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Keywords:

  • CB1;
  • endocannabinoid;
  • neuronal differentiation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The role of the CB1 cannabinoid receptor and endocannabinoid signalling has been widely studied in the adult nervous system. However, an emerging body of evidence suggests that the CB1 receptor may also play a role during development. Here we have scrutinized the expression profile of the CB1 receptor from the onset of neurogenesis in the chick embryo. We find that this gene exhibits a dynamic expression pattern that spatially and temporally follows neuronal differentiation in the early embryo.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The principal psychoactive component of Cannabis sativa exerts its effect in the brain by binding to a G-protein coupled receptor known as the CB1 cannabinoid receptor (Matsuda et al. 1990; Pertwee, 1997). The identification of this receptor was of great importance as it indicated the presence of an endocannabinoid signalling system within brain. Consequently, numerous anatomical and physiological studies have shown that endocannabinoids function as retrograde messengers acting on widely distributed presynaptic CB1 receptors to suppress neurotransmitter release at both excitatory and inhibitory synapses (Kreitzer & Regehr, 2001; Ohno-Shosaku et al. 2001; Wilson & Nicoll, 2001). Besides clearly playing an important role in the adult brain, there is also an accumulating body of evidence suggesting that endocannabinoid signalling is important for the development of the nervous system. Expression studies have demonstrated that the CB1 receptor is expressed from midgestation (E11) in the rat embryo (Buckley et al. 1998; Fernandez-Ruiz et al. 2000). Furthermore, it has recently been shown that CB1 receptor agonists can promote axonal growth in cultured postnatal cerebellar neurons (Williams et al. 2003).

Although there are indications that the CB1 receptor plays a significant role in the development of the nervous system, the nature of that role is far from clear. Thus, although it is known that CB1 expression is evident halfway through development, it is not as yet apparent where and when CB1 expression initiates in the developing nervous system. To address this issue we have scrutinized the expression of CB1 from the onset of neurogenesis in the chick embryo. We find that CB1 expression follows, spatially and temporally, neuronal differentiation at the stages analysed. Additionally, we also find that this gene is expressed in a region of the ventral forebrain as well as in the presomitic mesoderm.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In situ hybridization

Whole-mount in situ hybridization was carried out according to Mackenzie et al. (1998), using the chick CB1 probe.

Whole-mount immunohistochemistry

Following overnight fixation at 4 °C in 4% paraformaldehyde/PBS, embryos were incubated with anti-Hu antibody (1 : 300) and anti-NFM antibody (1 : 10 000) diluted in 2% milk/PBS/1% Triton for 4 days at 4 °C. After several washes with 2% milk/PBS/1% Triton, embryos were incubated overnight at 4 °C with HRP-conjugated anti-mouse secondary antibodies (DAKO) diluted (1 : 200) in 2% milk/PBS/1% Triton. Embryos were washed with PBS. The embryos were pre-incubated in a solution of inactive diaminobenzidine (DAB; 0.5 g L−1 in 0.1 m Tris-HCl, pH 7.4), for 3 h at 4 °C in the dark. The bound antibody was visualized using the same solution containing 0.03% hydrogen peroxide and the reaction was stopped with PBS once the colour had developed to the desired extent.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

