A cannabinoid analogue of Δ9-tetrahydrocannabinol disrupts neural development in chick

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<ul><li><p>Original Article</p><p>A Cannabinoid Analogue of D9-TetrahydrocannabinolDisrupts Neural Development in Chick</p><p>Delphine Psychoyos,1 Basalingappa Hungund,2,3,4 Thomas Cooper,2,3,4 and Richard H. Finnell1</p><p>1Center for Environmental and Genetic Medicine, Institute of Biosciences and Technology, Texas A&amp;M Health Science Center,Houston, Texas</p><p>2Department of Psychiatry, College of Physicians &amp; Surgeons, Columbia University, New York, New York3New York State Psychiatric Institute, New York, New York</p><p>4Nathan S. Kline Institute for Psychiatric Research, Orangeburg, New York</p><p>Marijuana is the most commonly abused illicit drug by pregnant women. Its major psychoactive constituent, D9-THC(D9-tetrahydrocannabinol), crosses the placenta and accumulates in the f!tus, potentially harming its development. Inhumans, marijuana use in early pregnancy is associated with miscarriage, a fetal alcohol-like syndrome, as well aslearning disabilities, memory impairment, and ADHD in the offspring. Classical studies in the 1970 s have reacheddisparate conclusions as to the teratogenic effects of cannabinoids in animal models. Further, there is very little knownabout the immediate effects of D9-THC on early embryogenesis. We have used the chick embryo as a model in order tocharacterize the effects of a water-soluble D9-THC analogue, O-2545, on early development. Embryos were exposed tothe drug (0.035 to 0.35mg/ml) at gastrulation and assessed for morphological defects at stages equivalent to 914somites. We report that O-2545 impairs the formation of brain, heart, somite, and spinal cord primordia. Shorterincubation times following exposure to the drug show that O-2545 interferes with the initial steps of head process andneural plate formation. Our results indicate that the administration of the cannabinoid O-2545 during earlyembryogenesis results in embryotoxic effects and serves to illuminate the risks of marijuana exposure during thesecond week of pregnancy, a time point at which most women are unaware of their pregnancies. Birth Defects Res (Part B)83:477488, 2008. r 2008 Wiley-Liss, Inc.</p><p>Key words: cannabinoids; anencephaly; brain malformation; CNSdevelopment; neural plate; neural folds; neural tube defects; chick embryo;animal model</p><p>INTRODUCTION</p><p>Marijuana is the most commonly abused illicit drug bypregnant women (NIDA, 2001; WHO, 1997). Its majorpsychoactive constituent, D9-THC (Gaoni and Mechou-lam, 1971), crosses the placenta and accumulates in thef!tus (Blackard and Tennes, 1984), potentially harmingits development. Marijuana use in early pregnancy isassociated with miscarriage (Day and Richardson, 1991),an increased prevalence of congenital malformations(Hecht et al., 1968; Carakushansky et al., 1969; Hingsonet al., 1982), as well as learning disability, memoryimpairment, and ADHD in the exposed offspring(Goldschmidt et al., 2004; Fried et al., 2005; Nolandet al., 2005). However, very little is known aboutmechanisms underlying the adverse effects of D9-THCin early pregnancy. Miscarriage itself could result fromseveral causes, including failure of blastocyst implanta-tion due to a nonreceptive endometrium, as demon-strated in rodent models (Paria et al., 1995), or secondaryto teratogenic effects during early organogenesis (weeks23 of human gestation). At such early stages, any severemalformations resulting from the marijuana exposure</p><p>would be lethal and result in spontaneous abortion, andthus the embryo would not be detectable for clinicalstudies. The adverse effects of marijuana use duringpregnancy are aggravated by the fact that the potency ofmarijuana preparations, in terms of contents of itspsychoactive