Treatment of pregnant Long Evans rats with a low dose of diazepam (1.25 mg/kg from gestational day 14-20) produced offspring suffering from suppression of cellular immune responses. Analogous effects were produced by clonazepam, a benzodiazepine (BDZ) with high affinity for the central-type, and Ro 5-4864, a BDZ with selective affinity for the peripheral-type BDZ receptor. Peripheral-type BDZ receptors are found to develop early in fetal life in peripheral organs including primary (thymus) and secondary (spleen) lymphoid organs, in the central nervous system and on immune cells (lymphocytes). In prenatally diazepam-exposed offspring the affinity constant is significantly changed. BDZ and PK 11195 also inhibit mitogen and alloantigen-induced T and B cell proliferation in vitro in adult murine lymphocytes. Diazepam, Ro 5-4864 and PK 11195 were found to be the most active compounds.

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Prenatal exposure to benzodiazepines (BDZ) can cause behavioral dysfunctions both in humans and in experimental animals. In addition, prolonged impairment of cellular immune functions is found in rats after low dose BDZ exposure (e.g., diazepam 1.25 mg/kg/day) during part of fetal life [gestational days (GD) 14-20]. Analysis of diazepam and its metabolites in maternal and fetal tissues revealed that in this rat model the drug is no longer present at birth, which excludes direct effects of diazepam during the postnatal period. The main target of BDZ in brain, the GABA(A) receptor complex, is structurally and functionally heterogeneous. Besides α- and β-subunits, γ2- or γ3-subunit should be coexpressed for a fully functional BDZ response. Signals of mRNAs encoding for α1, β2 and γ2 are detected in fetal rat spinal cord and lower brainstem by GD 14 and reach telencephalic regions in later fetal life, reminiscent of BDZ receptor ontogeny. Regional subunit distribution differs from the adult brain, one interesting feature being a preponderance of γ2 mRNA troughout fetal life. Since subunit composition influences the sensitivity to BDZ, these data suggest that prenatal effects of BDZ depend upon regional subunit compositions present at different developmental stages. The delayed depression of cellular immune responses in prenatally BDZ-exposed rat offspring during the first 2 postnatal months is accompanied by various changes in immune cell biology. Binding characteristics of the peripheral (ω3) type BDZ receptor are altered until adulthood (8 weeks). Membranes of spleen cell preparations containing mainly lymphocytes exhibit a decrease of affinity for the peripheral ligand [3H]PK11195, splenic marcrophage preparations a decrease of maximal binding capacity. Various defects in cytokine production by macrophages and T lymphocytes were observed: Mitogen-stimulated release of macrophage-derived tumor necrosis factor-α (TNF-α) and of the T cellderived interleukin-2 (IL-2) was drastically reduced at 2 and 4 weeks of life and recovered in young adulthood, exhibiting the same time course of depression as lymphocyte proliferation in response to immune stimuli. Interleukin-6 (IL-6) release remained diminished until adulthood. In female offspring, additional alterations were found in splenic noradrenaline turnover after immune stimulation. The mechanisms underlying the breakdown of the cytokine network in prenatally diazepam-exposed offspring, and the long-term consequences are as yet unknown.

The expression of mRNAs encoding the three GABAA receptor subunits that are associated with the most abundant benzodiazepine-sensitive GABAA receptor in adult cortex, that is, the alpha 1, beta 2, and gamma 2 subunits, was studied in rat cortex during pre- and postnatal development by means of in situ hybridization, gamma 2 and beta 2 mRNAs become detectable in neocortex at gestational day 16 (GD16), alpha 1 at GD18. gamma 2 mRNA exhibits the highest level of expression at early ages, while alpha 1 mRNA levels are low. beta 2 mRNA rises steeply during the last days of gestation. Around birth, it shows the highest expression of the three subunits studied in cortex, and increases further until postnatal day 15 (PD15). The expression of alpha 1 subunit mRNA also increases markedly shortly before birth and accelerates between PD8 and PD15, when it reaches higher levels than the other two subunits. Following the initial high expression, gamma 2 mRNA increases gradually and slowly until PD25. During prenatal development, highest expression of all three subunit mRNAs is found in the upper layers of cortex, that is, cortical plate and marginal zone. The subplate layer does not start to express GABAA receptor subunit mRNAs until GD18. At birth, all developing layers of the cortex express mRNAs for the three subunits, except the marginal zone. Highest levels are found in the upper part of the cortical plate. At the end of the first postnatal week (PD8), the laminar distribution of mRNA expression in neocortex becomes more differentiated. For all three subunit mRNAs, highest expression is then observed in neuron-like cells in layer IV in the granular areas, and over layers III and upper V in agranular areas. Subsequently, between PD8 and PD25, increasing levels of expression are observed over the pyramidal cell layer V. This regionally differentiated, developmental pattern suggests a close relationship between development of GABAA receptor subunits, ingrowth of thalamocortical projections, and maturation of neocortical circuitry.

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