Dose-dependent teratogenicity of the synthetic cannabinoid CP-55,940 in mice.

Potent synthetic cannabinoids (SCBs) are illegally distributed drugs of abuse that are frequently consumed in spite of their adverse consequences. This study was designed to determine if the toxicity observed in adults also extends to the prenatal period by examining the developmental toxicity/teratogenicity of one of these SCBs, CP-55,940, in a mammalian model. First, immunohistochemistry was employed for cannabinoid receptor 1 (CB1) localization within gestational day (GD) 8 mouse embryos; this receptor was identified in the cranial neural plate, suggesting that the endogenous cannabinoid system may be involved in normal development. Based on this information and on previous avian teratogenicity studies, the current investigation focused on cannabinoid exposure during neurulation. The treatment paradigm involved acute i.p. administration of vehicle, 0.0625, 0.125, 0.25, 0.5, 1.0, or 2.0mg/kg CP-55,940 to time-mated C57Bl/6J mice on their 8th day of pregnancy (n>10 litters per treatment group). On GD 17, litters were harvested and examined for numbers of live, dead, or resorbed fetuses, as well as for fetal weight, length, and gross morphological abnormalities. No effect on litter size, fetal weight, or crown rump length was seen at any of the CP-55,940 dosages tested. Major malformations involving the craniofacies and/or eyes were noted in all drug-treated groups. Selected fetuses with craniofacial malformations were histologically sectioned and stained, allowing investigation of brain anomalies. Observed craniofacial, ocular, and brain abnormalities in drug-treated fetuses included lateral and median facial clefts, cleft palate, microphthalmia, iridial coloboma, anophthalmia, exencephaly, holoprosencephaly, and cortical dysplasia. With the most commonly observed defects involving the eyes, the incidence and severity of readily identifiable ocular malformations were utilized as a basis for dose-response analyses. Ocular malformation ratings revealed dose-dependent CP-55,940 teratogenicity within the full range of dosages tested. While examination of additional critical periods and in depth mechanistic studies is warranted, the results of this investigation clearly show the dose-dependent teratogenicity of this SCB.

Developmental but not adult cannabinoid treatments persistently alter axonal and dendritic morphology within brain regions important for zebra finch vocal learning.

Prior work shows developmental cannabinoid exposure alters zebra finch vocal development in a manner associated with altered CNS physiology, including changes in patterns of CB1 receptor immunoreactivity, endocannabinoid concentrations and dendritic spine densities. These results raise questions about the selectivity of developmental cannabinoid effects: are they a consequence of a generalized developmental disruption, or are effects produced through more selective and distinct interactions with biochemical pathways that control receptor, endogenous ligand and dendritic spine dynamics? To begin to address this question we have examined effects of developmental cannabinoid exposure on the pattern and density of expression of proteins critical to dendritic (MAP2) and axonal (Nf-200) structure to determine the extent to which dendritic vs. axonal neuronal morphology may be altered. Results demonstrate developmental, but not adult cannabinoid treatments produce generalized changes in expression of both dendritic and axonal cytoskeletal proteins within brain regions and cells known to express CB1 cannabinoid receptors. Results clearly demonstrate that cannabinoid exposure during a period of sensorimotor development, but not adulthood, produce profound effects upon both dendritic and axonal morphology that persist through at least early adulthood. These findings suggest an ability of exogenous cannabinoids to alter general processes responsible for normal brain development. Results also further implicate the importance of endocannabinoid signaling to peri-pubertal periods of adolescence, and underscore potential consequences of cannabinoid abuse during periods of late-postnatal CNS development.

Novel song-stimulated dendritic spine formation and Arc/Arg3.1 expression in zebra finch auditory telencephalon are disrupted by cannabinoid agonism.

Cannabinoids are well-established to alter processes of sensory perception; however neurophysiological mechanisms responsible remain unclear. Arc, an immediate-early gene (IEG) product involved in dendritic spine dynamics and necessary for plasticity changes such as long-term potentiation, is rapidly induced within zebra finch caudal medial nidopallium (NCM) following novel song exposure, a response that habituates after repeated stimuli. Arc appears unique in its rapid postsynaptic dendritic expression following excitatory input. Previously, we found that vocal development-altering cannabinoid treatments are associated with elevated dendritic spine densities in motor-(HVC) and learning-related (Area X) song regions of zebra finch telencephalon. Given Arc's dendritic morphological role, we hypothesized that cannabinoid-altered spine densities may involve Arc-related signaling. To test this, we examined the ability of the cannabinoid agonist WIN55212-2 (WIN) to (1) acutely disrupt song-induced Arc expression, (2) interfere with habituation to auditory stimuli, and (3) alter dendritic spine densities in auditory regions. We found that WIN (3mg/kg) acutely reduced Arc expression within both NCM and Field L2 in an antagonist-reversible manner. WIN did not alter Arc expression in thalamic auditory relay nucleus ovoidalis (Ov), suggesting that cannabinoid signaling selectively alters responses to auditory stimulation. Novel song stimulation rapidly increased dendritic spine densities within auditory telencephalon, an effect blocked by WIN pretreatments. Taken together, cannabinoid inhibition of both Arc induction and its habituation to repeated stimuli, combined with prevention of rapid increases in dendritic spine densities, implicates cannabinoid signaling in modulation of physiological processes important to auditory responsiveness and memory.

Cannabinoid mitigation of neuronal morphological change important to development and learning: insight from a zebra finch model of psychopharmacology.

Normal CNS development proceeds through late-postnatal stages of adolescent development. The activity-dependence of this development underscores the significance of CNS-active drug exposure prior to completion of brain maturation. Exogenous modulation of signaling important in regulating normal development is of particular concern. This mini-review presents a summary of the accumulated behavioral, physiological and biochemical evidence supporting such a key regulatory role for endocannabinoid signaling during late-postnatal CNS development. Our focus is on the data obtained using a unique zebra finch model of developmental psychopharmacology. This animal has allowed investigation of neuronal morphological effects essential to establishment and maintenance of neural circuitry, including processes related to synaptogenesis and dendritic spine dynamics. Altered neurophysiology that follows exogenous cannabinoid exposure during adolescent development has the potential to persistently alter cognition, learning and memory.

Late-postnatal cannabinoid exposure persistently elevates dendritic spine densities in area X and HVC song regions of zebra finch telencephalon.

Centrally acting cannabinoids are well known for their ability to impair functions associated with both learning and memory but appreciation of the physiological mechanisms underlying these actions, particularly those that persist, remains incomplete. Our earlier studies have shown that song stereotypy is persistently reduced in male zebra finches that have been developmentally exposed to cannabinoids. In the present work, we examined the extent to which changes in neuronal morphology (dendritic spine densities and soma size) within brain regions associated with zebra finch vocal learning are affected by late-postnatal cannabinoid agonist exposure. We found that daily treatment with the cannabinoid agonist WIN55212-2 (WIN, 1mg/kg IM) is associated with 27% and 31% elevations in dendritic spine densities in the song regions Area X and HVC, respectively. We also found an overall increase in cell diameter within HVC. Changes in dendritic spine densities were only produced following developmental exposure; treatments given to adults that had completed vocal learning were not effective. These findings have important implications for understanding how repeated cannabinoid exposure can produce significant, lasting alteration of brain morphology, which may contribute to altered development and behavior.