Pregnenolone sulfate induces NMDA receptor dependent release of dopamine from synaptic terminals in the striatum

Pregnenolone sulfate (PS) is a neuroactive steroid that directly modulates glutamate and GABAA receptor function, suggesting that it may regulate the balance between excitatory and inhibitory neurotransmission (Farb and Gibbs 1996; Gibbs and Farb 2000). PS also modulates the activity of AMPA and kainate receptors (Wu et al. 1991), σ receptors (Monnet et al. 1995; Hayashi et al. 2000), and certain voltage-gated calcium channels (Bukusoglu and Sarlak 1996; Hige et al. 2006). Modulation of synaptic transmission by PS has been demonstrated in multiple experimental paradigms. PS potentiates spontaneously occurring excitatory postsynaptic currents (EPSCs) in hippocampal cell cultures (Park-Chung et al. 1997; Meyer et al. 2002) and in slices prepared from rat pre-limbic cortex (Dong et al. 2005), as well as evoked EPSCs from hippocampal (Schiess et al. 2006) and calyx of Held synapses (Hige et al. 2006). PS also augments NMDAR independent long term potentiation in the rat hippocampus via modulation of L-type Ca2+ channels and σ receptors (Sabeti et al. 2007), and a PS-like retrograde modulatory factor plays a role in plasticity of immature hippocampal synapses (Mameli et al. 2005). Enzymes for synthesis of pregnenolone from cholesterol (cytochrome P450 scc) and sulfation of pregnenolone to PS (neurosteroid sulfotransferase ST2A1, SULT2B1a) are present in neural tissue (Hojo et al. 2004; Kohjitani et al. 2006). Pregnenolone is inactive at both glutamate and GABAA receptors, indicating that the negatively charged sulfate group of PS is essential for its modulatory activity. Whereas pregnenolone is neutral and lipophilic, permitting rapid permeation across cell membranes, sulfation to form PS results in a negatively charged steroid that could be compartmentalized intracellularly. Pregnenolone sulfotransferase is present in rat C6 glioma cells, where its activity is regulated by AMPA receptors (Kohjitani et al. 2008). Steroid sulfatases are present in rodent, bovine, monkey, and human brain and offer a potential mechanism for inactivation of PS (Compagnone et al. 1997; Mellon et al. 2001; Plassart-Schiess and Baulieu 2001). Collectively, these observations indicate that PS satisfies several classical criteria for identification as a neurotransmitter or neuromodulator: it is synthesized in nervous tissue, has specific receptor pharmacology, and is inactivated by removal of the sulfate group, but the role of PS within the nervous system remains unresolved. Whether PS is stored and released at physiologically active concentrations by either neurons or glia has remained a controversial issue. The average tissue level of PS in extracts of postmortem aged human brain was found to be 2.8 nM in frontal cortex and 4.6 nM in cerebellum, while blood plasma contains about 380 nM free sulfated steroid. In rat, however, the average level of PS in adrenal tissue was 14 nM but was only 0.64 nM in two of five samples from anterior brain (limit of detection: 0.38 nM) and undetectable in other brain regions (Liere et al. 2004). Another study (Ebner et al. 2006) failed to detect PS in extracts of pooled whole rat brain, with a reported detection limit of 141 pM. Nevertheless, the inability to detect gross tissue levels of PS in various regions of rat brain does not exclude the possibility that significant amounts of this steroid might be present on a cellular level (Schumacher et al. 2008). Infusion of 10 nM PS into rat striatum via reverse microdialysis results in a significant increase in dopamine (DA) overflow in the striatum that involves NMDA receptor (NMDAR) function, but not σ receptor function (Sadri-Vakili et al. 2008; Gibbs et al. 2006). To ask whether this high affinity effect of PS was exerted via modulation of presynaptic NMDARs located on dopaminergic terminals, we investigated the effect of PS on DA release ex vivo from a preparation of striatal nerve terminals comprised of mixed synaptosomes and synaptoneurosomes (SSNs). We report that PS at concentrations as low as 25 pM induces [3H]DA release from striatal SSNs, while pregnenolone is without effect, suggesting that subnanomolar concentrations of PS could modulate nigro-striatal DA release. To our knowledge, this study is the first to report direct neurosteroid enhancement of NMDAR-dependent synaptosomal transmitter release at subnanomolar concentrations of steroid.