The cannabinoid receptor type 1 in the murine nervous system [Elektronische Ressource] : physiological roles and cross-talk with other receptor systems / Heike Hermann

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• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2004
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 32 Mo

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TECHNISCHE UNIVERSITÄT MÜNCHEN


Lehrstuhl für Biotechnologie der Nutztiere

Max-Planck-Institut für Psychiatrie, München





The cannabinoid receptor type 1 in the
murine nervous system: physiological roles
and cross-talk with other receptor systems


DIPL.-BIOL. UNIV. HEIKE HERMANN




Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung
des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation.




Vorsitzender: Univ.-Prof. Dr.rer.nat., Dr.rer.nat.habil. Jürgen Polster

Prüfer der Dissertation:
1. apl.Prof. Dr.agr., Dr.agr.habil. Oswald Rottmann
2. Univ.-Prof. Angelika Schnieke, Ph.D.
3. Priv.-Doz. Dr.sc.nat. Beat Lutz, Ludwig-Maximilians-Universität München





Die Dissertation wurde am 18.12.2003 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt am 09.04.2004 angenommen. I
Table of contents
1 INTRODUCTION 1
1.1 Overview of the cannabinoid receptor type 1 (CB1) 1
1.1.1 CB1 ligands (cannabinoids) 1
1.1.2 CB1-mediated signal transduction pathways 2
1.1.2.1 Regulation of adenylate cyclase 3
1.1.2.2 Modulation of ion channels 4
1.1.2.3 Regulation of intracellular calcium transients 5
1.1.2.4 Regulation of several kinases 6
1.1.3 The endocannabinoid system 7
1.1.4 Distribution of CB1 in the murine brain 9
1.1.5 Physiological functions and therapeutical implications of the cannabinoid
system 10
1.1.5.1 Neuroprotection 10
1.1.5.2 Nociception 12
1.1.5.3 Locomotion 12
1.1.5.4 Learning and memory 13
1.2 The cannabinoid system and cross-talk with other receptor systems 15
1.2.1 Interaction with the dopamine system 16
1.2.2 Interaction with the serotonin system 17
1.2.3 Interaction with the vanilloid system 18
1.2.4 Interaction with the CRH system 19
1.3 Aim of the thesis 20
2 MATERIAL AND METHODS 22
2.1 Drugs 22
2.2 Animals 22
2.3 In situ hybridization 22
2.3.1 Tissue preparation 22
2.3.2 Probe synthesis 23
2.3.3 Single-in situ hybridization 26
2.3.4 Double-in situ26
2.3.5 Numerical and densitometric evaluation of expression 27
2.4 Immunhistochemistry 28
2.4.1 Tissue preparation 28
2.4.2 Immunostaining
2.5 Cell culture 29
2.5.1 Cell lines 29
2.5.2 Primary cerebellar granule neurons
2.6 Establishment of CB1-expressing HT22 cells 30
2.6.1 Electroporation and selection 30 II
2.6.2 Northern blot analysis 30
2.6.3 cAMP accumulation assay 31
2.7 Establishment of CB1-VR1 expressing HEK-293 cells 31
2.7.1 Electroporation and selection 31
2.7.2 Northern blot analysis 32
2.7.3 Western blot analysis 32
2.7.4 cAMP accumulation assay 32
2.8 Intracellular calcium assay in CB1-VR1-expressing HEK-293 cells 33
2.9 Experiments in primary cerebellar granule neurons 34
2.9.1 34
2.9.2 Semi-quantitative RT-PCR 34
2.9.3 Enzyme-linked immunosorbent assay (ELISA) 35
2.10 Statistical analysis 35
3 RESULTS 36
3.1 CB1 and cross-talk with other receptor systems 36
3.1.1 Coexpression of CB1 with dopamine and serotonin receptors in the adult
mouse forebrain 36
3.1.1.1 CB1 and dopamine receptor D1 36
3.1.1.2 ine receptor D2 38
3.1.1.3 CB1 and serotonin receptor 5-HT1B 40
3.1.1.4 tor 5-HT3 41
3.1.2 Expression analysis of different marker genes in CB1-deficient mice 44
3.1.3 Expression of VR1 in the adult mouse forebrain 48
3.1.4 VR1-induced increase in intracellular calcium is differentially regulated
by CB1 activation 51
3.1.4.1 Double-transfected HEK-293 cells express functional CB1 receptors 51
3.1.4.2 Effect of HU210 on capsaicin response in CB1-VR1-HEK cells 53
3.1.4.3 Effect of various inhibitors on HU210 potentiation of capsaicin response 54
3.1.4.4 Effect of anandamide on CB1-VR1-HEK and VR1-HEK cells 55
3.1.4.5 Effect of HU210 on forskolin-induced potentiation of the capsaicin
response in CB1-VR1-HEK cells 56
3.1.5 Cross-talk of CB1 and CRHR1 receptors regulates BDNF expression 57
3.1.5.1 Coexpression of CB1 and CRHR1 in the adult mouse brain 58
3.1.5.2 Inhibition of CRH-mediated signaling by the CB1 agonist WIN55,212-2 61
3.1.5.3 Inhibition of CRH-mediated increases in BDNF expression by the
CB1 agonist WIN55,212-2 62
3.2 Neuroprotective and anti-inflammatory properties of the cannabinoid
