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Expression of the CB1 and CB2 receptor messenger RNAs during embryonic development in the rat
Pergamon
PII:
Neuroscience Vol. 82, No. 4, pp. 1131–1149, 1998 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00348-5
EXPRESSION OF THE CB1 AND CB2 RECEPTOR MESSENGER RNAS DURING EMBRYONIC DEVELOPMENT IN THE RAT N. E. BUCKLEY,*‡ S. HANSSON,† G. HARTA* and E u . MEZEY* *National Institute of Neurological Diseases and Stroke, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, U.S.A. †National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, U.S.A. Abstract––We mapped the distribution of CB1 and CB2 receptor messenger RNAs in the developing rat to gain insight into how cannabinoids may affect embryogenesis. In situ hybridization histochemistry studies were done using riboprobes specific for CB1 or CB2 receptor messenger RNAs. We found that CB1 and CB2 receptor messenger RNAs are expressed in the placental cone and in the smooth muscle of the maternal uterus at the earliest gestational periods studied [from eight days of gestation (E8) through E12]. In the embryo, as early as E11, CB1 receptor messenger RNA is expressed in some cells of the neural tube and, at later embryological stages (from E15 to E21), in several distinct structures within the central nervous system. In addition, high levels of CB1 receptor messenger RNA were also found in areas of the peripheral nervous system such as the sympathetic and parasympathetic ganglia, in the retina and in the enteric ganglia of the gastrointestinal tract. In addition to neural structures, high levels of the CB1 receptor messenger RNA were also present in two endocrine organs, the thyroid gland and the adrenal gland. On the other hand, CB2 receptor messenger RNA is expressed exclusively in the liver of the embryo as early as E13. The region-specific expression of CB1 and CB2 receptor messenger RNAs suggests that these receptors have a functional role during embryogenesis. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: cannabinoid receptors, embryogenesis, uterus, brain, liver.
Marijuana is one of the recreational drugs most commonly used during pregnancy in Western countries.9,11,42,53 It is known that cannabinoids can cross the placenta, but their effects on fetal and postnatal development are unclear. In humans, a longitudinal study has been carried out by Fried15 describing the effects of cannabinoids on children from mothers who used marijuana during pregnancy. He found that newborns exhibited an increase in tremors, prolonged startles and poor habituation to visual stimuli. These marijuana-induced effects were not observed again until the age of four, where children prenatally exposed to marijuana showed increased behavioral problems and decreased performance on visual perceptual tasks, language comprehension, sustained attention and memory.15 Furthermore, other investigators reported that, on average, children exposed prenatally to marijuana tend to have a lower IQ compared to unexposed children.10 In another study, however, it was ‡To whom correspondence should be addressed. Abbreviations: E, embryonic (gestational) day; EDTA, ethylenediaminetetra-acetate; ISHH, in situ hybridization histochemistry; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RT, reverse transcriptase; TH, tyrosine hydroxylase; THC, delta-9-tetrahydrocannabinol.
reported that prenatal exposure to marijuana did not affect gross motor development in preschool children.7 However, reliable comparisons between children prenatally exposed to marijuana and those who were not are very hard to make due to the great number of socioeconomic variables between individuals. In a recent review, Navarro et al.31 discussed data on the behavioral effects of rats exposed to cannabinoids during gestational and lactational periods. In adulthood, these animals displayed altered sexual and motor behaviors, as well as alteration in responses to social interactions and to novelty (for a review see Ref. 31). The same review also describes several neurobiological mechanisms which are affected by perinatal exposure to cannabinoids, such as the catecholaminergic and serotoninergic systems of developing offspring, the pituitary hormones, the sex steroids and the glucocorticoids. More recently, it was reported that in adult mice, which had been prenatally exposed to the endogenous cannabinoid anandamide, there was increased catalepsy, hypothermia and analgesia.14 In addition, these mice had an increased inflammatory response to arachidonic acid and lipopolysaccharide.14 To date, only two cannabinoid receptors are known. The CB1 receptor was first cloned from adult
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rat brain.25 The CB1 receptor mRNA is mainly expressed in the central nervous system26 and, to a much lesser extent, in the periphery: adrenal gland, heart, lung, prostate, uterus, ovary, testis, bone marrow, thymus and tonsils as determined by reverse transcriptase–polymerase chain reaction (RT– PCR),16 and in testis as also determined by northern blotting.18 The CB2 receptor mRNA is present in cells of the immune system.16,30 Expression of the two cannabinoid receptor mRNAs has not been studied throughout embryonic development, but recently it was reported that anandamide is in high levels in the uterus and that these levels are altered during implantation.46 In addition, it has been shown that preimplantation mouse embryos from the two-cell stage to the blastocyst stage express CB1 and CB2 receptors.33 In the present work, we used in situ hybridization histochemistry (ISHH) to demonstrate the expression of the CB1 and CB2 receptor mRNAs in rat embryonic tissues from gestational day 8 (E8) through gestational day 21 (E21). We also discuss the possible functional roles of these receptors during embryonic development. EXPERIMENTAL PROCEDURES
Preparation of rat embryo tissue Timed-pregnant Sprague–Dawley rats purchased from Taconic Farms (Germantown, NY, U.S.A.) were killed by CO2 inhalation. The uteri were dissected on ice. The day of the vaginal plug was designated as E1. Embryos plus uteri (E8–E13) or embryos alone (E13, E15, E17, E18, E19, E20 and E21) were frozen on dry ice and stored at "80)C until sectioning. Serial 12-µm-thick sections in sagittal and transversal planes were collected on silanized slides, briefly dried at 37)C and stored at "80)C until processed for ISHH. In situ hybridization histochemistry Probes. For the ISHH studies, two sets of probes were prepared complementary to two different non-overlapping regions of the rat CB1 receptor mRNA. The first CB1 riboprobe was synthesized from a cDNA template kindly provided by Dr Stephen Lolait (NIMH, Bethesda, MD, U.S.A.). A fragment (from bp 932 to 1226 from the reported CB1 receptor gene sequence25) was amplified by PCR. The following primers were used: CB1/1 (5*-CGC GCA ATT AAC CCT CAC TAA AGG GCT GCA ATC TGT TTG CTC GGA C-3*) and CB1/2 (5*-G CGC GTA ATA CGA CTC ACT ATA GGG GCC CCA GCA GAT GAT CAA CAC-3*). The second CB1 receptor riboprobe was synthesized from a cDNA template kindly provided by Dr Tom Bonner (NIMH). This fragment corresponds to bp 165–464 of the CB1 receptor gene sequence.25 The primers used for the amplification of this fragment were the following: CB1a (5*-CGC GCA ATT AAC CCT CAC TAA AGG CTA GAT GGC CTT GCA GAC ACC A-3*) and CB1b (5*-G CGC GTA ATA CGA CTC ACT ATA GGG CAT AAA GTT CTC CCC ACA CTG G-3*). The cDNA template for the rat CB2 riboprobe was kindly provided by Dr Sean Munro (MRC Laboratory of Molecular Biology, Hills Road, Cambridge, U.K.). The rat CB2 cDNA was a 595-bp fragment corresponding to bp 826– 1421 in the mouse CB2 gene sequence (Accession number U21681 in the Genebank). This cDNA was used as a template to transcribe a 595-bp CB2 riboprobe. In addition,
a PCR product corresponding to bp 916–1269 from the mouse CB2 gene sequence was obtained from this cDNA using the following primers: CB2a (5*-CGC GCA ATT AAC CCT CAC TAA AGG TGC TGT TGA CCG ATA CCT AT-3*) and CB2b (5*-G CGC GTA ATA CGA CTC ACT ATA GGG GCC AAC CTC ACG TCT AGC CG-3*). All primers contain specific sequences complementary to the cannabinoid receptor in addition to sequences corresponding to the T7 (bold) and T3 (italic) polymerase binding sites. The PCR amplification was done at 95)C for 1 min, 60)C for 1 min, 72)C for 2 min, and was carried out for 30 cycles using Taq Polymerase (Boehringer Mannheim, Indianapolis, IN, U.S.A.). The fragments were extended by incubating at 72)C for 10 min, after which they were stored at 4)C. The PCR fragments were purified and transcribed using [35áS]UTP (NEN, Boston, MA, U.S.A.) and the T7/T3 MAXIScript kit (AMBION, Austin, TX, U.S.A.). T7 polymerase was used to label the antisense probe and T3 polymerase to label the sense control probe. Hybridization. The slides were processed for ISHH as described previously.5 Briefly, the slides were treated with acetic anhydride, dehydrated, delipidated and incubated overnight at 55)C with the hybridization buffer [20 mM Tris, pH 7.4, 1 mM EDTA, 300 mM NaCl, 50% formamide, 10% dextran sulfate, 1#Denhardt’s, 100 µg/ml salmon sperm DNA, 250 µg/ml yeast total RNA, 250 µg/ml yeast tRNA, 100 mM dithiothreitol, 0.1% sodium dodecyl sulfate, 0.1% sodium thiosulfate] containing the probes (2#106 c.p.m./50 µl/slide) described above. The following day, the slides were washed to remove unbound probe and allowed to dry. The slides were exposed to Kodak Bio-Max MR (Kodak, Rochester, NY, U.S.A.) X-ray film for three days and then coated with undiluted NTB3 nuclear track emulsion (Kodak). The slides were developed three weeks later, counterstained with 1% Giemsa stain and viewed under a Leitz Dialux 20 microscope. Immunohistochemistry Affinity purified antibodies to rat CB1 receptor were kindly provided by Dr Ken Mackie (University of Washington, Seattle, WA, U.S.A.). The development of the antibody was supported by grant no. DA 08934 and has been described previously.48,49 Fresh frozen sections of E19 embryos were fixed by immersion into 4% formaldehyde in phosphate-buffered saline (PBS; pH 7.4) for 10 min at room temperature. The sections were rinsed several times and then blocked for 30 min in PBS containing 5% normal goat serum and 0.6% Triton X-100 (blocking solution). Following several rinses, the CB1 receptor antibody was applied to the sections at 1:500 or 1:5000 dilutions for fluorescence and 1:1000 dilution for the avidin–biotin–peroxidase complex method.20 The dilutions were made in the blocking solution. Prior to incubation with the primary antibody, the sections that were processed for the avidin–biotin–peroxidase complex method were treated with 1% H2O2 in methanol to block endogenous peroxidase activity. The sections were incubated with the antibody overnight at 4)C. The sections were then washed and fluorochrome-conjugated antibody was applied. The 1:5000 dilution was developed using the tyramide amplification procedure.21 The sections were then rinsed in PBS and briefly in distilled water, then air dried, mounted with cytoseal and coverslipped. Controls included immunostaining with the working dilution of the antibody that has been preabsorbed with 10 µg/ml of the antigen, no primary antibody or normal non-immune rabbit immunoglobulin G replacing the specific antibodies in the procedure. For more details of the techniques used see the ‘‘protocols’’ section in the following web site: http:// www.nimh.nih.gov/2snge/.
