Ascertaining in the mouse model the mechanisms of neural and behavioral birth defects induced by drugs of abuse and other agents and their reversal utilizing various techniques, mainly stem cell therapy - understanding the path by which the neural progenitors exert their therapeutic action. The study of the mechanism focuses on behaviors related to the septohippocampal cholinergic innervation. Our hypothesis is that abolishment in the hippocampus of cholinergic receptor-induced translocation/activation of PKCg represents a principal component in the mechanism by which various substances induce neurobehavioral birth defects.

Our parallel neurobehavioral birth defects model, which controls for maternal confounds, involves perturbation of imprinting in the chick related to defects in translocation/activation of PKCg in the IMHV nucleus

Pregnant female mice were exposed to heroin or other substances (e.g., nicotine or alcohol). Their offspring showed, upon maturity, an abolishment of cholinergic receptor-induced translocation/activation of PKCg in the septohippocampal cholinergic innervation, paralleled with defects in the hippocampus-related eight-arm maze behavior.

Understanding the synaptic mechanism of the behavioral defects enabled reversal of the neurobehavioral teratogenicity using the following therapies: a. manipulating the regulating pathways of the septohippocampal cholinergic innervation b. grafting of normal embryonic cholinergic cells into the impaired hippocampus c. clinically feasible nicotine therapy d. grafting of stem cells (neural progenitors) into the impaired hippocampus.

The mouse findings were confirmed in studies using the chick model, incubating eggs were injected with heroin or other substances. The hatching chicks suffered marked defects in their imprinting behavior, which was correlated with abolishment of cholinergic-induced translocation/activation of PKCg in their IMHV nucleus.

The study of the reversal of heroin neurobehavioral teratogenicity will serve as an example: Neural progenitors derived from normal developing brain were grafted into the hippocampus of mice offspring who were exposed prenatally to heroin and showed hippocampus related defects in behavior and PKC translocation/activation. The grafted cells grew in the host hippocampus as shown in the following figure.

Grafted BrdU-labeled neural precursors identified in the brain of the teratogen-impaired brain by immunofluorescent staining. Some of the transplanted cells acquire markers of specific neural lineages. In the present case double labeling BrdU+, GFAP+ astrocytes (confocal microscopy).

 Grafting of neural progenitors reversed the desensitization of hippocampal PKCg to the cholinergic agonist carbachol as can be seen in the following Figure.

Effect of prenatal treatment with heroin on cholinergic receptor-induced hippocampal PKCg activity and the effect of transplantation of neural progenitors on the heroin induced PKCg changes. 

Furthermore, grafting of neural progenitors restored maze behavior to normal levels as can be seen in the following Figure.

Effects of prenatal heroin exposure and subsequent grafting of neural progenitors on performance in the Morris water maze. Numbers represent the time spent to reach the platform (mean±SEM)

Control (pooled) = Offspring who were exposed prenatally to control injections and were sham grafted postnatally with medium (C-C) or with neural progenitors (C-NP).
H-C = Offspring exposed prenatally to heroin and were grafted postnatally with neural progenitors.
H-NP = Offspring exposed prenatally to heroin and were grafted postnatally with neural progenitors.
 

H-C  required more time than Control reach the platform (p=0.0336).

H-NP differed from H-C (p=0.0043) and did not from C-C and C-NP (p=0.29).

All interactions, prenatal-exposure X test-day, prenatal-exposure X day were not statistically significant.

Sample sizes (number of animals) are: Control, n=28; H-C, n=22; H-NP, n=23.

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