1% NGS overnight at 4°C. All of
the sections were then washed (3 ¥ 20 minutes) in
PBS-TX and cover-slipped with Glycergel mounting solution (Dako, Milan, Italy).
Free-floating slices were rinsed in distilled water for one
minute and placed in ammonium hydroxide (30%) in the
dark. After rinsing (one minute), the slices were placed in
Kodak Fix Film solution (Ilford, UK) for 40 minutes in
the dark, then the, slices were rinsed again (two minutes)
and fixed (for 10 minutes) with Kodak Paper fixative 1:7.
After rinsing and dehydration by alcohol ascendant scale
(from 50° to 100°), the slices were placed in xylene (three
minutes). Finally, the slices were mounted and coverslipped by Canada balsam.
NAcc Golgi-Cox staining
Image processing: surface rendering techniques
After perfusion, the anterior (NAcc-containing) part of
the brains (Fig. 1) were immediately rinsed (15 minutes ¥ 3 times) in 0.1 M PBS and immersed in a Golgi–
Cox solution (Glaser & Van der Loos 1981) composed by
5% potassium dycromate, 5% mercurium chloride and
5% potassium cromate (pH 6.5). The solution was
changed once after two days, and the brains were then
left in fresh Golgi–Cox solution for an additional 14 days.
After this period, the brains were cryoprotected with a
30% sucrose solution for two to three days. Beginning at
1.70 mm and ending at 0.70 mm from bregma, 50 mm
thick coronal slices, according to Paxinos & Watson
(1998) (Fig. 1b), were obtained with a cryostat.
Leica 4-D confocal laser scanning microscope (Leica
Microsystems, Heidelberg, Germany) with an argon–
krypton laser was used to analyze the TH-positive
neurons and the Golgi–Cox-stained sections. Confocal
images were generated using 40¥ oil (na = 1.00–0.5) and
100¥ oil (na = 1.3). Each frame was acquired eight times
and then averaged to obtain noise-free images. Optical
sections, usually at consecutive intervals of 0.5 mm in
z-axis, were imaged through the depth of the labelled
neurons and saved as image stacks as previously
described (Tredici et al. 1993; Spiga et al. 2003, 2005).
Maximum intensity algorithm (ImageJ) was used
for three-dimensional (3-D) reconstructions of TH-
Figure 1 Schematic illustration, modified from Paxinos & Watson (1998), of the location of sampled neurons. VTA-SNc (left panel) and
NAcc’s shell/core (right panel). Distance from bregma is indicated
© 2010 The Authors. Journal compilation © 2010 Society for the Study of Addiction
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Saturnino Spiga et al.
immunolabelled cells, while extended focus algorithm
was used for 3-D reconstructions of Golgi–Cox-stained
neurons (Bitplane Imaris V.5.7.2).
Morphometric determinations
Cell body morphometry
Morphometric analyses were performed by two independent observers blind to pharmacological treatments.
TH-immunolabelled somata (n = 80/group) were collected from a square area (approximately 200 mm/side).
When totally included in the sections, the TH-positive
neurons were reconstructed in 3-D and used for measurements and statistical analysis using Bioscan Optimas software (v 6.5.1; Media Cybernetics Inc., MD, USA). The cell
bodies were manually marked following their profile,
excluding all dendritc trunks, to measure their area (mm2),
perimeter, major length (MJ) and minor axis length (min).
Spines’ Counts
For each group (n = 80), dendritic segments (at least
20 mm long) of second-order dendrites were collected for
analysis from 0.7–1.70 mm from bregma (58) and identified by confocal-rendered cells. The spines’ density was
calculated by tracing a 10–15 mm long ‘sp.’ line along the
dendritic trunk and counting the number of spines
therein. The procedure was repeated along the entire dendritic length from the bifurcation from the first branch of
primary dendrites to the next bifurcation.
Statistical analysis was performed by means of oneway analysis of variance (ANOVA) followed by the
Student t-test for post hoc comparisons.
Computational modelling
For computational modelling, MSNs were reconstructed
using a modified version of Neuron Morpho plugin for
ImageJ v1.1.6 and Neuromantic v1.6.3.
