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+, positive modulation; -, negative modulation; ne, no effect; [ ], concentration range tested; G-p, indirect effect through activation of G-proteins.

+, positive modulation; -, negative modulation; ne, no effect; [ ], concentration range tested; G-p, indirect effect through activation of G-proteins.

regions. Ionotropic glutamate receptors are directly coupled to cation channels and are responsible for the majority of the excitatory synaptic responses in the mammalian CNS. These receptors are divided into three pharmacologically distinct classes of ion channels distinguished by sensitivity to the selective agonists N-menthyl-D-aspartate (NMDA), a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate. When activated, the NMDA receptor opens a pore permeable to Ca2+, Na+, and K+. AMPA and kainate receptors, when activated, allow Na+ and K+ ions to pass through, although some AMPA receptors are also permeable to Ca2+ (17).

Since the isolation of the first functional recombinant glutamate-receptor clone in 1989 (18), at least 34 other glutamate-receptor subunits have been cloned. On the basis of in situ hybridization and immunocytochemistry, these subunits have been differentially localized to various brain regions. These findings imply a molecular basis for functional differences in glutamate receptor pharmacology depending on brain region and subunit composition. Our laboratory demonstrated that ionotropic glutamate receptors can be directly modulated in both positive and negative directions by certain neuroactive steroids. This was described in electrophysiological studies showing that pregnenolone sulfate (PREGS) acts as a positive allosteric modulator of the NMDA receptor in chick embryo spinal-cord neurons (19). In these studies, 100 ^MPREGS increased the 30 ^M NMDA induced current by approximately200%. In contrast, responses to AMPA and kainate application were inhibited by approximately 30% in the presence of 100 ^M PREGS.

Calcium influx studies using microspectrofluorimetry in cultured rat hippocampal neurons subsequently confirmed and extended these electrophysiological findings by establishing that PREGS can also potentiate NMDA-activated calcium influx (20), and the results from a series of steroids showed that steroids can either potentiate or inhibit NMDA-activated calcium influx (21).

These studies are consistent with early studies of glutamate-receptor modulation using single-unit recordings from rat cerebellar neurons, which showed that systemic steroids can have profound effects on glutamate-receptor activity. Systemic administration of estradiol enhances neuronal excitation by iontophoretically applied glutamate (22), whereas systemic progesterone (PROG) rapidly attenuates the excitation produced by iontophoretic application of NMDA and non-NMDA agonists (23). These studies, however, were done in intact animals with systemically administered steroids using relatively nonspecific recording techniques.

Other electrophysiological studies, this time on dissociated rat hippocampal neurons, reported approximately 70% potentiation of NMDA responses and 6% inhibition of AMPA and kainate responses by 50 ^M PREGS (24). Single-channel studies of the mechanism of pregnenolone sulfate potentiation reported that PREGS increased both the mean single-channel open time and channel opening frequency, with no effect on the single-channel conductance (24,25).

Recent work from our own laboratory has focused on further understanding the mechanisms by which neuroactive steroids can modulate glutamate receptors. This work has taken three different approaches:

1. Modulation of excitatory amino acid-induced currents in mammalian neurons maintained in culture;

2. Modulation of excitatory amino acid-induced currents in recombinant receptors expressed in Xenopus oocytes; and

3. Modulation of excitatory amino acid-induced excitotoxicity.

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