To examine the role of the CB1 receptor during nervous system development, we have analysed its expression from the earliest stages of neuronal differentiation, stage 10 in the chick. At this stage, and more obviously one stage later, we find that CB1 is expressed within the developing hindbrain, restricted to a number of cells within rhombomere 4 and fewer cells within rhombomere 6 (Fig. 1A,B). In section, it can be seen that these CB1-positive cells are located midway along the dorso-ventral axis of the hindbrain (Fig. 1B). These cells are the projection interneurons of the hindbrain, which, as with other neuronal populations within the developing hindbrain, exhibit a segmental pattern of differentiation: differentiating first within rhombomere 4 and then in rhombomeres 6 and 2 (Eickholt et al. 2001). This differentiation pattern is mirrored by the expression of CB1. At stage 11, CB1 expression can also be detected in a group of cells that lie lateral to the developing brain and extend rostroventrally from the level of the midbrain, the neurons of the ophthalmic trigeminal placode (Fig. 1A) (Begbie et al. 2002). Sectioning of later embryos further reveals that this gene is expressed both by cells lying within the ectoderm of the placode and those that have moved internally to contribute to the forming trigeminal ganglion (Fig. 1F). As development proceeds, CB1 expression becomes evident in other neuronal populations. Thus, following the ophthalmic trigeminal placode, expression initiates in the cells of the vestibuloacoustic ganglion, and then those of the maxillomandibular portion of the trigeminal ganglion (Fig. 1C) (Begbie et al. 2002). Subsequently, CB1 expression becomes evident in the cells of the epibranchial ganglia: the geniculate, petrosal and nodose (Fig. 1C,H) (Begbie et al. 2002). Interestingly, although those neuronal populations such as the ophthalmic trigeminal neurons derive from placodes, this gene is not expressed in the ectoderm of these placodes but only in the cells that have reached the site of ganglion formation. The fact that CB1 is expressed in the ectoderm of the ophthalmic placode but not the others is significant as the ophthalmic placode is the only placode that directly generates post-mitotic neurons (Begbie et al. 2002). All the other placodes generate mitotically active neuronal cells, which then migrate internally and only become post-mitotic once they have reached the site of ganglion formation (Begbie et al. 2002).

image

Figure 1. Expression profile of CB1 in the early chick embryo. (A) Dorsal view of a stage 11 embryo showing CB1 expression in the differentiating interneurons of rhombomeres 4 (r4) and 6 (r6) of the hindbrain. Expression can also be seen in the cells of the ophthalmic trigeminal placode, marked by an arrowhead. Staining out of the plane of focus at the anterior of the embryo is in the ventral forebrain. (B) Transverse section through rhombomere 4 of a stage 11 embryo, showing the dorsoventral position of the projection interneurons that express CB1. (C) Side view of a stage 18 embryo showing expression of CB1 in the trigeminal and vestibuloacoustic ganglia, as well as in the epibranchial ganglia: the geniculate, petrosal and nodose, and also in the cells of the mesencephalic trigeminal nucleus (MTN). (D) Side view of a whole stage 21 embryo showing CB1 expression in the dorsal root ganglia (DRG). (E) Section through the developing eye of a stage 18 embryo showing CB1 expression in the differentiating retinal ganglion cells (RGC). Some expression is also seen in a portion of the ventral forebrain. (F) Transverse section through the anterior hindbrain and ophthalmic lobe of the trigeminal ganglion (T) of a stage 18 embryo. The ophthalmic trigeminal placode is indicated by an arrowhead. (G) Section through the midbrain of a stage 18 embryo showing expression of CB1 in the cells of the MTN, which lie either side of the dorsal midline. (H) Transverse section showing expression of CB1 in the forming vestibuloacoustic (VA) and geniculate (G) ganglia, and additionally in the motor neurons of the hindbrain, highlighted by an asterisk. (I) Longitudinal section through the trunk of a stage 22 embryo showing expression in the forming DRG. (J) Dorsal view of a stage 10 embryo showing CB1 expression in the ventral forebrain. (K) Dorsal view of the posterior region of a stage 10 embryo showing CB1 expression in the presomitic mesoderm (psm). The last formed somite is indicated (s).

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Strong expression of CB1 is also seen in the only centrally located sensory neurons of amniotes, the cells of the mesencephalic trigeminal nucleus (MTN) (Fig. 1C,G). These cells develop close to the dorsal midline of the mesencephalon extending from the isthmus to the midbrain–forebrain boundary (Hunter et al. 2001). In the trunk, CB1 expression is also clearly evident in the cells of the forming dorsal root ganglia (DRG) (Fig. 1D, I). With time, the expression of this gene becomes evident in other central neuronal populations, such as the retinal ganglion cells of the eye (Fig. 1E) and the motor neurons of the hindbrain (Fig. 1H). It is also apparent that CB1 expression can be lost from neuronal populations over time. Thus whereas this gene was expressed in the projection interneurons of the hindbrain at early stages (Fig. 1A,B) it is no longer expressed in these cells at later stages (Fig. 1H).