All simulations were carried out with the NEURON
program (v7, 61; Yale University, CT, USA). A custom 3-D
reconstruction of a medium spiny neuron composed of
519 segments, explicitly including 216 spines (accounting for 31% of the dendritic membrane), was used for all
simulations. Using the average experimental values for
the membrane time constant [7 ms, obtained from
nucleus accumbens shell neurons at physiological temperature (O’Donnell & Grace 1993) and a standard value
for the membrane capacitance (1 mF/cm2], the model
neuron resulted in an input resistance of 160 MW. To
model the basic active properties, we used channel
models downloaded from the public ModelDB repository
(http://senselab.med.yale.edu/ModelDB/), implemented
to reproduce the electrophysiological properties of NAcc
medium spiny neurons (Wolf et al. 2005). Excitatory
Figure 2 Histological sections depict sampled areas. TH-positive
neurons in the rat midbrain coronal section (top) and MSN Golgistained in NAcc (bottom). Abbreviations indicate: cp, cerebral
peduncle, basal part; VTA, vental tegmental area; SNc, substantia
nigra pars compacta
(amino - 3 - hydroxyl - 5 - methyl - 4 - isoxazole - propionate
[AMPA]) synaptic input was implemented on all synapses
using a double exponential conductance change mechanism with a rise and decay time constants of 0.5 and
3 ms, respectively. The same peak synaptic conductance
(0.15 nS) was used for all synapses, and different values
(0.2–0.6 nS) were tested obtaining the same qualitative
results (not shown). The up and down states observed in
these neurons in vivo (Gruber & O’Donnell 2009) were
implemented, activating the synapses asynchronously
and randomly (Poissonian) at an average frequency of
3 Hz for the down state (in the theta rhythm range) or at
40 Hz (in the gamma range). The up state was activated
at 0.8 Hz with a duration of 400 ms (Gruber & O’Donnell
2009). Average spiking rates under different conditions
were calculated from the last 20 seconds of simulations
lasting 22 seconds. A movie of the first four seconds from
simulations under control of after withdrawal is shown
as supplemental material (see Supporting Information
Movie S1). The complete model and simulation files are
available for public download at the ModelDB database.
RESULTS
VTA and SNc cells size
Confocal datasets of TH-immunolabelled neurons located
in the VTA and in the dorsomedial portion of SNc (Figs 1
& 2) (Gonzalez-Hernandez & Rodriguez 2000) were mor-
© 2010 The Authors. Journal compilation © 2010 Society for the Study of Addiction
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Mesolimbic dopamine system in cannabis dependence
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Table 1 Effect of treatments and one-way ANOVA results, on morphometrical changes in DA (a) neurons and (b) spine density of
accumbal MSN.
(a) Mesencephalic TH-positive neurons morphometry
SNC
CTRL
CP-Chr
THC-Chr
CP-Sw
THC-Sw
CP-SR
THC-SR
SR
VTA
CTRL
CP-Chr
THC-Chr
CP-Sw
THC-Sw
CP-SR
THC-SR
SR
Area (mm2) (%)
Perimeter (%)
MJ length (%)
Min length (%)
156.4 ! 3.4 (100.0)
158.2 ! 2.3 (101.2)
157.7 ! 4.5 (100.8)
156.6 ! 3.1 (100.1)
156.3 ! 3.9 (99.9)
157.1 ! 2.9 (100.4)
158.4 ! 2.8 (101.3)
150.6 ! 2.7 (96.3)
53.2 ! 0.7 (100.0)
52.8 ! 0.5 (99.2)
52.2 ! 0.8 (98.0)
53.3 ! 0.7 (100.1)
51.1 ! 0.7 (96.0)
52.9 ! 0.6 (99.4)
52.4 ! 0.6 (98.5)
52.0 ! 0.7 (97.7)
20.7 ! 0.3 (100.0)
21.2 ! 0.3 (102.8)
21.3 ! 0.4 (103.3)
20.9 ! 0.4 (101.1)