system 65
3.2.1 Cross-talk of CB1 with the glutamatergic system protects from kainic acid-
induced excitotoxicity in vitro and in vivo 65
3.2.1.1 CB1 expression is restricted to GABAergic interneurons in CB1
conditional knock-out mice 66
3.2.1.2 CB1 in glutamatergic neurons activates a protective signaling cascade
against kainic acid-induced excitotoxicity 68 III
3.2.1.3 CB1-dependent expression of BDNF protects against kainic acid-induced
excitotoxicity in organotypic hippocampal slice cultures 71
3.2.2 CB1 receptors in transfected HT22 cells are not involved in the
neuroprotective action of cannabinoids against oxidative stress 73
3.2.3 The endogenous cannabinoid system protects against colonic inflammation 77
3.2.3.1 CB1 mRNA is upregulated in the colon after DNBS-induced inflammation 78
3.2.3.2 Preproenkephalin mRNA is upregulated in the colon after DNBS-induced
inflammation 79
3.2.3.3 Number of neurons is unchanged between DNBS-treated and untreated
colons 80
4 DISCUSSION 82
4.1 Functional cross-talk of CB1 with other receptor systems 82
4.1.1 High coexpression levels of CB1 with dopamine and serotonin receptors
indicate functional interactions of the cannabinoid system with these
neurotransmitter systems 82
4.1.2 Expression levels of several marker genes are not affected in CB1-deficient
mice 85
4.1.3 CB1 regulates VR1 activity through modulation of multiple signaling
pathways 87
4.1.4 CB1 regulates BDNF expression via dampening of CRH-mediated signaling 91
4.2 The cannabinoid system protects against neurotoxic insults and
inflammation 95
4.2.1 CB1 in principal forebrain neurons activates a protective intracellular
cascade after kainic acid-induced excitotoxicity 95
4.2.2 Cannabinoids exert non-CB1-mediated antioxidant, neuroprotective effects 98
4.2.3 CB1 expression is important during the acute phase of inflammation 100
5 SUMMARY 102
6 ACKNOWLEDGEMENTS 104
7 LIST OF REFERENCES 105
8 APPENDIX 130
8.1 Abbreviations 130
8.2 Published articles of the thesis and data in preparation for publication 133 1 INTRODUCTION 1
1 INTRODUCTION
1.1 Overview of the cannabinoid receptor type 1 (CB1)
1.1.1 CB1 ligands (cannabinoids)
The plant Cannabis sativa (C. sativa), also known as Marihuana, is considered as one of the
very first plants grown for therapeutic and recreative purposes (reviewed in Peters and Nahas,
1999). First historical reports of the use of C. sativa were found in China nearly 5000 years
ago, where it was grown rather for fibers than for production of psychoactive extracts. From
China, C. sativa propagated to all continents over the ages and became more and more
important for medical applications besides its usage as pleasure-inducing drug. C. sativa
contains more than 60 compounds belonging to the chemical family of cannabinoids (Iversen,
9 92000), although the major psychoactive constituent is ∆ -tetrahydrocannabinol ( ∆ -THC)
8(Gaoni and Mechoulam, 1964). Other compounds found in C. sativa include ∆ -
9tetrahydrocannabinol, cannabidiol and cannabinol. Following the isolation of ∆ -THC from C.
9sativa, numerous synthetic cannabinoids, based on the structure of ∆ -THC, were synthesized.
These were shown to induce behavioral effects such as hypothermia, catalepsy and
9hypomobility, similar to the in vivo effects of ∆ -THC, when injected into animals (reviewed
in Howlett et al., 2002). Upon the identification and cloning of a specific cannabinoid receptor
9in the brain (CB1) that mediated the effects of ∆ -THC (Devane et al., 1988; Matsuda et al.,
1990), an endogenous agonist of this receptor, anandamide (AEA; Devane et al., 1992), was
identified. This suggested the presence of an endogenous cannabinoid system in the central
nervous system (CNS). Later, other endocannabinoids have also been isolated and shown to
be present in the CNS. A second cannabinoid receptor (CB2) was cloned from a leukaemic
cell line and has a relatively low sequence identity with CB1 (44% overall the whole protein,
68% in the transmembrane regions; Munro et al., 1993). Its expression is limited to cells and
organs of the immune system suggesting that the endocannabinoid system may also play a
role in modulating the immune system.
Cannabinoid receptor agonists can be classified into four groups: eicosanoid
cannabinoids, classical cannabinoids, nonclassical cannabinoids and aminoalkylindoles.
9Classical cannabinoids include compounds isolated fr