Brain Research Bulletin 61 (2003) 125–128

Neuromorphological background of cannabis addiction

Tibor Wenger a,∗ , Gabriella Moldrich a , Susanna Furst b
a Department of Human Morphology and Developmental Biology, Semmelweis University, Tuzolto u. 58, P.O. Box 95, Budapest, Hungary
b Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, Hungary

Received 27 February 2003; received in revised form 3 March 2003; accepted 5 March 2003

Abstract

The expression of central cannabinoid (CB1 ) receptors in tyrosine hydroxylase (TH) containing neurones was demonstrated. Co-localisation
was present in different brain areas responsible for reward-related mechanisms. The immunohistochemical investigations have shown that
co-localisation is present in parts of mesolimbic-mesocortical dopaminergic system like nucleus accumbens (Nacb), ventral tegmental area
(VTA), in the striatum, pyriform cortex, respectively. The results suggest a functional role of CB1 receptors in cannabis addiction by acting
directly on reward-related structures.
© 2003 Elsevier Science Inc. All rights reserved.

Keywords: Central cannabinoid receptor; Dopamine; Immunohistochemistry; Rat

1. Introduction The topographical distribution of CB1 receptors in

There are evidences that the mesolimbic-mesocortical
dopaminergic system is a major substrate for the rewarding
or reinforcing effects of drugs of abuse [6]. In the brain
these dopamine (DA) neurones are associated with median
forebrain bundle (MFB), ventral tegmental area (VTA), nu-
cleus accumbens (Nacb) or dorsal striatum [17]. It has been
proposed that DA is the main neurotransmitter involved in
responding to the motivational stimuli of addictive drugs.
GABAergic systems might play also an important role in
brain-reward mechanism [22]. DA released in dorsal stria-
tum and Nacb interact with excitatory neurotransmitters.
The action of drugs of abuse on rewarding-reinforcing
effects might be different, although they share common
features on crucial neural circuits [8]. The mechanism by
which cannabis acts on DA reward system is not yet clearly
known.
The discovery [4] and cloning of the central (CB1 ) and
peripheral (CB2 ) cannabinoid receptors [11,12] and their
endogenous ligands [2] has opened new avenues to inves-
tigate and understand the physiological role of endogenous
cannabinoids, and the mechanisms by which cannabis may
cause addiction [3].
∗ Corresponding author. Tel.: +36-1-215-6920/3682;
fax: +36-1-215-3064.
E-mail address: wenger@ana2.sote.hu (T. Wenger).
the brain has been elucidated [13,19]. The latero-dorsal
caudate-putamen, substantia nigra (pars reticulata), globus
pallidus are all populated with particularly high concentra-
tions of CB1 receptors. CB1 receptors are present in Nacb
and VTA, too. Within the basal ganglia CB1 receptors
may mediate endocannabinoid modulation of DA release
and/or uptake, suggesting that cannabinoids may modu-
late functions subserved by DA neuronal pathways [3,7].
Cannabinoids appear to pharmacologically interact with the
reward substrates in a manner not dissimilar to that of other
drugs with abuse potential. Thus, 9 -tetrahydrocannabinol
(THC), the psychoactive substance of marihuana, lowers
brain-reward threshold and augments Nacb extracellular
DA overflow [3,12,16]. From data collected using a wide
variety of neurobiological approaches, it has been previ-
ously argued that the site of action of the cannabinoids
on reward functions is likely to be within the Nacb, the
mesolimbic-dopaminergic system. It is worthy to note that
this postulation presume an opioid peptide mediation of
cannabinoid effects on the flow of reward-relevant signals
through the VTA-MFB-Nacb DA circuitry [6,8].
Although a functional interaction between the endo-
cannabinoid and DA systems have much data supporting
it, no morphological evidence on this point has heretofore
been available.
It is known that tyrosine hydroxylase (TH) is the first
and rate-limiting enzyme in catecholamine biosynthesis.