20.1 ! 0.3 (97.2)
21.3 ! 0.3 (102.9)
20.1 ! 0.3 (97.3)
20.7 ! 0.4 (100.2)
11.5 ! 0.2 (100.0)
11.1 ! 0.2 (97.0)
11.3 ! 0.2 (98.8)
11.1 ! 0.2 (96.7)
11.5 ! 0.2 (100.4)
10.9 ! 0.2 (95.2)
11.6 ! 0.2 (100.6)
11.0 ! 0.3 (96.2)
181.5 ! 3.6 (100)
177.4 ! 3.5 (97.7)
177.2 ! 4.2 (97.6)
116.3 ! 2.4 (64.1)
130.7 ! 3.0 (72.0)
124.1 ! 2.3 (68.4)
124.7 ! 2.9 (68.7)
128.4 ! 2.5 (70.7)
56.2 ! 0.7 (100)
56.5 ! 1.0 (100.7)
54.4 ! 0.7 (96.8)
44.5 ! 0.6 (79.2)
46.6 ! 0.8 (83.0)
45.4 ! 0.5 (80.9)
45.4 ! 0.6 (80.9)
46.1 ! 0.9 (82.1)
21.9 ! 0.4 (100)
21.2 ! 0.6 (96.8)
20.4 ! 0.3 (93.2)
17.2 ! 0.4 (78.2)
18.7 ! 0.3 (85.4)
17.5 ! 0.5 (79.7)
17.7 ! 0.2 (80.6)
17.7 ! 0.4 (80.7)
12.4 ! 0.3 (100)
12.5 ! 0.2 (101.0)
12.6 ! 0.3 (102.2)
10.0 ! 0.2 (80.6)
10.3 ! 0.2 (83.5)
10.2 ! 0.3 (82.4)
10.1 ! 0.3 (82.1)
10.4 ! 0.2 (84.2)
(b) MSN Secondary dendrites spines density (spine/10 mm)
CTRL
CP-Chr
THC-Chr
CP-Sw
THC-Sw
CP-SR
THC-SR
SR
Shell (%)
Core (%)
8.065 ! 0.096 (100)
8.431 ! 0.164 (106.54)
7.885 ! 0.151 (97.76)
5.383 ! 0.132 (66.75)
5.121 ! 0.186 (63.49)
4.611 ! 0.144 (57.17)
6.113 ! 0.121 (75.79)
5.898 ! 0.149 (73.12)
10.741 ! 0.138 (100)
10.565 ! 0.143 (98.36)
10.590 ! 0.156 (98.59)
10.433 ! 0.162 (97.13)
10.443 ! 0.148 (97.22)
10.706 ! 0.216 (99.67)
10.617 ! 0.173 (98.85)
10.427 ! 0.131 (97.08)
Data are expressed as mean ! standard error.
phometrically analyzed in order to evaluate the effects of
treatments. In accordance with previous reports, these
neurons exhibited considerable variability in shape and
size (ovoid, polygonal or fusiform) (Grace & Bunney
1983; Oades & Halliday 1987; Tepper, Sawyer & Groves
1987; Spiga et al. 2003) in both areas (Table 1, Fig. 3).
ANOVA showed anatomical differences between the
experimental groups in the VTA of the mean calculated
area (F639 = 82.83; P < 0.0001), perimeter (F639 =
71.35; P < 0.0001), MJ (F639 = 37.71; P < 0.0001) and
min (F639 = 32.76; P < 0.0001) length. Post hoc analysis
revealed that cell bodies in the VTA exhibited smaller
somata after both withdrawal conditions. In particular,
a mean reduction as compared with CTRL was
found for area [(t158 = 15.2: P < 0.0001); (t158 = 10.9;
P < 0.0001)], perimeter [(t158 = 14.3 P < 0.0001);
(t158 = 11.6; P < 0.0001)], MJ [(t158 = 11.6; P < 0.0001);
(t158 = 7.6; P < 0.0001)] and min length [(t158 = 8.43
P < 0.0001); (t158 = 6.88; P < 0.0001)] of CP-Sw and
THC-Sw, respectively. Further, we found similar results
for precipitated withdrawal with the CB1 antagonist
SR-treated rats after chronic CP [area (t158 = 12.3
P < 0.0001), perimeter (t158 = 12.8 P < 0.0001), MJ
(CP-SR t158 = 9.45, P < 0.0001), min (CP-SR t158 = 7.06,
P < 0.0001] and THC [area (t158 = 13.5, P < 0.0001),
perimeter (t158 = 12.8, P < 0.0001), MJ (t158 = 11.1;
P < 0.0001), min (t158 = 7.22; P < 0.0001)]. Specifically,
CP-SR and THC-SR showed a statistically significant
reduction of morphometric parameters compared with
CTRL. Unexpectedly, we also found changes in the SR
group qualitatively similar with the other withdrawal
conditions [area (t158 = 12.1, P < 0.0001), perimeter
(t158 = 13.2 P < 0.0001), MJ (t158 = 10.4, P < 0.0001),
min (t158 = 6.9, P < 0.0001)]. On the contrary, no significant changes were observed in both the CP-Chr [area
(t158 = 0.82, P = 0.41), perimeter (t158 = 0.39, P = 0.69),
© 2010 The Authors. Journal compilation © 2010 Society for the Study of Addiction
Addiction Biology
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Saturnino Spiga et al.