Fig. 1. CB1 receptor mRNA is expressed in the placenta, in the maternal uterus and in the embryo. The embryo was hybridized with the antisense probe for CB1 receptor mRNA and then stained with Giemsa stain as indicated under Experimental Procedures. Panels A, C, E and G show the bright field, panels B, D, F and H the dark field. Panels A–D correspond to an E11 embryo, panels E and F to an E12 embryo and panels G and H to an E13 embryo. The E11 embryo, boxed in panels A and B, is shown in more detail in panels C and D. The arrowheads in panels A and B show heavily labeled cells within the intervillous space of the placenta. The arrows in panels C–H indicate the neural tube, spinal and sympathetic ganglia. The asterisk labels the lumen of the telencephalic region. Scale bars=400 µm (A, B), 100 µm (C–F), 200 µm (G, H).
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Fig. 2. CB2 receptor mRNA is expressed in the placenta and maternal uterus. Hybridization to CB2 receptor mRNA-specific probes is shown in the placenta at gestational stage E8 (A, B), and in the placenta and maternal uterus at gestational stage E11 (C, D). Panels A and C show the bright field, panels B and D the dark field. The arrows indicate cells within the ectoplacental cone. The arrowheads show the labeled uterine wall. Scale bars=400 µm.
Cannabinoid receptor mRNA expression in rat embryo
Fig. 3. CB1 receptor mRNA expression in an E18 embryo. A midsagittal section of an E18 embryo was hybridized with the antisense probe for CB1 receptor mRNA (shown in the dark field) and then stained with Giemsa stain (shown in the bright field) as indicated under Experimental Procedures. Very strong mRNA expression is present throughout the nervous system, and there is also clear signal in the thyroid gland. The embryo is shown at #10 magnification.
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Fig. 4.
Cannabinoid receptor mRNA expression in rat embryo
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Fig. 5. CB1 receptor mRNA expression in cranial nerve ganglia. ISHH was done on coronal brain sections of an E21 embryo using CB1 receptor mRNA-specific probes. Panels A, C and E show the bright field, panels B, D and F the dark field.Abbreviations: BST, brain stem; V, trigeminal ganglia; VIII, acoustic nerve; C, carotid; SCG, superior cervical ganglia; OG, otic ganglia; Ch, cochlea. Scale bars=200 µm. RESULTS
The following are the results obtained using the antisense probes. No signal was obtained when using the sense probes and are therefore not shown.
CB1 and CB2 receptor messenger RNAs are expressed in the placenta and maternal uterus From gestational stages E8–E12, the earliest stages studied, CB1 and CB2 receptor mRNAs are expressed
Fig. 4. CB1 receptor mRNA expression in the brain. Using CB1 receptor mRNA-specific probes for ISHH in coronal brain sections of an E21 embryo, we observe an almost identical distribution of CB1 receptor mRNA to that published earlier for adult rats.26 Panels A, C, E, G, I and K show bright field, panels B, D, F, H, J and L show dark field. Abbreviations: C, cortex; B, basal neuroepithelium; S, subventricular neuroepithelium; R, rhinencephalon; A, amygdala; Su, subiculum; H, hypothalamus; IC, inferior colliculus; D, dorsal tegmentum; CB, cerebellum; V, vestibular nuclei; IO, inferior olive. The ventricular space is labeled with an asterisk. Scale bars=400 µm.
Fig. 6.
Cannabinoid receptor mRNA expression in rat embryo
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Fig. 7. CB1 receptor mRNA expression in the digestive tract. ISHH using CB1 receptor mRNA-specific probes were done on the gut of E15 (A, B), E17 (C, D) and E20 (E, F) embryos. Panels A, C and E show the bright field, panels B, D and F the dark field. The arrows indicate the myenteric and the arrowheads the submucosal plexus; the asterisk labels the intestinal lumen. Scale bars=100 µm.
in the ectoplacental cone and in the outer longitudinal layer and the inner circular layer of myometral smooth muscles of the mother’s uterus (Figs
1, 2, respectively). In addition, CB1 receptor mRNA is expressed in the intervillous space of the placenta (Fig. 1, arrowheads).
Fig. 6. CB1 receptor mRNA expression in the eye. ISHH using CB1 receptor mRNA-specific probes was done on sections of eyes from E13 (A, B), E15 (C, D), E17 (E, F) and E20 (G, H) embryos. The bright field is shown in panels A, C, E and G, the dark field in panels B, D, F and H. At E13, the expression of CB1 receptor mRNA is observed within a region of the retina (A, B). Based on cellular location and number, the cell layer expressing CB1 receptor mRNA by E15 and E17 is the ganglionic cell layer (C–F). By E20, CB1 receptor mRNA is also expressed in another cell layer within the retina. Abbreviations: el, eyelid; c, cornea; l, lens; asterisk, vitreous body. Scale bars=100 µm (A, B), 200 µm (C–H).
Fig. 8.
