The GABAA receptor is found all over the brain and mediates most of the fast inhibitory neurotransmission. A notable feature of the receptor is that it can be modulated by a wide range of compounds. Various anxiolytic and anaesthetic agents including benzodiazepines, barbiturates and anaesthetic steroids work by binding to different sites on this receptor (for review see Hevers & Lüddens, 1998; Mehta & Ticku, 1999).
Over the last decade considerable evidence has emerged that various progesterone metabolites, which are active and in some cases can be synthesised in the brain (Akwa et al. 1991), act directly on the GABAA receptor (Majewska et al. 1986; Turner et al. 1989; for review see Baulieu, 1997) in a stereospecific manner (Harrison & Simmonds, 1984). The most potent neurosteroids reported to date are 5α-pregnane-3α-ol-20-one (tetrahydroprogesterone, THP) and 5α-pregnane-3α,21-diol-20-one (tetrahydrodeoxycorticosterone, THDOC). The enhancing effects of such neurosteroids on GABAergic currents have recently led to the development of related compounds with the aim of developing improved anticonvulsants for clinical use as an alternative therapy to benzodiazepines (Carter et al. 1997; Rupprecht & Holsboer, 1999).
In this study we observe the effect of bath applied tetrahydrodeoxycorticosterone (THDOC) on GABA released synaptically onto GABAA receptors. It is thus not relevant to this study whether the source of THDOC, in studies to which we refer, is from the breakdown of peripherally produced steroids or from synthesis in the brain. To avoid complication, we will thus refer to steroids which have stereoselective modulatory actions on GABAA receptors as neurosteroids throughout this study, irrespective of their putative source in different reported studies.
As well as their clinical relevance, the effects of neurosteroids on GABAA receptors are likely to have important physiological significance. For example, levels of steroid hormones rise in relation to acute stress, (e.g. Barbaccia et al. 1996) and, conversely, fluctuation of such hormones, due to other causes such as the menstrual cycle (Bixo et al. 1997; Bicikova et al. 1998), can cause fluctuation in mood and changes in stress-like tension (Dennerstein et al. 1985; Smith et al. 1998). Moreover injection of THDOC has been shown to increase exploratory behaviour in mice between a dark and light chamber and to inhibit the effects of application of mild electric shocks in rats (Majewska, 1990).
Other examples of modulators of the GABAA receptors which occur physiologically are various cations, in particular H+ ions (Pasternack et al. 1996) and Zn2+ (e.g. Westbrook & Mayer, 1987), both of which certainly vary under normal or pathological conditions and are dependent in their effects on the specific subunit combination of the receptor. Neurosteroids are, however, probably the first physiologically occurring substances to be considered as potential therapeutic agents in this context.
While it seems clear that fluctuations in neurosteroids in the brain result in changes in stress-related behaviours, the mechanism is far from clear. Various steroid hormones have been shown to have genomic effects under chronic conditions but others exhibit non-genomic effects, such as the direct effect on GABAA receptors and these are probably particularly important under acute conditions of hormonal imbalance. Under conditions of acute stress, various neurosteroids have been detected in rat brain up to about 20 nm (e.g. Purdy et al. 1991), though the highest levels measured were not in stress but rather during the 3rd trimester of pregnancy (100 nm THP; Paul & Purdy, 1992). The types of stress which can reasonably be imposed under experimental conditions are, however, relatively mild and it is likely that much higher levels of neurosteroids could occur in situations which cause extreme pain or other stress related conditions.
Various studies have investigated the actions of neurosteroids on receptors of the central nervous system and it has been repeatedly shown with both biochemical and electrophysiological assays that, at micromolar concentrations, neurosteroids greatly increase chloride flux through the GABAA receptor-channel (Majewska et al. 1986; Harrison et al. 1987). On the single channel level this effect has been shown to be due to an increase in the receptor mean open time rather than channel conductance (Twyman & Macdonald, 1992) as a result of binding to a steroid-specific site on the GABAA receptor (Gee et al. 1988; Lan et al. 1990). This effect, which is rapid and fully reversible, has been suggested to be due to a change in the kinetics of a desensitised state (Zhu & Vicini, 1997). Twyman & Macdonald (1992) also showed an increase in open frequency of single channels during steady state application of GABA but this is probably not relevant to the brief pulse of GABA seen by postsynaptic receptors which are largely saturated at the peak of a synaptic current (Edwards et al. 1990).