MJ (t158 = 0.29, P = 0.77), min (t158 = 0.39, P = 0.7)] and
the THC-Chr [area (t158 = 0.70, P = 0.48), perimeter
(t158 = 1.83, P = 0.068), MJ (t158 = 1.92, P = 0.055), min
(t158 = 0.77, P = 0.44)] groups. No statistical differences
were found for the TH-positive neurons from SNc
[area (F639 = 0.58; P = 0.77), perimeter (F639 = 1.17;
P = 0.31), MJ (F639 = 1.68; P = 0.11), Min (F639 = 1.41;
P = 0.19)] (Fig. 4, Table 1).
NAcc MSN spines density
Figure 5 show the effects of all the treatments on dendritic spines density of accumbal MSN (shell and core)
versus CTRL. The spine densities of the CTRL groups were
significantly different between shell and core (Fig. 6). A
one-way ANOVA revealed a significant effect on the
shell (F639 = 107.2; P < 0.0001) but not on the core
Figure 3 Representative confocal picture of TH-positive neurons
in theVTA, obtained by a projection of 53 scans for a total of 26.5 mm
in the z-axis
(F639 = 0.58; P = 0.77) on spine density in the experimental groups. Post hoc analysis showed a selective
reduction on spine density for spontaneous withdrawal
[CP-Sw (t158 = 16.4 P < 0.0001), THC-Sw (t158 = 14.4
P < 0.0001)] and pharmacologically precipitated
[CP-SR (t158 = 19.9 P < 0.0001), THC-SR (t158 = 12.6
P < 0.0001)] as well as SR(t158 = 12.2; P < 0.0001)
groups in respect to CTRL. These reductions were found
to be about 33% for CP-Sw, 36 % for THC-Sw, 42 % for
CP-SR, 24% for THC-SR and 26% for SR. Further, the post
hoc analysis failed to reveal any significant difference
between spine density counts in the shell MSN for the
CP-Chr (t158 = 1.2; P = 0.056) and the THC-Chr (t158 = 1;
P = 0.32) groups versus CTRL.
Computational modelling
The possible functional consequences of the morphological changes were investigated using a realistic computational model of NAcc shell neurons to study how the
average firing rate could be modified after drug withdrawal. To model the effects of drug withdrawal, we compared the simulation results under a control condition
assuming that all synapses were active, with the results
from two simulations in which 30% randomly chosen
synaptic compartments were deleted from the morphology. Furthermore, to model the well-known changes in
the dopaminergic D2 input, and especially its inhibitory
effect on the AMPA currents (up to a 15% reduction of
the peak current, Hernández-Echeagaray et al. 2004), we
tested the possible effects of different amounts of reduction in the dopaminergic input by increasing the peak
synaptic conductances up to 115% of the control value.
Typical traces are shown in Fig. 7b, and the modelling
results are summarized in Fig. 7c. A 30% reduction in the
active synapses caused a ~45% decrease in the average
Figure 4 Morphometric measures of VTA
TH-positive neurons. Data are presented as
% of control ! SEM. * indicates P < 0.05
versus CTRL
© 2010 The Authors. Journal compilation © 2010 Society for the Study of Addiction
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Figure 5 Histograms
represent
the
mean ! SEM of dendritic spines densities
(number of spines/10 mm of second-order
dendrites) of NAcc shell and core MSN. *
indicates P < 0.05 versus CTRL (student
t-test post hoc analysis)
Figure 6 Representative confocal reconstructions synthesizing major findings in the
NAcc. (a) Golgi–Cox-stained MSNs from
core CTRL group, (b) MSN from shell
CTRL group and (c) MSN from shell the
THC-Sw group, using extended focus algorithm (left part) by Voxblast v3 (SGI Iris).
Right panels show the same reconstructed
neurons on the left, using ‘Surpass’ and ‘Filament Tracer’ by Bitplane Imaris v5.7.2 software. ‘Filament Tracer’ is used for the
automatic detection of filaments in 3-D
objects (in confocal microscopy), and it is
based on interactive thresholding and anatomical and geometrical properties (length
and radius) of the filaments as a dendritic
branches. From the resulting database, it is
possible to extract various information
including the ‘terminal points’ number. This
feature can offer a representation of dendritic spines (green dots) of entire scanned
neurons
firing rate (Fig. 7c, 100% DA input). However, the model
suggests that this reduction could be progressively
balanced by a reduction in the DA input because
this would also reduce the inhibitory effect of the
activation of D2 receptors on the AMPA channels
(Hernández-Echeagaray et al. 2004). The reduction of
the dopaminergic input after withdrawal could thus be
considered as a kind of homeostatic signalling (reviewed
in Davis 2006) to maintain the functional role of these
neurons under pathological conditions.
