Abstracts

Rationale: During the period of active synaptogenesis, the immature brain is vulnerable to enhanced neuronal apoptosis (ENA) induced by a variety of drugs. In rodents, this vulnerability is manifest during the first two postnatal weeks in response to alcohol, anesthetics, and certain antiepileptic drugs, such as phenobarbital (PB). In addition, long-term behavioral deficits have been observed following neonatal exposure to these drugs, suggesting that drug-induced ENA may contribute to adverse behavioral outcomes. It has been previously demonstrated that a single administration of these drugs is sufficient to cause ENA in a number of brain regions, including striatum, thalamus, hippocampus, and multiple cortical areas. It remains unclear whether clinically relevant repeated drug exposure will further exacerbate ENA or the drugs target only a limited number of vulnerable neurons. During the period of vulnerability to ENA, most brain areas exhibit a low level of naturally occurring apoptosis, presumably affecting neurons that are at a competitive disadvantage. This raises the possibility that only a subset of neurons may be vulnerable to ENA and once they are cleared by the initial drug exposure, no further ENA should be seen following repeated drug exposure. In the present study, we tested this hypothesis. Methods: Rat pups were exposed to PB (75 mg/kg/d, i.p) daily from postnatal (P) days 5-8. To detect ENA following each subsequent PB dose, we utilized quantification of active caspase-3 immunoreactivity (aCa3 IR) which peaks between 4-8 h following PB injection and is significantly diminished by the time of the next drug exposure. The difference between repeated exposure (e.g., at T=0h and T=24h) determined 8h after initial drug administration (e.g., at T=32h), and the remaining aCa3 IR 32h following exposure at T=0h alone was used to assess the effects of the second PB dose on ENA. The same strategy was used to determine the effects of subsequent PB administrations (at T36h and T48h). Matching injection(s) of the vehicle (saline) were given to control groups. The dorsomedial portion of anterior striatum, an area highly vulnerable to PB-induced ENA, was examined. Results: We found that a single PB dose on P5 had no effect on ENA, while single exposure on either P6 or P7 increased ENA ~2.5 fold and was comparable to the extent of ENA following repeated (P5-6) PB. However, there was no significant difference in the number of aCa3 positive neurons after repeated doses of PB on P6-7 and following a single P6 exposure when measured on P7. Conclusions: Our data indicate that repeated administration of PB does not cause a cumulative increase in ENA in striatum, and support the hypothesis that ENA occurs in a limited number of vulnerable neurons. Our results also suggest that possible contribution of ENA to long-term adverse outcomes may be limited to the initial drug exposure during the critical period of brain development. Support: NIH grant MH079991 and the research grant from the March of Dimes.