constituent D9-THC, has increased nearly25-fold since 1970, when the content of D9-THC inmarijuana was 1.25%; it now reaches 15%30% in somepreparations (NIDA, 2001). Thus, the modern cannabissmoker may be exposed to doses of D9-THC many timesgreater than his or her counterpart in the 1960 s and 1970 s(WHO, 1997). This fact is important, because the effects ofD9-THC are dose related, and most of the research on</p><p>Published online in Wiley InterScience (www.interscience.wiley.com)DOI: 10.1002/bdrb.20166</p><p>Additional supporting information may be found in the online version ofthis article.</p><p>*Correspondence to: Delphine Psychoyos, Center for Environmentaland Genetic Medicine, Institute of Biosciences and Technology, TexasA&amp;M Health Science Center, Houston, Texas 77030.E-mail: dpsychoyos@ibt.tamhsc.eduReceived 7 July 2008; Accepted 15 August 2008</p><p>Birth Defects Research (Part B) 83:477488 (2008)&amp; 2008 Wiley-Liss, Inc.</p></li><li><p>cannabis was carried out in the 1970 s using doses thatreflected cannabis intake at the time (WHO, 1997).Classical studies in rodents show that the develop-</p><p>mental stage at which D9-THC is administered is acritical factor in determining the degree of D9-THCembryotoxicity. The period of greatest susceptibility tothe embryotoxic effects of D9-THC occurs during earlyorganogenesis (E6.08.0 in mouse). During this period,D9-THC administration results in a high incidence ofembryonic death and congenital malformations. Theincidence of fetal loss varies between 40% and 100%,depending on the route of administration, the species,</p><p>and the dosage used (Harbison and Mantilla-Plata, 1972;Mantilla-Plata et al., 1973; Uyeno, 1973; Fleischman et al.,1975; Wright et al., 1976). Surviving embryos presentwith malformations in the enteric and nervous systems(holoprosencephaly, anencephaly, exencephaly, cleft pa-late, degenerating spinal cord, and spina bifida), defec-tive skeletogenesis (absent or reduced sternebrae, extraor fused ribs, abnormal vertebral ossification) (Geber andSchramm, 1969; Mantilla-Plata et al., 1975; Jonega, 1976,1977; Rosenkrantz, 1978), and cognitive deficiencies inthe adult (Gianutsos and Abbatiello, 1972; Antonelli,2005). By contrast, exposure to D9-THC prior to</p><p>Fig. 1. Dose related effects of cannabinoid O-2545. (A) Structure of O-2545: The terminal carbon atom of the side chain of D9-THC issubstituted by an imidazole group in O-2545. This substitution results in a CB1 agonist which produces the same spectrum ofpharmacological effects in the mouse model as D9-THC. (BG) Representative embryos treated with vehicle alone (BD; PBS), or O-2545(+CB) at 0.035 mg/ml (E), 0.07 mg/ml (F), and 0.35 mg/ml (G). Embryos were treated at stages (HH) 4+ (B,E), 4 (C,F) and 4 (D,G).Control embryos reached the equivalent of stages 11 (B), 8+ (C), and 10 (D). Whole-mount in situ hybridization was performed withKrox20 (B,E), or Otx2 and Delta-1 (C,D,F,G). Red arrow in (E): failure of the neural tube to close in treated embryo (see text). All embryosare shown in dorsal view. Abbreviations: aip, anterior intestinal portal; fb, primordium for the forebrain; fg, margin of the foregut; hn,Hensens node; mb, primordium for the midbrain; hb, primordium for the hindbrain; nt, neural tube; psm, presomitic mesoderm; s,somites. Scale bar 500 mm in C,D,F,G; 400 mm in B,E.</p><p>478 PSYCHOYOS ET AL.