Cannabinoid receptor mRNA expression in rat embryo
CB1 receptor messenger RNA expression in the nervous system As seen in Fig. 1D, at E11, the marginal layer of the neural tube and at E12 and E13 the telencephalic region (neocortical neuroepithelium) express CB1 receptor mRNA (Fig. 1F, H). In addition, by E13, the spinal ganglia and sympathetic ganglia also express CB1 receptor mRNA (Fig. 1H). From E15 to birth, CB1 receptor mRNA can be seen in a very similar distribution to the adult:26 neocortex, olfactory bulb, thalamus, hypothalamus, hippocampus, superior colliculus, the differentiating cerebellum, pons and spinal cord (Figs 3, 4). In addition, the ganglia of the sympathetic chain and the parasympathetic ganglia express CB1 receptor mRNA. Furthermore, CB1 receptor mRNA is detected in the ganglia of brain nerves V, VIII (Fig. 5B, D, respectively), IX and X. CB1 receptor messenger RNA expression in the eye At E12, the invagination of the ectoderm giving rise to the lens vesicle, as well as the inner (neural) layer of the optic cup (future nervous layer of the retina), express CB1 receptor mRNA. By E13, CB1 receptor mRNA is present in the retina (Fig. 6A, B). By E15 and E17, the ganglion cell layer in the retina (single layer) expresses the mRNA for CB1 receptor (Fig. 6C–F). At E20 and E21, the retina expresses CB1 receptor mRNA in the ganglion cells and also in another cell layer within the retina (Fig. 6G, H). CB1 receptor messenger RNA expression in the digestive tract By E15, CB1 receptor mRNA is expressed all along the digestive tract. Histologically, the regions within the gut expressing the CB1 receptor mRNA correspond to Meissner’s submucosal plexus and Auerbach’s myenteric plexus. This pattern of expression does not change up to E21 (Fig. 7). CB1 receptor messenger RNA expression in endocrine organs We detected CB1 receptor mRNA expression in the thyroid at the later embryological stages, E18, E20 and E21. Figures 3 and 8 show expression of CB1 receptor mRNA in the thyroid as detected by ISHH. CB1 receptor mRNA is also expressed in the adrenal gland. At E17, CB1 receptor mRNA is expressed in the cortex of the adrenal gland (the zona glomerulosa and the zona fasciculata; Fig. 9A, B). At
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E18, E20 and E21, the level of expression is higher in the zona glomerulosa (Fig. 9C, D). CB1 receptor messenger RNA expression in the lung The expression of CB1 receptor mRNA in the lung appears at the later gestational stages. At E15, CB1 receptor mRNA expression is seen only in a few airway passages (Fig. 10A, B). However, by E17, CB1 receptor mRNA expression becomes more widespread throughout the bronchi (Fig. 10C, D). The highest expression of CB1 receptor mRNA in the bronchi is seen by E20 and E21 (Fig. 10E, F). The trachea also expresses CB1 receptor mRNA at E21, as seen in Fig. 8A and B. The blood vessels do not express CB1 receptor mRNA. CB2 receptor messenger RNA expression in the liver In the embryo, the liver was the only tissue expressing CB2 receptor mRNA. This expression occurs at E13 and lasts throughout gestation (E21–E22). The CB2 receptor mRNA level of expression declines as development progresses (Fig. 11). The cells expressing the CB2 receptor mRNA seem to be Kupffer cells by their number and histological appearance. The results described are representative of four (for CB1 receptor mRNA expression) or six (for CB2 receptor mRNA expression) ISHH experiments performed. Immunohistochemical detection of CB1 receptor To confirm that the expression of CB1 receptor mRNA parallels the presence of the CB1 receptor protein, we immunostained an E19 rat embryo. We used a specific antibody that recognizes rat CB1 receptor.48,49 We found a perfect correlation with the mRNA expression pattern. The receptor was present in the brain (Fig. 12A), ganglia (Fig. 12B), gastrointestinal tract (Fig. 12D, E) and adrenal gland (Fig. 12C). No specific staining was seen when the primary antibody was preabsorbed with the antigen or when no primary antibody was applied. DISCUSSION
Although exogenous cannabinoids have been associated with teratogenic effects, the role of endogenous cannabinoids remains largely unknown. In the present work, we have studied the fetal
Fig. 8. CB1 receptor mRNA expression in the thyroid gland and respiratory tract. ISHH using CB1 receptor mRNA-specific probes was done on an E21 embryo. Panels A and C show the bright field, panels B and D the dark field. The boxed region in panels A and B is the thyroid gland, which is shown at a higher magnification in panels C and D. The arrows indicate individually labeled cells. Abbreviations: t, trachea; oe, oesophagus. Scale bars=200 µm (A, B), 100 µm (C, D).
Fig. 9.
Cannabinoid receptor mRNA expression in rat embryo
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Fig. 10. CB1 receptor mRNA expression in the lung. ISHH using CB1 receptor mRNA-specific probes was done on E15 (A, B), E17 (C, D) and E20 (E, F) embryos. The signal that we observed in the airway epithelium seemed to grow stronger with age. Abbreviations: asterisk, bronchus; arrows, bronchioli; arrowheads, vessels. Scale bars=100 µm.
distribution of the two known cannabinoid receptor mRNAs, as well as the presence of these receptor mRNAs in the uterus and placenta. The expression of these receptor mRNAs during development suggests that exogenous as well as endogenous
cannabinoids may affect fetal development. Recently, the presence of CB1 receptor, but not CB2 receptor, mRNA was detected by RT–PCR in the mouse pregnant uterus.8 It was also shown that the cannabinoids are functional in the uterus during
Fig. 9. CB1 receptor mRNA expression in the adrenal gland. ISHH using CB1 receptor mRNA-specific probes was done on E17 (A, B) and E20 (C, D) embryos. Panels A and C show the bright field, panels B and D the dark field. Note that the signal in the adrenal is present in scattered cells at day 17 (B), while at day 20 most of the signal is localized in the glomerular zone (D). Abbreviations: a, adrenal gland; k, kidney. Scale bars=100 µm (A, B), 200 µm (C, D).
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Fig. 11. CB2 receptor mRNA expression in the liver. The embryo was hybridized with the antisense probe for CB2 receptor mRNA and then stained with Giemsa stain as indicated under Experimental Procedures. Panels A and B correspond to an E13 embryo, panels C and D to an E15 embryo, and panels E and F to an E18 embryo. Panels A, C and E show the bright field, panels B, D and F the dark field. The arrow indicates the liver in the E13 embryo. The expression of CB2 receptor mRNA weakens at the later gestational periods. Abbreviations: st, stomach; s, sinusoid. Scale bars=200 µm (A, B), 100 µm (C–F).
peri-implantation,35 and that the uterus itself is capable of synthesizing and metabolizing the endogenous cannabinoid, anandamide.8,34,35,46 Our results confirm these findings, since we find both receptor mRNAs in the uterus as well as in the placenta of 8-, 9-, 10- and 13-day pregnant rats. It has also been found that both cannabinoid receptor mRNAs are present in the preimplantation
mouse embryos,33 and that cannabinoids inhibit preimplantation mouse embryo development, presumably via CB1 receptor.52 This suggests a regulatory role for cannabinoid receptors during this stage of embryonic development. Our findings show that CB1 receptor mRNA is more widely expressed than CB2 receptor mRNA during the embryonic development of the rat.
Cannabinoid receptor mRNA expression in rat embryo
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Fig. 12. Immunohistochemistry using an anti-CB1 receptor antibody. Immunohistochemistry was performed on an E19 embryo as described under Experimental Procedures. The brain is shown in panel A. Note the staining in many cortical neurons. The otic ganglion is shown in panel B (asterisk), along with the cochlea (arrows). In panel C, while the antibody stains the medulla of the adrenal gland (a), the kidney (k) remains unstained. Intestinal sections are depicted in panels D and E, where the anti-CB1 receptor antibody stains cells and fibers of the myenteric (arrowheads) and the submucosal plexus (arrows). Abbreviations: c, cortex; ch, choroid plexus; asterisk, ventricle. Scale bars=100 µm (A–C), 50 µm (D, E).