During the stages of development described here, there are two additional sites of expression in the embryo where CB1 is not expressed in individual neurons. The first is a broad region of the ventral forebrain (Fig. 1J), which at this stage is not producing neurons. The second site of expression is the presomitic mesoderm (Fig. 1K). The expression of the CB1 receptor in this tissue is particularly interesting as it demonstrates that although this gene is thought to be exclusively expressed in the nervous system, this is not true in the early embryo.

The expression profile of CB1 in the developing nervous system is particularly interesting as it seems to correlate with neuronal differentiation. The fact that this gene is expressed in the ectoderm of the ophthalmic placode but not the other placodes would support this view. However, to look at this more closely we have compared the onset of CB1 expression in neurons of the cranial sensory ganglia with that of Hu immunoreactivity, which marks peripheral neurons immediately before neuronal birthdays (Marusich et al. 1994). We have focused on these neuronal cells because they display a reproducible sequence of maturation. The cells of the ophthalmic trigeminal placode differentiate first, followed by those of the vestibuloacoustic ganglion, then the cells of the maxillomandibular ganglion and finally those of the epibranchial ganglia: the geniculate, petrosal and nodose (Begbie et al. 2002). Interestingly, we find that in every case CB1 expression in these cells commences after Hu immunoreactivity. Thus although the cells of the maxillomandibular portion of the trigeminal ganglion are Hu immunopositive by stage 15 of development (Fig. 2B), these cells do not express CB1 until stage 16 (Fig. 2C). Similarly, although the epibranchial ganglia are highlighted by Hu staining at stage 16 (Fig. 2D), these neurons do not express CB1 until stage 17 (Fig. 2E). These results demonstrate that CB1 expression follows neuronal differentiation.

image

Figure 2. CB1 is expressed after neuronal birthdays. (A) Side view of a stage 15 embryo showing CB1 expression in the ophthalmic portion of the trigeminal ganglion (Top), as well as expression initiating in the vestibuloacoustic (VA) ganglion. (B) Side view of a stage 15 embryo showing Hu immunoreactivity in the ophthalmic and maxillomandibular (Tmm) portions of the trigeminal and additionally in the vestibuloacoustic ganglion. (C) Side view of a stage 16 chick embryo revealing CB1 expression in both the ophthalmic and the maxillomandibular portions of the trigeminal ganglion as well as in the vestibuloacoustic ganglion. (D) Side view of a stage 16 embryo showing Hu immunopositivity in the cells of the trigeminal ganglion, in the vestibuloacoustic ganglion and now additionally in the epibranchial ganglia: the geniculate (g), the petrosal (p) and the nodose (n). (E) Side view of a stage 18 embryo showing expression of CB1 in the trigeminal and vestibuloacoustic ganglia, as well as in the epibranchial ganglia: the geniculate, petrosal and nodose, and also in the cells of the mesencephalic trigeminal nucleus (MTN). (F) Side view of a stage 18 embryo showing Hu immunopositivity in the cells of the trigeminal ganglion, in the vestibuloacoustic ganglion and the epibranchial ganglia: the geniculate, the petrosal and the nodose. Scale bars = 200 µm.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In this study we have demonstrated that the expression of the endocannabinoid receptor, CB1, initiates in the first born neurons of the central nervous system, the projection interneurons of the hindbrain, and subsequently in the earliest born neurons of the peripheral nervous system, the cells of the ophthalmic trigeminal placode. As development proceeds, CB1 expression also becomes apparent in other differentiating neuronal populations. We have also closely scrutinized the timing of onset of CB1 expression, and we find that this occurs after neuronal differentiation in the early embryo.

Interestingly, it has also been demonstrated that CB1 receptor agonists can promote axonal growth in cultured postnatal cerebellar neurons. Furthermore, an inhibitor of the lipase that is responsible for the synthesis of 2-arachidonyl-glycerol, which for some authors is the true endogenous ligand for the CB1 receptor (Sugiura et al. 1999; Schmid et al. 2002), stalls axonal growth in the developing Xenopus retinal nerve (Lom et al. 1998) and perturbs axonal guidance in the developing mouse retina (Brittis et al. 1996). Collectively, these studies, together with ours, demonstrating that CB1 expression follows neuronal differentiation, suggest that it is likely that the CB1 receptor plays an important role in directing axonal outgrowth in newly born neurons.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was funded by the Medical Research Council (UK). We would like to thank David Wilkinson for the CB1 probe.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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