0361-9230/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved.
doi:10.1016/S0361-9230(03)00081-9
126 T. Wenger et al. / Brain Research Bulletin 61 (2003) 125–128
TH immunohistochemistry has been used for visualising
catecholamine-containing neurones and pathways. When
comparing the TH immunoreactivity (TH-IR) with the
well-known DA containing brain structures we can account
the TH immunopositivity for the presence of DA. In a pre-
vious paper, CB1 receptors and TH-IR were observed in
the same regions by double in situ hybridisation histochem-
istry but no fine resolution or exact cellular localisation
was detected [7]. In the present paper, we describe some
brain areas where TH and DA co-localisation is present and
discuss its possible functional significance.
2. Materials and methods
Male Sprague–Dawley rats (Charles River, Hungary)
weighing 240–260 g were used. The rats were housed in
controlled temperature (20 ± 2 ◦ C) and light (on 07:00 h, off
19:00 h) conditions and were provided with water and food
(rat pellets) ad lib. All experimental procedures conformed
to the Revised Guide for the Care and Use of Laboratory
Animals (NCR, Washington, DC, 1996).
Fixation and tissue preparation were performed as de-
scribed before [20,21]. Briefly the animals were perfused
with fixative (4% paraformaldehyde and 0.04% picric acid
in 0.1 M, pH 7.4 phosphate buffer) following deep anaesthe-
sia (sodium pentobarbiturate, 60 mg/kg). After perfusion the
brains were quickly removed and postfixed in the same so-
lution by immersion. Having washed thoroughly with buffer
the specimens were embedded in paraplast. Seven to ten mi-
crometers serial sections were cut between the rostral pole
of the brain (before olfactory bulb) and spinal cord. The sec-
tions were individually mounted on slides. Adjacent sections
were collected and treated with either anti-CB1 antibody
(kindly supplied by Dr. A. Howlett) or anti-TH antibody
(purchased from Institute Jacques Boy S.A., Reims, France).
Fig. 1. The figure shows two consecutive slides of zona inserta (A13 area) (A) and of the VTA (B). TH: tyrosine hydroxylase, CB-1: CB1 receptor
expression. The arrows show the same neurones (perikaryon or fibre). 3V: third cerebral ventricle. Scale bar represents 40 ␮m.
A third adjacent section was stained with hematoxylin–eosin
to facilitate the identification of the location of IR. Anti-CB1
antibody was used at a 1:5000 and anti-TH antibody at
1:10,000 dilution, respectively. CB1 receptor IR using the
same antibody has been already detected not only in the
brain [13], but in the anterior pituitary [18,20] and in the
Leydig cells of the testis [21], too.
The primary antibody was raised against the CB1
aminoterminus 1–14, modified by a cysteine at the carboxyl
terminus to facilitate coupling to protein carrier. We used
this antibody because the juxtamembrane terminus of the
receptor is critical for G protein activation [5]. The speci-
ficity of the antibody was determined as described [5]. For
specificity controls of the immunohistochemical reaction,
we used either incubation with buffer only or with saturated
antibody on randomly chosen sections.
In both cases biotin-conjugated goat anti-rabbit was used
as second antibody in 1:2000 dilution, followed by peroxi-
dase-conjugated streptavidin (both purchased from Jackson
Immunoresearch, West Grove, PA). The visible reaction
product was detected by diamino-benzidine (DAB) or by
DAB-nickel sulphate combination. DAB was purchased
from Sigma-Aldrich, Hungary.
For the evaluations of the results a ZEISS Axiophot mi-
croscope was used. The atlas of Paxinos and Watson [15]
served to identify relevant brain structures.
3. Results
CB1 immunoreactivity (CB1 -IR) and TH-IR were de-
tected alike it had been demonstrated earlier in different
brain structures [13,14,19]. None of the controls gave visible
reactions. When comparing the consecutive slides, we were
able to observe co-localisation of CB1 -IR and TH-IR mainly
in mesolimbic-mesocortical areas. In the diencephalon in the
 T. Wenger et al. / Brain Research Bulletin 61 (2003) 125–128 127