CB1 receptor messenger RNA expression in the central nervous system Previous studies in humans have suggested that prenatal exposure to marijuana caused an increase in tremor in the newborn.15 In infants, there is an enrichment of CB1 receptor in the striatum, as seen by ISHH and binding studies.24 Since in the striatum the processing of cortical input is modulated by dopaminergic transmission from the substantia nigra, the cannabinoids may alter the dopaminergic pathway. In adult rats, perinatal exposure to delta-9-tetrahydrocannabinol (THC) caused altered motor behavior in rearing, grooming and sniffing.44 Recently, functional studies in the striatum in rats suggested that cannabinoids interacting with striatal
CB1 receptor may modulate the dopaminergic neuronal pathways.6 Furthermore, the effects of cannabinoids on tyrosine hydroxylase (TH) activity, the enzyme involved in the synthesis of dopamine, have been studied. Perinatal exposure of rats to cannabinoids caused an alteration in TH activity, an effect mainly observed in males.17,43 Prenatal exposure to THC in rats affects the expression of the TH gene and TH activity in brain catecholaminergic neurons during early fetal brain development.3,4 TH activity was increased three-fold at E14 in THC-exposed fetuses as compared to control.3,4 At E18, THC-exposed female embryos exhibited increased amounts of TH mRNA over control, while a decrease in TH mRNA was detected in male embryos.3
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Our findings that, in embryonic rat, CB1 receptor mRNA is present within the basal ganglia, and particularly within regions that process motor functions and regulate sensorimotor information, suggest that these events may be mediated through CB1 receptor. CB1 receptor messenger RNA expression in the eye In humans, it has been indicated that prenatal exposure to marijuana decreases visual perception,15 and in animals, prenatal exposure to cannabinoids disrupts the habituation and reactivity to different illumination conditions.31 Since the presence of cannabinoid receptors in the eye has not been studied previously, there have been no indications that these effects are receptor mediated. Here we show that CB1 receptor mRNA is present in the retina of rat embryos, suggesting the possibility that cannabinoids have the potential to interact with receptors within the retina during development. Recently, the presence of cannabinoid receptors was suggested in isolated adult guinea-pig retina, since cannabinoids inhibited dopamine release45 in these preparations. This finding, along with ours, is particularly interesting, since it has been shown that cannabinoids are capable of reducing intraocular pressure, a major problem in patients with glaucoma.1,27,29,36,37,47,50 The mechanism by which cannabinoids cause the reduction in intraocular pressure is unknown. Perhaps the receptors found in the eye play a role in this effect. CB1 receptor messenger RNA expression in the digestive system The earliest embryonic stage at which CB1 receptor mRNA is detected in the gut is at E15 and continues throughout E21. Histologically, the region of the digestive tract expressing CB1 receptor mRNA corresponds to the ganglionic cells of Meissner’s submucosal and of Auerbach’s myenteric plexi. It has been shown that the myenteric plexus– longitudinal muscle preparation of guinea-pig small intestine contains functional cannabinoid binding sites.38–41 Our findings indicate that these receptors are already present during embryogenesis. CB1 receptor messenger RNA expression in the lung The levels of CB1 receptor mRNA expression significantly increase and are highest right before birth (at E21–E22). Perhaps CB1 receptor is involved in preparing the embryo for breathing immediately after birth. CB1 receptor messenger RNA expression in the thyroid gland Fetal thyroid hormones play a crucial role in fetal growth and development of certain individual tis-
sues.13 In the rat, the thyroid is fully developed by E17 and is able to synthesize and secrete hormones by E18.12,32 It has been reported that, in adult rat, acute treatment with THC decreases serum levels of thyrotropin and the thyroid hormones triiodothyronine and thyroxine.19 In our study, CB1 receptor mRNA is present in the thyroid by E18. Perhaps cannabinoids modulate thyroid hormone production during embryogenesis at this stage. CB1 receptor messenger RNA expression in the adrenal gland In our studies, we found CB1 receptor mRNA expression in the adrenal cortex by E17. Glucocorticoids are synthesized in the zona fasciculata. Glucocorticoids have major effects during embryonic development. In the period immediately before birth, glucocorticoids have a critical role in the maturation of many fetal tissues, such as lung, gut and liver.13 In rats, the enzymes responsible for the synthesis of glucocorticoids are present in the fetal adrenal even before zonation occurs (i.e. E9).28 Perinatal exposure to THC causes an alteration in production of corticotropin-releasing factor and corticosterone. Female rats exposed perinatally to THC had higher levels of corticotropinreleasing factor and corticosterone, while males had lower levels of these hormones.44 We suggest that cannabinoids may modulate glucocorticoid production at the level of the adrenal cortex. Mineralocorticoids are synthesized in the zona glomerulosa. The characteristic mineralocorticoid is aldosterone. The enzyme responsible for the synthesis of aldosterone is expressed at very low levels during embryonic development, suggesting that aldosterone does not play a role in the development of the fetus.23,28 In postnatal life, however, aldosterone plays a role in electrolyte balance.51 We found CB1 receptor mRNA expression to be highest in the zona glomerulosa at the latest gestational stages (i.e. E18– E21). Perhaps CB1 receptor has a modulatory effect on aldosterone synthesis shortly before birth and in preparation for birth. CB2 receptor messenger RNA expression in the embryonic liver In the embryonic rat, from E12 through E16, the liver is almost exclusively the fetal hematopoietic organ. Kupffer cells, the phagocytic cells in the liver, are already present by E11.2 In our studies, we observe CB2 receptor mRNA expression in what appear to be the Kupffer cells by their histological appearance. The earliest embryological stage at which we detect the expression of CB2 receptor mRNA is E13 and continues through E21. The level of expression of the CB2 receptor mRNA decreases at the later gestational stages, and in the adult rat liver we were unable to detect the CB2 receptor
Cannabinoid receptor mRNA expression in rat embryo
mRNA by ISHH. The expression of CB2 receptor mRNA was first determined in adult rat spleen,30 and CB2 receptor mRNA is now known to occur in other tissues of the immune system, such as the thymus and tonsils.16 Our observation that CB2 receptor mRNA occurs in phagocytic cells during embryogenesis may suggest an immunoregulatory effect in the fetus, as has been suggested in the adult.22 Recently, using the highly sensitive RT–PCR technique, Galiegue et al. 16 found that the expression of CB1 receptor mRNA in adult humans occurred predominantly in the central nervous system and, to a lesser extent, in adrenal gland, heart, lung, prostate, uterus, ovary, testis, bone marrow, thymus and tonsils. They reported that CB2 receptor mRNA is predominantly present in the tissues of the immune system.16 Our data confirm most of their findings. However, we did not detect cannabinoid receptor mRNA in all the tissues they described. This may be due to species difference or to the difference in the expression of this receptor during development versus postnatal life or to the
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sensitivity of the techniques used, or to a combination of the above. CONCLUSIONS
Our findings may provide some insight into the functional role of endogenous cannabinoids and into possible targets of exogenous cannabinoids during pregnancy. In addition, our findings may also provide information for possible medicinal uses of CB1 versus CB2 agonists in a variety of known diseases where marijuana seems beneficial. Marijuana has been used for the treatment of ailments associated with cancer chemotherapy, glaucoma, AIDS, multiple sclerosis and other diseases (for a review see Refs 27 and 50). Acknowledgements—We wish to thank Dr Stephen Lolait and Dr Tom Bonner for the the CB1 receptor plasmid templates, and Dr Sean Munro for the CB2 receptor plasmid template. We also thank Dr Ken Mackie for providing us with the rat CB1 antibody. Finally, we would like to thank Ricardo Dreyfuss for the photomicrography.
