Perinatal flunitrazepam exposure causes persistent alteration of parvalbumin-immunoreactive interneuron localization in rat prefrontal cortex A. Chistina Grobin * , Jeffrey A. Lieberman, A. Leslie Morrow Department of Psychiatry, Pharmacology and Bowles Center for Alcohol Studies, CB #7160, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7178, USA Received 15 September 2003; received in revised form 4 December 2003; accepted 20 December 2003 Abstract GABA regulates proliferation via GABA A receptors during development of the neocortex. We recently demonstrated that the endogenous GABA A receptor modulator allopregnanolone plays a role in regulating normal neurodevelopment in prefrontal cortex. Benzodiazepine exposure during early development produces marked behavioral changes in adult rats. To determine if exposure to benzodiazepines during development alters GABAergic interneurons in prefrontal cortex (PFC), rat pups were exposed to flunitrazepam (2.5 mg/kg) on postnatal day (P) 2 and assayed for parvalbumin- and calbindin-immunoreactivity on P80. The ratio of parvalbumin labeled cells in deep vs. superficial layers increased five-fold; calbindin-immunoreactivity and total cell number were not altered. These data are consistent with altered distribution of a subset of interneurons after benzodiazepine exposure and suggest a role for GABA A receptor modulation in normal development of GABAergic systems in PFC. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Calbindin; GABA A receptor; Benzodiazepine; Prefrontal cortex; Neurodevelopment; Lamination GABA is the principle inhibitory neurotransmitter in adult mammalian CNS. However, early in development, GABA A receptor-mediated signaling is excitatory; GABA functions as a chemoattractant, serves as a neurotrophic factor [15] and facilitates neurite outgrowth through GABA A receptor mechanisms [1–3]. Moreover, laminar placement of devel- oping cortical interneurons is sensitive to levels of allopregnanolone, an endogenous GABA A receptor modu- lator [8]. The cortical GABAergic system plays a crucial role in refining and focusing signal processing in mamma- lian cerebral cortex via localization of different types of interneurons relative to one another and to principal cells. GABAergic interneurons may be classified into distinct subpopulations based on morphology and expression of Ca þþ binding proteins. Unlike pyramidal cells that follow an inside-out development pattern, GABAergic inter- neurons migrate tangentially from the thalamus [17]. They display a distinctive protracted developmental pattern from E16 to P10 at both the neuron and synapse level [18] and thus may be preferentially vulnerable. However, mechan- isms modulating laminar placement of subpopulations of cortical GABA interneurons are not well documented. The genetic null mutant rodents used to study tangential migration are generally embryonic lethal, therefore post- natal development events are difficult to study. It is possible that GABA A receptor stimulation modulates the localization of a subset of cortical interneurons during development. GABAergic neurotransmission is a potential site of considerable developmental regulation, yet very little is known about the role of allosteric modulators at GABA A receptors. Arguably, gross malformations are not evident after developmental exposure to GABA A receptor modu- lators (ethanol exposure being the most notable exception). However, a wide range of behavioral and biochemical anomalies are associated with developmental exposure to GABA A modulators, with some effects not evident until maturity. Gestational exposure to diazepam increases seizure susceptibility in adult offspring [4] and sex-specific behavioral deficits at maturity [7,13]. Diazepam exposure early in development results in reduced social interaction 0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.12.124 Neuroscience Letters 359 (2004) 9–12 www.elsevier.com/locate/neulet * Corresponding author. Tel.: þ 1-919-843-3794; fax: þ1-919-966-9604. E-mail address: grobinac@med.unc.edu (A.C. Grobin).