Prenatal Cocaine Exposure Affects Forebrain Development
This research project in Dr. Crandall’s laboratory has been investigating the effects of prenatal cocaine exposure on dopamine influences during development of the mouse forebrain. Dopaminergic inputs reach the developing mouse striatum by embryonic day 13 (E13) when dopamine levels also become detectable. Dopamine receptor mRNA and binding sites appear by E13/E14. Large-scale migration 
of neurons within and from the striatum to the neocortex is robust by E13. Prenatal cocaine exposure disrupts the aminergic system, especially the dopaminergic system, and leads to deficits in cortical structure and function that may be related to disrupted neurogenesis and neuronal migration.
The major route of neuronal migration to the cerebral cortex for projection neurons is considered to be radial, from the ventricular zone (VZ) outwards to the cortical plate. Recent investigations on the origin of the other major class of cortical neuron, the local inhibitory neurons that express gamma-aminobutyric acid (GABA), suggest that many arise from the ganglionic eminences (GE) and migrate from an alternative tangential route (see diagram left) into other regions of the developing forebrain, including the striatum (STR) and the cerebral cortex (NCX).
In collaboration with Drs. Pradeep Bhide and Barry Kosofsky of the Massachusetts General Hospital and Harvard Medical School, coronal slices of the forebrain from E15 mice exposed either to saline or cocaine for 1 week beginning on E8 onwards were cultured on polycarbonate membranes in defined medium (see diagram above). Microscopic glass shards coated with a fluorescent dye, DiI, were placed in the ganglionic eminence/striatum. At the end of the 2-day culture period, the slices were fixed and the position of DiI-labeled neurons was recorded using a grid system to determine the extent of neuronal migration. In addition, the number of DiI-labeled cells entering the cerebral cortex was estimated.
Prenatal cocaine exposure significantly decreased the distance of neuronal migration within the striatum as well as the number of neurons reaching the cortex (compare above figure). Many Di-I labeled cells have migrated from the ganglionic eminence (GE) into the cerebral cortex (CX) of a 250µm thick E15 forebrain slice from the saline control (left) after two days in vitro compared to the cocaine-exposed embryo (right). Arrows indicate several clearly definable neurons with processes indicative of migrating cells. Dashed lines represent the outer edge of the cerebral cortex. Brain slices are oriented such that dorsal is up and medial is to the left.
These in vitro results are being corroborated by additional studies using immunocytochemical markers to study the extent to which GABAergic neurons are affected by prenatal cocaine exposure. Preliminary comparisons indicate (see figure above) that this critical population of cells in the prenatal brain are decreased by as much as 50% in some layers of the developing cerebral cortex. The distribution of GABA-expressing cells in developing cortical layers appears to be selectively decreased following prenatal cocaine exposure.
Coronal brain sections from E15 littermates obtained from the different prenatal exposure groups used for slice cultures were immunostained for GABA. The distribution of GABAergic profiles (darkly stained cellular images) were quantified in the different embryonic cortical laminae (MZ - marginal zone, CP - cortical plate, IZ - intermediate zone and SVZ/VZ - subventricular and ventricular zones).
Prenatal cocaine exposure significantly decreased the number of GABAergic profiles by approximately half in presumptive primary somatosensory cortex. Medial prefrontal cortex where effects of cocaine addiction have been extensively studied pharmacologically and behaviorally, although clearly at a more immature stage, appear to be similarly affected. The GABAergic population in all embryonic cerebral layers except the marginal zone are decreased. This suggests that migration from the lateral ganglionic eminence and not the medial ganglionic eminence may be selectively affected by maternal cocaine exposure.
In summary, by using an in vitro slice culture system and in vivo immunocytochemistry to assay cell migration in this mouse model of prenatal cocaine exposure, cell migration from the ganglionic eminence to the cerebral cortex is significantly changed: 1) substantially fewer cells reach the cerebrum from the ganglionic eminence after 1 week of prenatal cocaine exposure, 2) cells migrated significantly less distance from the ganglionic eminence following prenatal cocaine exposure, 3) the distribution of GABA-expressing neurons in developing cortical layers is selectively decreased following prenatal cocaine exposure, and 4) dopamine signaling appears to influence neuronal migration from the ganglionic eminence into the cerebral cortex of the mammalian forebrain.