</p><p>Birth Defects Research (Part B) 83:477488, 2008</p></li><li><p>organogenesis (peri-implantation stages), results in 100%embryo death before the embryo can reach organogen-esis (Paria et al., 1995; Persaud and Ellington, 1967,1968a,b). Administration of D9-THC after organogenesis(E1014) no longer results in a high incidence of death orcongenital malformations (Mantilla-Plata et al., 1975;Banerjee et al., 1975; Haley et al., 1975; Harbison et al.,1977; Fleischman et al., 1980).There is clearly a developmental period of suscept-</p><p>ibility to the embryotoxic effects of D9-THC. Thiswindow of susceptibility coincides with the period ofearly organogenesis. In the present studies, we havetargeted this developmental window using the chickembryo in culture model system (stages 31 to 12). Thissystem enables the continuous monitoring of drug-induced teratogenic effects over time. Thus, any mal-formations occurring during the initial stages of embryo-nic development can be easily detected without resultingin embryo death (Gebhardt, 1972; Kotwani, 1998).Furthermore, the separation of the embryo frommaternalinfluences permits the evaluation of a response attributedsolely to the effect of the drug applied to the embryo(Wilson, 1978; Jelinek, 1982). Finally, the chick embryorepresents a long-standing model of embryonic verte-brate development (Romanoff, 1960; Balinsky, 1975;Stern, 2002), with close similarities to the human embryoduring early organogenesis (Romanoff, 1960; Balinsky,1975; Rahilly and Muller, 1987; Sulik, 2008).D9-THC is highly lipophilic and thus requires solubi-</p><p>lisation with either DMSO or surfactant agents (Tween80t, Emulphort), all of which are potentially embry-otoxic. To overcome this problem, we have used a water-soluble analogue of D9-THC, termed O-2545 (Martinet al., 2006). In this cannabinoid, the terminal carbon ofthe side chain of D9-THC is substituted by an imidazolegroup (Fig. 1A). This substitution results in a cannabi-noid receptor 1 (CB1) agonist that produces the samespectrum of pharmacological effects in the mouse modelas D9-THC (Martin et al., 2006). Yet, O-2545 is soluble inPBS and thus readily applicable to the chick embryo inculture.</p><p>METHODS</p><p>Chick Embryo Culture and Drug Administration</p><p>Chick embryos (Charles River, CT, USA) were ex-planted in culture (New, 1955; Psychoyos and Stern,1996a,b), and grouped according to developmental stage(Hamburger and Hamilton, 1951): group 1 (stage 31 to4; 39 controls, 63 treated), group 2 (stage 4 to 41; 39controls, 36 treated), group 3 (stage 5 to 6; 38 controls, 42treated) and group 4 (stage 7 to 8; 8 controls, 13 treated).These stages correspond to days 1319 (weeks 2 to 3) ofhuman pregnancy (Nishimura et al., 1974). Embryoswere treated with a single exposure of O-2545 in PBS(10 ml) at a given dose: Initially, O-2545 was solubilized inPBS at 0.035mg/ml and kept in aliquots at 201C. Forsubsequent experiments, O-2545 was diluted with thesame vehicle to obtain concentrations of 0.070mg/mland 0.35mg/ml and kept in aliquots at 201C. O-2545was thawed immediately before use for all experiments.The drug or vehicle alone for synchronous controls wasgently placed just above the surface of the embryos(ventral side up), using a 10ml Lambda tip (Corning LifeSciences, Corning, NY, USA). Embryos were cultured for</p><p>1822hr at 381C, or until controls (vehicle alone) hadreached stages 911. Embryos were fixed overnight in 4%PFA in depc-PBS, examined for morphological defects aspreviously described (Psychoyos and Stern, 1996b), andprocessed for immunohistochemistry or whole-mount insitu hybridization.</p><p>Endpoints, Morphometric Analysis, andMorphological Score</p><p>Each embryo was given a morphological score basedon the following criteria: (1) abnormal brain primordia;(2) abnormal foregut and/or heart primordia, as visua-lized by abnormal anterior intestinal portal, abnormaldorsal and ventral margins of the foregut, abnormalprecardiac mesoderm, and abnormal heart, wheneverpresent; (3) abnormal neural tube posterior to the levelequivalent to the hindbrain/first somite; (4) fragmentedor absent notochord; and (5) abnormal somitogenesis.For each embryo, 1 point was given for each abnormality,and the points were cumulative. Normal embryos scored0; abnormal embryos scored 1 or above. Embryos withthe highest number of malformations scored 5.