zona inserta (A13 dopaminergic cell group according to

Dahlström and Fuxe [1]) cells containing both IR were
present (Fig. 1A). The anterior cingulate cortex and Nacb
showed co-localisation, too. Both IR were present in the
VTA area (Fig. 1B) and in the pyriform cortex. Both
the perikaria and nerve processes show co-localisation
(Fig. 1). We were unable to localise IR in the synapses
with the used methods. The olfactory bulb, which is rich
in both CB1 receptors and dopaminergic neurones, did not
show co-localisation. No co-localisation was seen in the
tubero-infundibular area either (Fig. 2). Fig. 3 shows the
mapping of our results. On this schematic drawing well seen
is that the co-localisation is present mainly in forebrain.

4. Discussion

We present here, for the first time, strong evidence for

Fig. 2. The figure shows two consecutive slides at the level of infundibulum
of the hypothalamus and pituitary stalk. CB-1: CB1 receptor expression,
TH: tyrosine hydroxylase. Note that no CB1 receptor expression is seen,
thus at this level no co-localisation is present. Scale bar represents 60 ␮m.
co-localisation of CB1 receptors and the DA neuronal marker
TH. CB1 receptors and DA co-localisation is seen only in
a limited number of CNS loci, including VTA, Nacb, zona
inserta and subdomains of both striatum and hippocampus.
Strikingly, most of these CNS loci are tied together by a com-
mon functional theme-reward and reward-related functions.

Fig. 3. Schematic drawing of the rat brain to show the localisation of CB1 receptor expression (CB-1, red) and tyrosine hydroxylase (TH, blue). The
co-localisation places (Coloc, pink) are seen in the olfactory tubercle, pyriform cortex, anterior cingulate cortex, Nacb, some parts of the striatum, zona
inserta (A13), respectively.
128 T. Wenger et al. / Brain Research Bulletin 61 (2003) 125–128
The presence of cannabinoid receptors in TH expressing
neurones (most probably dopaminergic neurones [14]) might
mean that the endocannabinoid system has direct influence
on dopaminergic reward mechanisms.
The addictive behaviour appears to derive from pos-
itive reinforcing (rewarding) effects produced by drugs
of abuse. In turn these rewarding effects appear to de-
rive from pharmacological action of addictive drugs on a
common, albeit multifactorial, neuroanatomical and neuro-
chemical brain-reward substrate [6,8,17,22]. Biochemical,
behavioural and electrophysiological data demonstrate that
cannabinoids activate the brain’s reward system in a similar
manner to other drugs of abuse [3]. Moreover, in CB1 re-
ceptor knockout mice, the reinforcing effects of opioids and
the severity of withdrawal syndrome after chronic morphine
usage were both reduced [9]. An increase of extracellular
DA in the Nacb was not observed after morphine treatment
in these mice [10,23]. These later also suggest that the
CB1 receptors are involved in the reinforcing effect as well
as the development of dependence on opioids, too. The
changes in midbrain mesolimbic-mesocortical DA would
be essential for the reinforcing effects. It was suggested
that the functional role of CB1 receptors in the CNS areas
involved in opioid dependence is only through the action
on opioid system [23]. The presence of CB1 receptors in
DA neurones strongly supports and gives morphological
background to the postulation that CB1 receptor activation
modulates physical as well as psychological dependence
producing effects through the dopaminergic system itself,
too. Further research is needed to elucidate the exact role of
endogenous cannabinoids in the brain-reward mechanisms.
Acknowledgements
The support of OTKA T 032736 (to S.F.) and T 034365
(to T.W.) and of ETT 156/2000 (to S.F.) and of NIH-NIDA
(to T.W.) is fully acknowledged. The authors thank Professor
A. Howlett for the gift of CB1 receptor antibody.
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