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N. E. Buckley et al. Hillard C. J., Farber N. E., Hagen T. C. and Bloom A. S. (1984) The effects of delta 9-tetrahydrocannabinol on serum thyrotropin levels in the rat. Pharmac. Biochem. Behav. 20, 547–550. Hsu S. and Raine L. (1981) Protein A, avidin, and biotin in immunohistochemistry. J. Histochem. Cytochem. 29, 1349–1353. Hunyady B., Krempels K., Harta G. and Mezey E. (1996) Immunohistochemical signal amplification by catalyzed reporter deposition and its application in double immunostaining. J. Histochem. Cytochem. 44, 1353–1362. Kaminski N. E. (1996) Immune regulation by cannabinoid compounds through the inhibition of the cyclic AMP signaling cascade and altered gene expression. Biochem. Pharmac. 52, 1133–1140. Keeney D. S., Jenkins C. M. and Waterman M. R. (1995) Developmentally regulated expression of adrenal 17A-hydroxylase cytochrome P450 in the mouse embryo. Endocrinology 136, 4872–4879. Mailleux P. and Vanderhaeghen J. J. (1992) Localization of cannabinoid receptor in the human developing and adult basal ganglia. Higher levels in the striatonigral neurons. Neurosci. Lett. 148, 173–176. Matsuda L., Lolait S. J., Brownstein M. J., Young A. C. and Bonner T. I. (1990) Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 346, 561–564. Matsuda L. A., Bonner T. I. and Lolait S. J. (1993) Localization of cannabinoid receptor mRNA in rat brain. J. comp. Neurol. 327, 535–550. Mechoulam R. and Feigenbaum J. J. (1987) Towards cannabinoid drugs. In Progress in Medicinal Chemistry (eds Ellis G. P. and West G. B.), pp. 159–200. Elsevier Science, Oxford. Mellon S. H., Compagnone N., Sander M., Cover C., Ganten D. and Djavidani B. (1995) Rodent models for studying steroids and hypertension: from fetal development to cells in culture. Steroids 60, 59–64. Muchtar S., Almog S., Torracca M. T., Saettone M. F. and Benita S. (1992) A submicron emulsion as ocular vehicle for delta-8-tetrahydrocannabinol: effect on intraocular pressure in rabbits. Ophthalm. Res 24, 142–149. Munro S., Thomas K. L. and Abu-Shaar M. (1993) Molecular characterization of a peripheral receptor for cannabinoids. Nature 365, 61–65. Navarro M., Rubio P. and Rodriguez de Fonseca F. (1995) Behavioural consequences of maternal exposure to natural cannabinoids in rats. Psychopharmacology 122, 1–14. Obregon M. J., Mallol J., Pastor R., Morreale de Escobar G. and Escobar del Rey F. (1984) -Thyroxine and 3,5,3*-triiodo--thyronine in rat embryos before onset of fetal thyroid function. Endocrinology 114, 305–307. Paria B. C., Das S. K. and Dey S. K. (1995) The preimplanatation mouse embryo is a target for cannabinoid ligand-receptor signaling. Proc. natn. Acad. Sci. U.S.A. 92, 9460–9464. Paria B. C., Deutsch D. D. and Dey S. K. (1996) The uterus is a potential site for anandamide synthesis and hydrolysis: differential profiles of anandamide synthase and hydrolase activities in the mouse uterus during the periimplantation period. Molec. Reprod. Devl. 45, 183–192. Paria B. C., Yang Z. M. and Dey S. K. (1996) Cannabinoids arrest preimplantation mouse embryo development via brain-type cannabinoid receptors. Biol. Reprod. 54, 96. Pate D. W., Jarvinen K., Urtti A., Jarho P., Fich M., Mahadevan V. and Jarvinen T. (1996) Effects of topical anandamides on intraocular pressure in normotensive rabbits. Life Sci. 58, 1849–1860. Pate D. W., Jarvinen K., Urtti A., Jarho P. and Jarvinen T. (1995) Ophthalmic arachidonylethanolamide decreases intraocular pressure in normotensive rabbits. Curr. Eye Res. 14, 791–797. Paterson S. J. and Pertwee R. G. (1993) Characterization of the cannabinoid binding site in the guinea-pig. Br. J. Pharmac. 109, 82P. Pertwee R. G., Fernando S. R., Griffin G., Ryan W., Razdan R. K., Compton D. R. and Martin B. R. (1996) Agonist–antagonist characterization of 6*-cyanohex-2*-yne-delta-8-tetrahydrocannabinol in two isolated tissue preparations. Eur. J. Pharmac. 315, 195–201. Pertwee R. G., Fernando S. R., Nash J. E. and Coutts A. A. (1996) Further evidence for the presence of cannabinoid CB1 receptors in guinea-pig small intestine. Br. J. Pharmac. 118, 2199–2205. Pertwee R. G., Stevenson L. A., Elrick D. B., Mechoulam R. and Corbett A. D. (1992) Inhibitory effects of certain enantiomeric cannabinoids in the mouse vas deferens and the myenteric plexus preparation of guinea-pig small intestine. Br. J. Pharmac. 105, 980–984. Robins L. M. and Mills J. L. (1993) Effects of in utero exposure to street drugs. Am. J. Public Health 83, 9–31. Rodriguez de Fonseca F., Hernandez M. L., De Miguel R., Fernandez-Ruiz J. J. and Ramos J. A. (1992) Early changes in the development of dopaminergic neurotransmission after maternal exposure to cannabinoids. Pharmac. Biochem. Behav. 41, 469–474. Rubio P., Rodriguez de Fonseca F., Munoz R. M., Ariznavarreta C., Martin-Calderon J. L. and Navarro M. (1995) Long-term behavioral effects of perinatal exposure to a delta-9-tetrahydrocannabinol in rats: possible role of pituitary–adrenal axis. Life Sci. 56, 2169–2176. Schlicker E., Timm J. and Gothert M. (1996) Cannabinoid receptor-mediated inhibition of dopamine release in the retina. Naunyn-Schmiedeberg’s Arch. Pharmac. 354, 791–795. Schmid P. C., Paria B. C., Krebsbach R. J., Schmid H. H. O. and Dey S. K. (1997) Changes in anandamide levels in mouse uterus are associated with uterine receptivity for embryo implantation. Proc. natn. Acad. Sci. U.S.A. 94, 4188–4192. Sugrue M. F. (1989) The pharmacology of antiglaucoma drugs. Pharmac. Ther. 43, 91–138. Tsou K., Brown S., Mackie K. and Walker J. M. (1997) The distribution of the CB1 receptor in rat brain as determined by a CB1 antibody. Neuroscience (In press). Twitchell W., Brown S. and Mackie K. (1997) Cannabinoids inhibit N and P/Q-type calcium currents in cultured rat hippocampal neurons. J. Neurophysiol. 78, 43–50. Voth E. A. and Schwartz R. H. (1997) Medicinal applications of delta-9-tetrahydrocannabinol and marijuana. Ann. intern. Med. 126, 791–798. Weiss L. (1983) Cell and Tissue Biology, A Textbook of Histology. Urban & Schwarzenberg, Baltimore, MD. Yang Z., Paria B. and Dey S. (1996) Activation of brain-type cannabinoid receptors interferes with preimplantation mouse embryo development. Biol. Reprod. 55, 756–761.
Cannabinoid receptor mRNA expression in rat embryo 53.
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Zuckerman B., Frank D. A., Hingson R., Amaro H., Levenson S. M., Kayne H., Parker S., Vinci R., Aboagye K., Fried L. E., Cabral H. and Timperi R. (1989) Effects of maternal marijuana and cocaine use on fetal growth. New Engl. J. Med. 320, 762–768. (Accepted 10 July 1997)
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Neuroscience Vol. 82, No. 4, pp. 1131–1149, 1998 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00348-5
EXPRESSION OF THE CB1 AND CB2 RECEPTOR MESSENGER RNAS DURING EMBRYONIC DEVELOPMENT IN THE RAT N. E. BUCKLEY,*‡ S. HANSSON,† G. HARTA* and E u . MEZEY* *National Institute of Neurological Diseases and Stroke, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, U.S.A. †National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, U.S.A. Abstract––We mapped the distribution of CB1 and CB2 receptor messenger RNAs in the developing rat to gain insight into how cannabinoids may affect embryogenesis. In situ hybridization histochemistry studies were done using riboprobes specific for CB1 or CB2 receptor messenger RNAs. We found that CB1 and CB2 receptor messenger RNAs are expressed in the placental cone and in the smooth muscle of the maternal uterus at the earliest gestational periods studied [from eight days of gestation (E8) through E12]. In the embryo, as early as E11, CB1 receptor messenger RNA is expressed in some cells of the neural tube and, at later embryological stages (from E15 to E21), in several distinct structures within the central nervous system. In addition, high levels of CB1 receptor messenger RNA were also found in areas of the peripheral nervous system such as the sympathetic and parasympathetic ganglia, in the retina and in the enteric ganglia of the gastrointestinal tract. In addition to neural structures, high levels of the CB1 receptor messenger RNA were also present in two endocrine organs, the thyroid gland and the adrenal gland. On the other hand, CB2 receptor messenger RNA is expressed exclusively in the liver of the embryo as early as E13. The region-specific expression of CB1 and CB2 receptor messenger RNAs suggests that these receptors have a functional role during embryogenesis. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: cannabinoid receptors, embryogenesis, uterus, brain, liver.
Marijuana is one of the recreational drugs most commonly used during pregnancy in Western countries.9,11,42,53 It is known that cannabinoids can cross the placenta, but their effects on fetal and postnatal development are unclear. In humans, a longitudinal study has been carried out by Fried15 describing the effects of cannabinoids on children from mothers who used marijuana during pregnancy. He found that newborns exhibited an increase in tremors, prolonged startles and poor habituation to visual stimuli. These marijuana-induced effects were not observed again until the age of four, where children prenatally exposed to marijuana showed increased behavioral problems and decreased performance on visual perceptual tasks, language comprehension, sustained attention and memory.15 Furthermore, other investigators reported that, on average, children exposed prenatally to marijuana tend to have a lower IQ compared to unexposed children.10 In another study, however, it was ‡To whom correspondence should be addressed. Abbreviations: E, embryonic (gestational) day; EDTA, ethylenediaminetetra-acetate; ISHH, in situ hybridization histochemistry; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RT, reverse transcriptase; TH, tyrosine hydroxylase; THC, delta-9-tetrahydrocannabinol.