</p><p>Short-Term Incubation Following Exposure toCannabinoid</p><p>Embryos in culture were exposed to 0.070mg/ml O-2545, as described above (47 embryos in total; n5 31 withdrug; n5 16 with vehicle alone). Embryos were incu-bated for a shorter period of time (45 hr or 68 hr insteadof 1822 hr), after which they were fixed, grosslyanalyzed, and processed as above. Morphometric criteriaincluded presence of a thickened primitive streak andHensens node, as well as changes in the morphologyof the nascent neural plate and head fold (whenidentifiable).</p><p>Double Whole-Mount in situ Hybridization</p><p>In situ hybridization was performed as previouslydescribed (Wilkinson, 1992; Henrique et al., 1995), withminor modifications: Following rehydration from metha-nol, embryos were incubated in 50mg/ml Proteinase Kfor a shorter period (1min for stages up to 8, 2min forstage 9, and 3min for stages 1012). For the analysis oftwo genes, embryos were hybridized simultaneouslywith digoxigenin (DIG)- and fluorescein (fluorescein-12-UTP, FITC)-conjugated probes (Roche Diagnostics). Afterhybridization at 661C, embryos were washed in hybridi-zation buffer, followed by washes in 100mM maleatebuffer containing 0.1% Tween 20 (MABT). Embryos werethen blocked in MABT containing 2% Blocking Reagent(Roche Diagnostics) and 20% sheep serum (JacksonImmunoResearch), and incubated overnight with anti-DIG antibody coupled with alkaline phosphatase (1:2000;Roche Diagnostics) at 41C. After several washes inMABT, anti-DIG antibody-coupled alkaline phosphataseactivity was revealed with BCIP (5-bromo-4-chloro-3-indolyl phosphate)/NBT (nitroblue tetrazolium chloride)substrate (Roche Diagnostics) in staining solution (0.1MTris-HCl pH9.5; 0.1M NaCl). In order to inactivatealkaline phosphatase activity, embryos were pinned ona sylgard-coated dish containing PBS, fixed overnight in4% PFA, placed in a scintillation vial with 100mM EDTAin MABT buffer, and incubated at 651C for 30min. The</p><p>479CANNABINOID ANALOGUE</p><p>Birth Defects Research (Part B) 83:477488, 2008</p></li><li><p>embryos were subsequently washed in MABT, andblocked in the same blocking buffer as above. Embryoswere incubated in alkaline phosphatase-coupled anti-FITC-antibody (1:500) overnight (41C), and washed inMABT for up to 3 hr. Embryos were washed in Trisbuffer (100mM Tris-HCl pH 8.45 with 0.1% Tween-20),and the color reaction was performed with Vector RedSubstrate Kit (Vector Research) in Tris buffer. Embryoswere transferred to PBS, fixed in 4% paraformaldehyde,and further examined morphologically prior to beingprocessed for photography. The following probes wereused at 200ng/ml: Delta-1 (Henrique et al., 1995); Krox20(Irving et al., 1996); Lef-1 (Schmidt et al., 2004); Noggin(Connolly et al., 1997); Otx2 (Bally-Cuif et al., 1995); Pax6(Goulding et al., 1993); Shh (Levin et al., 1995); Sox2 (Rexet al., 1997); to determine alterations in expressionpatterns secondary to the drug treatment.</p><p>Photomicroscopy and Histology</p><p>Embryos were photographed as whole mount pre-parations with a stereo microscope (model MZ9.5; LeicaMicrosystems Ltd, Switzerland), equipped with a 5.0megapixel CDD 35mm digital camera (model DFC480;Leica Microsystems Ltd, Switzerland). Image acquisitionwas controlled via the MetaView software. Embryoswere processed for sectioning as described previously(Psychoyos and Stern, 1996b). To ensure signal preserva-tion, embryos were dehydrated in absolute methanol, inpropan-2-ol and cleared in 1,2,3,4-tetrahydronaphtalene(Sigma Aldrich) prior to xylene and wax treatment.10 mm Sections were photographed on an uprightmicroscope (model Axioskop 40 A Pol; Carl Zeiss MicroImaging, Inc., Thornwood, NY, USA) equipped witha 1.4 megapixel CDD digital camera (m...</p></li></ul>