© 2010 The Authors. Journal compilation © 2010 Society for the Study of Addiction
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Saturnino Spiga et al.
(a)
(b)
(c)
Figure 7 Simulation results. (a) The 3-D reconstruction of a NAcc
medium spiny shell neuron used in all simulations; (b) typical
5-second somatic traces during simulations under control conditions
(top), and after 30% reduction of the active synapses (bottom); (c)
average number of APs (with respect to control) as a function of
dopaminergic input
DISCUSSION
The results of the present study show that withdrawal
from a regimen of chronic cannabinoid administration
profoundly affects the morphological characteristics of
TH-positive neurons of the rat VTA and spines’ density of
dendrites of MSN of NAcc shell. In contrast, SNc TH positive neurons and core spines density were unaffected.
In particular, withdrawal from chronic administration of the synthetic CB1 agonist CP determined the
shrinkage of the somatic region of TH-positive neurons of
the VTA as assessed by the decrease of area, perimeter, MJ
and min. These changes were paralleled by a reduction of
the spines’ density in the NAcc shell. Thus, spontaneous
withdrawal appears responsible for the morphological
changes detected in the VTA and NAcc shell. The specificity of these effects can be reasonably attributed to
abrupt removal of the chronic cannabinoid because
when chronic administration was pharmacologically
interrupted, by administration of the CB1 antagonist SR,
such morphological measures were affected similarly.
Further supporting this contention, THC’s spontaneous
and precipitated withdrawal yielded similar results both
in the VTA and NAcc shell. In contrast, chronic administration of both compounds fails to affect the morphology
of VTA neurons and spines’ density in the NAcc, thus
pointing to a critical role of cannabinoid withdrawal in
the shrinkage of mesencephalic neurons and the spines’
loss in the NAcc shell.
The spine loss described in the present study is in line
and significantly extends previous findings (Kolb et al.
2006) that reported an elongation of the dendrites as
well as the number of dendritic branches in the shell of
the nucleus accumbens and in the medial prefrontal
cortex but not in other brain areas. This further strengthens the view that withdrawal from chronic cannabinoids
exerts powerful and long-lasting (Kolb et al. 2006)
changes in key brain structures affected by addicting
compounds. In particular, the spines’ loss observed here,
besides contributing to reduce the already abated DA
transmission, could help in explaining the down regulation of CB1 receptors after THC-withdrawal (Breivogel
et al. 2003) as well as the CB1-mediated inhibition of
excitatory synaptic transmission at the excitatory synapses between the prefrontal cortex and the NAcc (Mato
et al. 2005).
Administration of the CB1 antagonist SR in salinetreated rats produced effects qualitatively similar to those
observed in the subjects treated with the exogenous cannabinoids CP and THC in both areas examined (i.e. VTA
and NAcc shell). This unexpected finding would seem to
suggest that endogenous cannabinoids are involved in
the trophic control of key elements of the mesolimbic
system such as VTA neurons and their physiological
postsynaptic site. While further experiments are needed
to corroborate this notion, the present finding supports
the idea of an endocannabinoid trophic and protective
role (Galve-Roperh et al. 2006, 2007) at the level of the
DA system (Melis et al. 2006). Accordingly, endocannabinoids modulate synaptic plasticity in the VTA (Melis et al.
2004), SN pars reticulata (Szabo et al. 2000) and striatum (Gerdeman & Lovinger 2001). Alternatively, the possibility that SR might be acting as an inverse agonist
(Rubino et al. 2000; Bass et al. 2002) should be considered and further experiments will be needed to clarify this
issue.