reported that prenatal exposure to marijuana did not affect gross motor development in preschool children.7 However, reliable comparisons between children prenatally exposed to marijuana and those who were not are very hard to make due to the great number of socioeconomic variables between individuals. In a recent review, Navarro et al.31 discussed data on the behavioral effects of rats exposed to cannabinoids during gestational and lactational periods. In adulthood, these animals displayed altered sexual and motor behaviors, as well as alteration in responses to social interactions and to novelty (for a review see Ref. 31). The same review also describes several neurobiological mechanisms which are affected by perinatal exposure to cannabinoids, such as the catecholaminergic and serotoninergic systems of developing offspring, the pituitary hormones, the sex steroids and the glucocorticoids. More recently, it was reported that in adult mice, which had been prenatally exposed to the endogenous cannabinoid anandamide, there was increased catalepsy, hypothermia and analgesia.14 In addition, these mice had an increased inflammatory response to arachidonic acid and lipopolysaccharide.14 To date, only two cannabinoid receptors are known. The CB1 receptor was first cloned from adult
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rat brain.25 The CB1 receptor mRNA is mainly expressed in the central nervous system26 and, to a much lesser extent, in the periphery: adrenal gland, heart, lung, prostate, uterus, ovary, testis, bone marrow, thymus and tonsils as determined by reverse transcriptase–polymerase chain reaction (RT– PCR),16 and in testis as also determined by northern blotting.18 The CB2 receptor mRNA is present in cells of the immune system.16,30 Expression of the two cannabinoid receptor mRNAs has not been studied throughout embryonic development, but recently it was reported that anandamide is in high levels in the uterus and that these levels are altered during implantation.46 In addition, it has been shown that preimplantation mouse embryos from the two-cell stage to the blastocyst stage express CB1 and CB2 receptors.33 In the present work, we used in situ hybridization histochemistry (ISHH) to demonstrate the expression of the CB1 and CB2 receptor mRNAs in rat embryonic tissues from gestational day 8 (E8) through gestational day 21 (E21). We also discuss the possible functional roles of these receptors during embryonic development. EXPERIMENTAL PROCEDURES
Preparation of rat embryo tissue Timed-pregnant Sprague–Dawley rats purchased from Taconic Farms (Germantown, NY, U.S.A.) were killed by CO2 inhalation. The uteri were dissected on ice. The day of the vaginal plug was designated as E1. Embryos plus uteri (E8–E13) or embryos alone (E13, E15, E17, E18, E19, E20 and E21) were frozen on dry ice and stored at "80)C until sectioning. Serial 12-µm-thick sections in sagittal and transversal planes were collected on silanized slides, briefly dried at 37)C and stored at "80)C until processed for ISHH. In situ hybridization histochemistry Probes. For the ISHH studies, two sets of probes were prepared complementary to two different non-overlapping regions of the rat CB1 receptor mRNA. The first CB1 riboprobe was synthesized from a cDNA template kindly provided by Dr Stephen Lolait (NIMH, Bethesda, MD, U.S.A.). A fragment (from bp 932 to 1226 from the reported CB1 receptor gene sequence25) was amplified by PCR. The following primers were used: CB1/1 (5*-CGC GCA ATT AAC CCT CAC TAA AGG GCT GCA ATC TGT TTG CTC GGA C-3*) and CB1/2 (5*-G CGC GTA ATA CGA CTC ACT ATA GGG GCC CCA GCA GAT GAT CAA CAC-3*). The second CB1 receptor riboprobe was synthesized from a cDNA template kindly provided by Dr Tom Bonner (NIMH). This fragment corresponds to bp 165–464 of the CB1 receptor gene sequence.25 The primers used for the amplification of this fragment were the following: CB1a (5*-CGC GCA ATT AAC CCT CAC TAA AGG CTA GAT GGC CTT GCA GAC ACC A-3*) and CB1b (5*-G CGC GTA ATA CGA CTC ACT ATA GGG CAT AAA GTT CTC CCC ACA CTG G-3*). The cDNA template for the rat CB2 riboprobe was kindly provided by Dr Sean Munro (MRC Laboratory of Molecular Biology, Hills Road, Cambridge, U.K.). The rat CB2 cDNA was a 595-bp fragment corresponding to bp 826– 1421 in the mouse CB2 gene sequence (Accession number U21681 in the Genebank). This cDNA was used as a template to transcribe a 595-bp CB2 riboprobe. In addition,
a PCR product corresponding to bp 916–1269 from the mouse CB2 gene sequence was obtained from this cDNA using the following primers: CB2a (5*-CGC GCA ATT AAC CCT CAC TAA AGG TGC TGT TGA CCG ATA CCT AT-3*) and CB2b (5*-G CGC GTA ATA CGA CTC ACT ATA GGG GCC AAC CTC ACG TCT AGC CG-3*). All primers contain specific sequences complementary to the cannabinoid receptor in addition to sequences corresponding to the T7 (bold) and T3 (italic) polymerase binding sites. The PCR amplification was done at 95)C for 1 min, 60)C for 1 min, 72)C for 2 min, and was carried out for 30 cycles using Taq Polymerase (Boehringer Mannheim, Indianapolis, IN, U.S.A.). The fragments were extended by incubating at 72)C for 10 min, after which they were stored at 4)C. The PCR fragments were purified and transcribed using [35áS]UTP (NEN, Boston, MA, U.S.A.) and the T7/T3 MAXIScript kit (AMBION, Austin, TX, U.S.A.). T7 polymerase was used to label the antisense probe and T3 polymerase to label the sense control probe. Hybridization. The slides were processed for ISHH as described previously.5 Briefly, the slides were treated with acetic anhydride, dehydrated, delipidated and incubated overnight at 55)C with the hybridization buffer [20 mM Tris, pH 7.4, 1 mM EDTA, 300 mM NaCl, 50% formamide, 10% dextran sulfate, 1#Denhardt’s, 100 µg/ml salmon sperm DNA, 250 µg/ml yeast total RNA, 250 µg/ml yeast tRNA, 100 mM dithiothreitol, 0.1% sodium dodecyl sulfate, 0.1% sodium thiosulfate] containing the probes (2#106 c.p.m./50 µl/slide) described above. The following day, the slides were washed to remove unbound probe and allowed to dry. The slides were exposed to Kodak Bio-Max MR (Kodak, Rochester, NY, U.S.A.) X-ray film for three days and then coated with undiluted NTB3 nuclear track emulsion (Kodak). The slides were developed three weeks later, counterstained with 1% Giemsa stain and viewed under a Leitz Dialux 20 microscope. Immunohistochemistry Affinity purified antibodies to rat CB1 receptor were kindly provided by Dr Ken Mackie (University of Washington, Seattle, WA, U.S.A.). The development of the antibody was supported by grant no. DA 08934 and has been described previously.48,49 Fresh frozen sections of E19 embryos were fixed by immersion into 4% formaldehyde in phosphate-buffered saline (PBS; pH 7.4) for 10 min at room temperature. The sections were rinsed several times and then blocked for 30 min in PBS containing 5% normal goat serum and 0.6% Triton X-100 (blocking solution). Following several rinses, the CB1 receptor antibody was applied to the sections at 1:500 or 1:5000 dilutions for fluorescence and 1:1000 dilution for the avidin–biotin–peroxidase complex method.20 The dilutions were made in the blocking solution. Prior to incubation with the primary antibody, the sections that were processed for the avidin–biotin–peroxidase complex method were treated with 1% H2O2 in methanol to block endogenous peroxidase activity. The sections were incubated with the antibody overnight at 4)C. The sections were then washed and fluorochrome-conjugated antibody was applied. The 1:5000 dilution was developed using the tyramide amplification procedure.21 The sections were then rinsed in PBS and briefly in distilled water, then air dried, mounted with cytoseal and coverslipped. Controls included immunostaining with the working dilution of the antibody that has been preabsorbed with 10 µg/ml of the antigen, no primary antibody or normal non-immune rabbit immunoglobulin G replacing the specific antibodies in the procedure. For more details of the techniques used see the ‘‘protocols’’ section in the following web site: http:// www.nimh.nih.gov/2snge/.
Fig. 1. CB1 receptor mRNA is expressed in the placenta, in the maternal uterus and in the embryo. The embryo was hybridized with the antisense probe for CB1 receptor mRNA and then stained with Giemsa stain as indicated under Experimental Procedures. Panels A, C, E and G show the bright field, panels B, D, F and H the dark field. Panels A–D correspond to an E11 embryo, panels E and F to an E12 embryo and panels G and H to an E13 embryo. The E11 embryo, boxed in panels A and B, is shown in more detail in panels C and D. The arrowheads in panels A and B show heavily labeled cells within the intervillous space of the placenta. The arrows in panels C–H indicate the neural tube, spinal and sympathetic ganglia. The asterisk labels the lumen of the telencephalic region. Scale bars=400 µm (A, B), 100 µm (C–F), 200 µm (G, H).
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Fig. 2. CB2 receptor mRNA is expressed in the placenta and maternal uterus. Hybridization to CB2 receptor mRNA-specific probes is shown in the placenta at gestational stage E8 (A, B), and in the placenta and maternal uterus at gestational stage E11 (C, D). Panels A and C show the bright field, panels B and D the dark field. The arrows indicate cells within the ectoplacental cone. The arrowheads show the labeled uterine wall. Scale bars=400 µm.
Cannabinoid receptor mRNA expression in rat embryo
Fig. 3. CB1 receptor mRNA expression in an E18 embryo. A midsagittal section of an E18 embryo was hybridized with the antisense probe for CB1 receptor mRNA (shown in the dark field) and then stained with Giemsa stain (shown in the bright field) as indicated under Experimental Procedures. Very strong mRNA expression is present throughout the nervous system, and there is also clear signal in the thyroid gland. The embryo is shown at #10 magnification.
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Fig. 4.
Cannabinoid receptor mRNA expression in rat embryo
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Fig. 5. CB1 receptor mRNA expression in cranial nerve ganglia. ISHH was done on coronal brain sections of an E21 embryo using CB1 receptor mRNA-specific probes. Panels A, C and E show the bright field, panels B, D and F the dark field.Abbreviations: BST, brain stem; V, trigeminal ganglia; VIII, acoustic nerve; C, carotid; SCG, superior cervical ganglia; OG, otic ganglia; Ch, cochlea. Scale bars=200 µm. RESULTS
The following are the results obtained using the antisense probes. No signal was obtained when using the sense probes and are therefore not shown.
CB1 and CB2 receptor messenger RNAs are expressed in the placenta and maternal uterus From gestational stages E8–E12, the earliest stages studied, CB1 and CB2 receptor mRNAs are expressed
Fig. 4. CB1 receptor mRNA expression in the brain. Using CB1 receptor mRNA-specific probes for ISHH in coronal brain sections of an E21 embryo, we observe an almost identical distribution of CB1 receptor mRNA to that published earlier for adult rats.26 Panels A, C, E, G, I and K show bright field, panels B, D, F, H, J and L show dark field. Abbreviations: C, cortex; B, basal neuroepithelium; S, subventricular neuroepithelium; R, rhinencephalon; A, amygdala; Su, subiculum; H, hypothalamus; IC, inferior colliculus; D, dorsal tegmentum; CB, cerebellum; V, vestibular nuclei; IO, inferior olive. The ventricular space is labeled with an asterisk. Scale bars=400 µm.
