The endocannabinoid receptor, CB1, is required for normal axonal growth and fasciculation

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Abstract

Endocannabinoids are retrograde neurotransmitters, which act upon the presynaptically located, G-protein coupled receptor CB1, to modulate synaptic transmission in the adult brain. Recently, however, a number of lines of evidence have suggested that endocannabinoid signalling may play an important role in early neuronal development. In this study, we show that the CB1 receptor has a wide expression pattern in the developing nervous system and that its expression follows neuronal differentiation in the embryo from the earliest stages. We also show that the enzymes involved in 2-AG synthesis are expressed in an overlapping manner at these stages. We further show that interfering with CB1 function using a pharmacological inhibitor causes problems in axon pathfinding and fasciculation. Similarly, CB1 gene knock down in the zebrafish by morpholino injection results in defects in axonal growth and fasciculation in these embryos. Thus CB1 function is required in the early embryo for axonal growth and fasciculation.

Introduction

The cannabinoid receptor CB1 is one of the most abundant G-protein coupled receptors in the adult nervous system (Herkenham et al., 1991, Matsuda et al., 1993, Tsou et al., 1998). It not only mediates the psychoactive effects of marijuana, but also plays a key role in the endocannabinoid signalling system. The recent cloning of the sn-1 specific diacylglycerol lipases (DAGLα and DAGLβ) that make 2-arachidonoylglycerol (2-AG), the most abundant endogenous ligand for the CB1 receptor, has provided evidence that this second messenger can be synthesised at CB1 positive synapses throughout the adult brain (Bisogno et al., 2003, Lafourcade et al., 2007, Mátyás et al., 2008, Uchigashima et al., 2007, Yoshida et al., 2006). In this context, the postsynaptic localisation of the enzymes allows 2-AG to serve as a retrograde synaptic messenger that following release, can activate the presynaptically located CB1 receptors. (Harkany et al., 2007, Wilson and Nicoll, 2002). This results in the suppression of neurotransmitter release from both inhibitory and excitatory synapses. Thus endocannabinoid signalling exerts a potent influence on synaptic transmission and enables the endocannabinoids to modulate behaviours such as fear, anxiety and feeding.

Over the last few years, however, there has been a growing body of evidence that points towards endocannabinoid signalling also playing key roles in the development of the nervous system. We, and others, have shown that the CB1 receptor is expressed by neurons during development (Begbie et al., 2004, Buckley et al., 1998). Furthermore, we have also shown in culture that a number of cell adhesion molecules stimulate axonal growth by activating an FGF receptor signalling cascade and that this involves a DAGL dependent activation of neuronal CB1 receptors; importantly, CB1 agonists will stimulate the growth of axons, while CB1 antagonists inhibit this process (Williams et al., 2003). Other studies, in the developing cortex, have suggested that endocannabinoid signalling can exert both chemoattractive and chemorepulsive effects upon cortical interneurons (Berghuis et al., 2005, Berghuis et al., 2007). Finally, endocannabinoids have also been suggested to play roles in stimulating the proliferation of neural progenitor cells as well as being involved in the differentiation of these cells towards both glial and neuronal fates (Aguado et al., 2005Aguado et al., 2006, Jiang et al., 2005, Jin et al., 2004).

While, collectively, these studies point towards endocannabinoid signalling playing multiple roles during the development of the nervous system, they have dealt with what are relatively late stages of neuronal development. In an attempt to gain an insight into some of the earliest roles of endocannabinoid signalling during neuronal development, we previously analysed the expression of CB1 in the early chick embryo (Begbie et al., 2004). We found that CB1 was expressed in the earliest born neurons of the central and peripheral nervous systems; the reticulospinal neurons of the hindbrain and the neurons contributing to the ophthalmic portion of the trigeminal ganglion. We also found that, as embryogenesis progressed, CB1 expression became established in other neuronal groups. Finally, we noted that, at least with respect to the development of the placodally-derived neurons of the cranial sensory ganglia, CB1 was only expressed in post-mitotic neurons (Begbie et al., 2004).

In this present study, we have extended our analysis of the CB1 receptor and other components of the endocannabinoid signalling system in the embryonic nervous system. We find that, as embryogenesis proceeds, many neuronal populations in the chick express CB1, and that the expression of this receptor follows neuronal birthdays. During development, the levels of 2-AG greatly exceed those of other putative endocannabinoids (e.g. anandamide), suggesting that 2-AG is more relevant for developmental processes (Fernandez-Ruiz et al., 2000). We have therefore also analysed the expression patterns of the DAGLs, which synthesise 2-AG, and of monoglyceride lipase, MGL, which catabolises 2-AG, in the chick embryo at early stages of nervous system development (Bisogno et al., 2003, Dinh et al., 2002). We find that DAGLα and DAGLβ are expressed widely in the developing nervous system from early stages, while MGL is only expressed at high levels in neural tissue at later stages. We have also analysed the requirement for CB1 receptor function during early neuronal development. We find that in two model systems interfering with CB1 function results in abnormal axonal growth most obviously characterised by a failure in fasciculation. Thus, we conclude that endocannabinoid signalling is required in early neuronal development for the establishment of axonal tracts.

Section snippets

CB1 expression follows neuronal differentiation

Here we have extended our analysis of CB1 in the developing nervous system to gain further insights into the dynamics of expression of this gene. An analysis of whole mount in situ hybridisations clearly shows a temporal maturation of CB1 expression in the peripheral nervous system (PNS) from rostral to caudal. Thus at stage 19, CB1 is expressed in the trigeminal and vestibuloaccoustic ganglia (Figs. 1A, B). However, by stage 20, CB1 expression can be seen to be maintained in these ganglia and

Discussion

There are a number of important conclusions that can be drawn from our study. Firstly, we present further evidence that, in the early embryo, CB1 expression in neurons in both the CNS and PNS follows terminal mitosis. We find no evidence of CB1 expression in the neuronal progenitor cells. We also demonstrate that the key synthetic enzymes for the generation of 2-AG, DAGLα and DAGLβ, are expressed at the same time and in the vicinity of CB1 expressing cells, which would be consistent with this

Whole mount in situ hybridisation and immunohistochemistry

Fertilised hen eggs were incubated in a humidified chamber at 37 °C and staged according to Hamburger and Hamilton (1992). Whole mount in situ hybridisation was performed according to Henrique et al. (1995). Immunostaining was done using standard whole mount immunostaining protocol (Mackenzie et al., 1998). Primary antibodies used were: mouse anti-neurofilament medium chain (RMO-270) at 1:10,000 (Zymed); and secondary antibody anti-mouse Alexa 488, used at 1:1000 (Molecular Probes). For

Acknowledgements

We are particularly indebted to Corinne Houart for her invaluable assistance with the zebrafish work. This work was funded by the MRC (UK).

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