Irrespective of the mechanisms underlying
SR-induced changes, the structural observations
reported here are likely to have profound consequences
on dopaminergic transmission in the shell of the NAcc,
both pre- and postsynaptic levels. Indeed, the reduced DA
© 2010 The Authors. Journal compilation © 2010 Society for the Study of Addiction
Addiction Biology
Mesolimbic dopamine system in cannabis dependence
firing (Diana et al. 1998) is accompanied by a ‘shrinkage’
of the somatic region, thereby rendering the cell more
excitable in line with the ‘size principle’ (Shepherd 1994;
Spiga et al. 2003). At the postsynaptic side, the reduced
number of spines would reduce total membrane surface
(Gray 1959; Rall 1962), thereby decreasing membrane
resistance (Wilson 1984) eventually leading to altered
excitability. This possibility is in line with classical theoretical predictions (Chang 1952), and subsequent confirmatory experimental tests (Wilson 1984), which
ascribed to the spine an attenuating effect on synaptic
potentials. Importantly, recent studies (Hoffman et al.
2003) employing a chronic regimen of THC very similar
to that employed here have reported that long-term exposure and subsequent withdrawal (recordings were performed 24 hours after last treatment) of the active
ingredient of marijuana blocks synaptic plasticity in the
NAcc and reduces the sensitivity of GABAergic and
glutamatergic synapses. What could then be the functional consequence of losing about 30% of the synapses?
There are two mechanisms directly related with the
reduction in the number of synapses that can affect the
overall firing rate of the neuron. On the one hand, the loss
of neuronal membrane associated with the loss synapses
(~9% of the total membrane in our model morphology)
increases the input resistance of the neuron, and in principle, this results in a more excitable neuron; on the other
hand, the neuron may decrease its firing rate because the
overall excitatory input is reduced. The model’s result
suggests that the reduced excitatory input upon withdrawal might be the dominant mechanism, at least under
the conditions tested in this work. However, the model
also suggests a possible compensatory mechanism:
because of the reduced dopaminergic input on these
neurons upon withdrawal, the inhibitory effect of D2
receptor activation on AMPA channels will also be
reduced, amplifying the effect of the remaining synapses
(Fig. 7).
Overall, the present data suggest that the altered
architecture of the DA system projecting to the shell of
the NAcc documented here would profoundly alter the
synaptic equilibrium affecting various neurotransmistters involved in the neurobiological mechanisms of cannabis dependence (Pulvirenti & Diana 2001; Melis et al.
2005).
On the basis of the present and previous findings
(Spiga et al. 2003, 2005; Diana 1996; Diana et al. 1995,
1999, 2006), we suggest that shrinkage of DA neurons
and reduction of spines’ densities in their postsynaptic
elements (i.e. MSN) upon withdrawal from chronic cannabinoids, might represent a morphological correlate of
the functional deficits detected by electrophysiological
(Diana et al. 1998) and neurochemical (Tanda et al.
1999) means that ultimately may contribute to negative
9
motivational properties of withdrawal from addictive
drugs (Koob & Le Moal 2001; Di Chiara 2002; Melis et al.
2005).
In general, the present data lends further support to
the notion that drug addiction can be seen as a chronic
drug-induced, aberrant form of neural plasticity (Nestler
& Aghajanian 1997; Nestler 2001; Pulvirenti & Diana
2001; Robinson & Kolb 2004; Melis et al. 2005), whereby
DA neurons originating in the VTA represent major cellular substrate involved at molecular (Nestler 2001), cellular (White 1996; Pulvirenti & Diana 2001; Diana &
Tepper 2002) and behavioural levels (Koob & Le Moal
1997, 2001; Berridge & Robinson 1998; Di Chiara 1999;
Robinson & Berridge 2001) and are coherent with recent
hypothesis (Melis et al. 2005) that underscores a
hypodopaminergic state as a distinctive feature of the
‘addicted brain’.
Disclosure/Conflict of Interest
The authors declare that over the past three years MD has
received compensation from ASMED srl and ESSEX Italia
for work unrelated to the material presented here.
Acknowledgements
This work was supported, in part, by a grant from MIUR
(PRIN. N°2004052392) to M.D. The authors wish to
thank G. Di Chiara for a gift of THC and William Dunn III
for proofreading the article.
Authors Contribution
SS and AL performed research and analysed the date. MM
contributed the computational evaluation and analysed
the data. MD designed, researched and wrote the paper.
All authors have critically reviewed content and
approved final version submitted for publication.
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the
online version of this article:
Movie S1 Movie of the first four seconds from two simulations modelling control conditions (left plots) and after
withdrawal (right plots). Top traces are somatic membrane potential, bottom plots illustrate the time course of
the membrane potential over the entire neuron. Different
colours correspond to different membrane potentials
according to the colour scale
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© 2010 The Authors. Journal compilation © 2010 Society for the Study of Addiction
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