Fig. 6.
Cannabinoid receptor mRNA expression in rat embryo
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Fig. 7. CB1 receptor mRNA expression in the digestive tract. ISHH using CB1 receptor mRNA-specific probes were done on the gut of E15 (A, B), E17 (C, D) and E20 (E, F) embryos. Panels A, C and E show the bright field, panels B, D and F the dark field. The arrows indicate the myenteric and the arrowheads the submucosal plexus; the asterisk labels the intestinal lumen. Scale bars=100 µm.
in the ectoplacental cone and in the outer longitudinal layer and the inner circular layer of myometral smooth muscles of the mother’s uterus (Figs
1, 2, respectively). In addition, CB1 receptor mRNA is expressed in the intervillous space of the placenta (Fig. 1, arrowheads).
Fig. 6. CB1 receptor mRNA expression in the eye. ISHH using CB1 receptor mRNA-specific probes was done on sections of eyes from E13 (A, B), E15 (C, D), E17 (E, F) and E20 (G, H) embryos. The bright field is shown in panels A, C, E and G, the dark field in panels B, D, F and H. At E13, the expression of CB1 receptor mRNA is observed within a region of the retina (A, B). Based on cellular location and number, the cell layer expressing CB1 receptor mRNA by E15 and E17 is the ganglionic cell layer (C–F). By E20, CB1 receptor mRNA is also expressed in another cell layer within the retina. Abbreviations: el, eyelid; c, cornea; l, lens; asterisk, vitreous body. Scale bars=100 µm (A, B), 200 µm (C–H).
Fig. 8.
Cannabinoid receptor mRNA expression in rat embryo
CB1 receptor messenger RNA expression in the nervous system As seen in Fig. 1D, at E11, the marginal layer of the neural tube and at E12 and E13 the telencephalic region (neocortical neuroepithelium) express CB1 receptor mRNA (Fig. 1F, H). In addition, by E13, the spinal ganglia and sympathetic ganglia also express CB1 receptor mRNA (Fig. 1H). From E15 to birth, CB1 receptor mRNA can be seen in a very similar distribution to the adult:26 neocortex, olfactory bulb, thalamus, hypothalamus, hippocampus, superior colliculus, the differentiating cerebellum, pons and spinal cord (Figs 3, 4). In addition, the ganglia of the sympathetic chain and the parasympathetic ganglia express CB1 receptor mRNA. Furthermore, CB1 receptor mRNA is detected in the ganglia of brain nerves V, VIII (Fig. 5B, D, respectively), IX and X. CB1 receptor messenger RNA expression in the eye At E12, the invagination of the ectoderm giving rise to the lens vesicle, as well as the inner (neural) layer of the optic cup (future nervous layer of the retina), express CB1 receptor mRNA. By E13, CB1 receptor mRNA is present in the retina (Fig. 6A, B). By E15 and E17, the ganglion cell layer in the retina (single layer) expresses the mRNA for CB1 receptor (Fig. 6C–F). At E20 and E21, the retina expresses CB1 receptor mRNA in the ganglion cells and also in another cell layer within the retina (Fig. 6G, H). CB1 receptor messenger RNA expression in the digestive tract By E15, CB1 receptor mRNA is expressed all along the digestive tract. Histologically, the regions within the gut expressing the CB1 receptor mRNA correspond to Meissner’s submucosal plexus and Auerbach’s myenteric plexus. This pattern of expression does not change up to E21 (Fig. 7). CB1 receptor messenger RNA expression in endocrine organs We detected CB1 receptor mRNA expression in the thyroid at the later embryological stages, E18, E20 and E21. Figures 3 and 8 show expression of CB1 receptor mRNA in the thyroid as detected by ISHH. CB1 receptor mRNA is also expressed in the adrenal gland. At E17, CB1 receptor mRNA is expressed in the cortex of the adrenal gland (the zona glomerulosa and the zona fasciculata; Fig. 9A, B). At
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E18, E20 and E21, the level of expression is higher in the zona glomerulosa (Fig. 9C, D). CB1 receptor messenger RNA expression in the lung The expression of CB1 receptor mRNA in the lung appears at the later gestational stages. At E15, CB1 receptor mRNA expression is seen only in a few airway passages (Fig. 10A, B). However, by E17, CB1 receptor mRNA expression becomes more widespread throughout the bronchi (Fig. 10C, D). The highest expression of CB1 receptor mRNA in the bronchi is seen by E20 and E21 (Fig. 10E, F). The trachea also expresses CB1 receptor mRNA at E21, as seen in Fig. 8A and B. The blood vessels do not express CB1 receptor mRNA. CB2 receptor messenger RNA expression in the liver In the embryo, the liver was the only tissue expressing CB2 receptor mRNA. This expression occurs at E13 and lasts throughout gestation (E21–E22). The CB2 receptor mRNA level of expression declines as development progresses (Fig. 11). The cells expressing the CB2 receptor mRNA seem to be Kupffer cells by their number and histological appearance. The results described are representative of four (for CB1 receptor mRNA expression) or six (for CB2 receptor mRNA expression) ISHH experiments performed. Immunohistochemical detection of CB1 receptor To confirm that the expression of CB1 receptor mRNA parallels the presence of the CB1 receptor protein, we immunostained an E19 rat embryo. We used a specific antibody that recognizes rat CB1 receptor.48,49 We found a perfect correlation with the mRNA expression pattern. The receptor was present in the brain (Fig. 12A), ganglia (Fig. 12B), gastrointestinal tract (Fig. 12D, E) and adrenal gland (Fig. 12C). No specific staining was seen when the primary antibody was preabsorbed with the antigen or when no primary antibody was applied. DISCUSSION
Although exogenous cannabinoids have been associated with teratogenic effects, the role of endogenous cannabinoids remains largely unknown. In the present work, we have studied the fetal
Fig. 8. CB1 receptor mRNA expression in the thyroid gland and respiratory tract. ISHH using CB1 receptor mRNA-specific probes was done on an E21 embryo. Panels A and C show the bright field, panels B and D the dark field. The boxed region in panels A and B is the thyroid gland, which is shown at a higher magnification in panels C and D. The arrows indicate individually labeled cells. Abbreviations: t, trachea; oe, oesophagus. Scale bars=200 µm (A, B), 100 µm (C, D).
Fig. 9.
Cannabinoid receptor mRNA expression in rat embryo
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Fig. 10. CB1 receptor mRNA expression in the lung. ISHH using CB1 receptor mRNA-specific probes was done on E15 (A, B), E17 (C, D) and E20 (E, F) embryos. The signal that we observed in the airway epithelium seemed to grow stronger with age. Abbreviations: asterisk, bronchus; arrows, bronchioli; arrowheads, vessels. Scale bars=100 µm.
distribution of the two known cannabinoid receptor mRNAs, as well as the presence of these receptor mRNAs in the uterus and placenta. The expression of these receptor mRNAs during development suggests that exogenous as well as endogenous
cannabinoids may affect fetal development. Recently, the presence of CB1 receptor, but not CB2 receptor, mRNA was detected by RT–PCR in the mouse pregnant uterus.8 It was also shown that the cannabinoids are functional in the uterus during
Fig. 9. CB1 receptor mRNA expression in the adrenal gland. ISHH using CB1 receptor mRNA-specific probes was done on E17 (A, B) and E20 (C, D) embryos. Panels A and C show the bright field, panels B and D the dark field. Note that the signal in the adrenal is present in scattered cells at day 17 (B), while at day 20 most of the signal is localized in the glomerular zone (D). Abbreviations: a, adrenal gland; k, kidney. Scale bars=100 µm (A, B), 200 µm (C, D).
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Fig. 11. CB2 receptor mRNA expression in the liver. The embryo was hybridized with the antisense probe for CB2 receptor mRNA and then stained with Giemsa stain as indicated under Experimental Procedures. Panels A and B correspond to an E13 embryo, panels C and D to an E15 embryo, and panels E and F to an E18 embryo. Panels A, C and E show the bright field, panels B, D and F the dark field. The arrow indicates the liver in the E13 embryo. The expression of CB2 receptor mRNA weakens at the later gestational periods. Abbreviations: st, stomach; s, sinusoid. Scale bars=200 µm (A, B), 100 µm (C–F).
peri-implantation,35 and that the uterus itself is capable of synthesizing and metabolizing the endogenous cannabinoid, anandamide.8,34,35,46 Our results confirm these findings, since we find both receptor mRNAs in the uterus as well as in the placenta of 8-, 9-, 10- and 13-day pregnant rats. It has also been found that both cannabinoid receptor mRNAs are present in the preimplantation
mouse embryos,33 and that cannabinoids inhibit preimplantation mouse embryo development, presumably via CB1 receptor.52 This suggests a regulatory role for cannabinoid receptors during this stage of embryonic development. Our findings show that CB1 receptor mRNA is more widely expressed than CB2 receptor mRNA during the embryonic development of the rat.
Cannabinoid receptor mRNA expression in rat embryo
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Fig. 12. Immunohistochemistry using an anti-CB1 receptor antibody. Immunohistochemistry was performed on an E19 embryo as described under Experimental Procedures. The brain is shown in panel A. Note the staining in many cortical neurons. The otic ganglion is shown in panel B (asterisk), along with the cochlea (arrows). In panel C, while the antibody stains the medulla of the adrenal gland (a), the kidney (k) remains unstained. Intestinal sections are depicted in panels D and E, where the anti-CB1 receptor antibody stains cells and fibers of the myenteric (arrowheads) and the submucosal plexus (arrows). Abbreviations: c, cortex; ch, choroid plexus; asterisk, ventricle. Scale bars=100 µm (A–C), 50 µm (D, E).
CB1 receptor messenger RNA expression in the central nervous system Previous studies in humans have suggested that prenatal exposure to marijuana caused an increase in tremor in the newborn.15 In infants, there is an enrichment of CB1 receptor in the striatum, as seen by ISHH and binding studies.24 Since in the striatum the processing of cortical input is modulated by dopaminergic transmission from the substantia nigra, the cannabinoids may alter the dopaminergic pathway. In adult rats, perinatal exposure to delta-9-tetrahydrocannabinol (THC) caused altered motor behavior in rearing, grooming and sniffing.44 Recently, functional studies in the striatum in rats suggested that cannabinoids interacting with striatal
CB1 receptor may modulate the dopaminergic neuronal pathways.6 Furthermore, the effects of cannabinoids on tyrosine hydroxylase (TH) activity, the enzyme involved in the synthesis of dopamine, have been studied. Perinatal exposure of rats to cannabinoids caused an alteration in TH activity, an effect mainly observed in males.17,43 Prenatal exposure to THC in rats affects the expression of the TH gene and TH activity in brain catecholaminergic neurons during early fetal brain development.3,4 TH activity was increased three-fold at E14 in THC-exposed fetuses as compared to control.3,4 At E18, THC-exposed female embryos exhibited increased amounts of TH mRNA over control, while a decrease in TH mRNA was detected in male embryos.3
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Our findings that, in embryonic rat, CB1 receptor mRNA is present within the basal ganglia, and particularly within regions that process motor functions and regulate sensorimotor information, suggest that these events may be mediated through CB1 receptor. CB1 receptor messenger RNA expression in the eye In humans, it has been indicated that prenatal exposure to marijuana decreases visual perception,15 and in animals, prenatal exposure to cannabinoids disrupts the habituation and reactivity to different illumination conditions.31 Since the presence of cannabinoid receptors in the eye has not been studied previously, there have been no indications that these effects are receptor mediated. Here we show that CB1 receptor mRNA is present in the retina of rat embryos, suggesting the possibility that cannabinoids have the potential to interact with receptors within the retina during development. Recently, the presence of cannabinoid receptors was suggested in isolated adult guinea-pig retina, since cannabinoids inhibited dopamine release45 in these preparations. This finding, along with ours, is particularly interesting, since it has been shown that cannabinoids are capable of reducing intraocular pressure, a major problem in patients with glaucoma.1,27,29,36,37,47,50 The mechanism by which cannabinoids cause the reduction in intraocular pressure is unknown. Perhaps the receptors found in the eye play a role in this effect. CB1 receptor messenger RNA expression in the digestive system The earliest embryonic stage at which CB1 receptor mRNA is detected in the gut is at E15 and continues throughout E21. Histologically, the region of the digestive tract expressing CB1 receptor mRNA corresponds to the ganglionic cells of Meissner’s submucosal and of Auerbach’s myenteric plexi. It has been shown that the myenteric plexus– longitudinal muscle preparation of guinea-pig small intestine contains functional cannabinoid binding sites.38–41 Our findings indicate that these receptors are already present during embryogenesis. CB1 receptor messenger RNA expression in the lung The levels of CB1 receptor mRNA expression significantly increase and are highest right before birth (at E21–E22). Perhaps CB1 receptor is involved in preparing the embryo for breathing immediately after birth. CB1 receptor messenger RNA expression in the thyroid gland Fetal thyroid hormones play a crucial role in fetal growth and development of certain individual tis-
sues.13 In the rat, the thyroid is fully developed by E17 and is able to synthesize and secrete hormones by E18.12,32 It has been reported that, in adult rat, acute treatment with THC decreases serum levels of thyrotropin and the thyroid hormones triiodothyronine and thyroxine.19 In our study, CB1 receptor mRNA is present in the thyroid by E18. Perhaps cannabinoids modulate thyroid hormone production during embryogenesis at this stage. CB1 receptor messenger RNA expression in the adrenal gland In our studies, we found CB1 receptor mRNA expression in the adrenal cortex by E17. Glucocorticoids are synthesized in the zona fasciculata. Glucocorticoids have major effects during embryonic development. In the period immediately before birth, glucocorticoids have a critical role in the maturation of many fetal tissues, such as lung, gut and liver.13 In rats, the enzymes responsible for the synthesis of glucocorticoids are present in the fetal adrenal even before zonation occurs (i.e. E9).28 Perinatal exposure to THC causes an alteration in production of corticotropin-releasing factor and corticosterone. Female rats exposed perinatally to THC had higher levels of corticotropinreleasing factor and corticosterone, while males had lower levels of these hormones.44 We suggest that cannabinoids may modulate glucocorticoid production at the level of the adrenal cortex. Mineralocorticoids are synthesized in the zona glomerulosa. The characteristic mineralocorticoid is aldosterone. The enzyme responsible for the synthesis of aldosterone is expressed at very low levels during embryonic development, suggesting that aldosterone does not play a role in the development of the fetus.23,28 In postnatal life, however, aldosterone plays a role in electrolyte balance.51 We found CB1 receptor mRNA expression to be highest in the zona glomerulosa at the latest gestational stages (i.e. E18– E21). Perhaps CB1 receptor has a modulatory effect on aldosterone synthesis shortly before birth and in preparation for birth. CB2 receptor messenger RNA expression in the embryonic liver In the embryonic rat, from E12 through E16, the liver is almost exclusively the fetal hematopoietic organ. Kupffer cells, the phagocytic cells in the liver, are already present by E11.2 In our studies, we observe CB2 receptor mRNA expression in what appear to be the Kupffer cells by their histological appearance. The earliest embryological stage at which we detect the expression of CB2 receptor mRNA is E13 and continues through E21. The level of expression of the CB2 receptor mRNA decreases at the later gestational stages, and in the adult rat liver we were unable to detect the CB2 receptor
Cannabinoid receptor mRNA expression in rat embryo
mRNA by ISHH. The expression of CB2 receptor mRNA was first determined in adult rat spleen,30 and CB2 receptor mRNA is now known to occur in other tissues of the immune system, such as the thymus and tonsils.16 Our observation that CB2 receptor mRNA occurs in phagocytic cells during embryogenesis may suggest an immunoregulatory effect in the fetus, as has been suggested in the adult.22 Recently, using the highly sensitive RT–PCR technique, Galiegue et al. 16 found that the expression of CB1 receptor mRNA in adult humans occurred predominantly in the central nervous system and, to a lesser extent, in adrenal gland, heart, lung, prostate, uterus, ovary, testis, bone marrow, thymus and tonsils. They reported that CB2 receptor mRNA is predominantly present in the tissues of the immune system.16 Our data confirm most of their findings. However, we did not detect cannabinoid receptor mRNA in all the tissues they described. This may be due to species difference or to the difference in the expression of this receptor during development versus postnatal life or to the
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sensitivity of the techniques used, or to a combination of the above. CONCLUSIONS
Our findings may provide some insight into the functional role of endogenous cannabinoids and into possible targets of exogenous cannabinoids during pregnancy. In addition, our findings may also provide information for possible medicinal uses of CB1 versus CB2 agonists in a variety of known diseases where marijuana seems beneficial. Marijuana has been used for the treatment of ailments associated with cancer chemotherapy, glaucoma, AIDS, multiple sclerosis and other diseases (for a review see Refs 27 and 50). Acknowledgements—We wish to thank Dr Stephen Lolait and Dr Tom Bonner for the the CB1 receptor plasmid templates, and Dr Sean Munro for the CB2 receptor plasmid template. We also thank Dr Ken Mackie for providing us with the rat CB1 antibody. Finally, we would like to thank Ricardo Dreyfuss for the photomicrography.
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