Very few studies have observed the effects of levels of neurosteroids at concentrations likely to occur physiologically. However, Belelli et al. (1996) have demonstrated nanomolar sensitivity to a range of neurosteroids of recombinant human GABAA receptors expressed in Xenopus oocytes. At even lower concentrations Dayanithi & Tapia-Arancibia (1996) have reported increases in [Ca2+]i in response to picomolar concentrations of allopregnanolone in primary cultures of fetal rat hypothalamic neurones. They suggest this is due to enhancement of the effects of background GABA in the culture.
Few studies have reported the effects of neurosteroids on synaptic signals. One report in toad spinal cord (Reith & Sillar, 1997) demonstrated that micromolar concentrations of the steroid 5β-pregnan-3α-ol-20-one increased both frequency and decay time of GABAergic potentials without affecting glycinergic signals. The observed effect on decay time was in agreement with an earlier study by Harrison et al. (1987), who showed in rat that the synthetic steroid alphaxalone increased the decay time of GABAergic synaptic currents in hippocampal cultures with no effect on rise time or amplitude. A more recent study showed an enhancement of a long-lasting depolarising component of the IPSP in adult rat hippocampus with very high concentrations of THDOC (10-20 μm, Burg et al. 1998). Another functional study (Brussaard et al. 1997, 1999) showed that in the hypothalamus the neurosteroid allopregnanolone decreased the firing rate of female rat magnocellular neurones, which would be expected to result in a decrease in oxytocin release. At micromolar concentrations it also increased the decay time constant of spontaneous IPSCs, during pregnancy, but not after parturition. This change was attributed to a subunit switch in the GABAA receptor. In contrast to these findings, Poisbeau et al. 1997 observed an increase in both amplitude and frequency of spontaneous GABAergic synaptic currents in cocultures of hypothalamic and intermediate lobe pituitary neurones. This latter effect was observed at the physiological relevant concentrations of 10 nm but no change in kinetics of the currents was observed (Poisbeau et al. 1997). The authors concluded this to be a presynaptic effect of the neurosteroid.
Thus although the previous studies clearly show a non-genomic effect of neurosteroids and related compounds via the GABAA receptor, which is likely to be relevant to the anaesthetic effects of such compounds as alphaxalone, it is not at all clear whether endogenous neurosteroids have such effects under physiological conditions. The only study of synaptic currents which uses a physiologically relevant concentration of a neurosteroid was performed on cultured cells at a single concentration. Moreover in the light of recent evidence that different GABAA receptors congregate under synaptic specialisations from those which are found extrasynaptically (Nusser et al. 1998), it seems particularly important to study the synaptic signal in situ in brain slices.
In the present study we address this question by observing the effects of the neurosteroid THDOC on GABAergic synaptic transmission, both at concentrations likely to occur physiologically (50 and 100 nm) and at the higher concentrations used in previous studies (1 and 2 μm). Further, in the light of many studies showing the wide range of GABA receptors which can be expressed in the brain at different stages of development and in different cell types, dependent on the different subunits expressed (e.g. Wisden et al. 1992), we have selected cell types which show different subunit distributions between the cell types and, in the case of hippocampal granule cells, different subtypes over development. We thus investigate whether our observations are general or specific to particular cell types by observing the effect of this neurosteroid in three cell types and at two stages of postnatal development.
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The results of this study clearly indicate that at concentrations of the neurosteroid THDOC that are likely to occur under physiological conditions, GABAA receptor-mediated synaptic currents can be potentiated in the rat brain via an increase in the slow decay time constant. Moreover as this effect is reversible and stereospecific, it is highly unlikely that the mechanism is genomic but rather demonstrates that it is the effect of binding to a specific membrane receptor. It is interesting to note that the fast decay time constant was unaffected by the application of neurosteroids. Although the reason for the double exponential decays of GABA synaptic currents is still controversial; a possible explanation is that the fast decay is related to the rate of initial closing within a burst, perhaps due to rapid desensitisation of the channel, while the slow decay is determined by inactivation of the channel after dissociation of the transmitter (Jones & Westbrook, 1995; McClelland & Twyman, 1999). The latter component may be controlled in part by recovery from desensitisation but also by the unbinding kinetics in the non-desensitised state. Thus the present results suggest that the neurosteroids affect the long closed-time components of channel kinetics rather than the short closed intervals. Note that the fast decay in the average synaptic current will be particularly susceptible to dendritic filtering and thus will be measured as being slower than the within-burst openings measured in outside-out patches. Where the selection criteria for rise time are more stringent the first decay time constant is faster (see discussion below and Edwards et al. 1990).
The effect of neurosteroids is, however, not uniform, with cerebellar Purkinje cells showing a greater sensitivity at concentrations which might be expected to occur physiologically than hippocampal granule cells which, though somewhat sensitive at 10 days, become virtually insensitive to low concentrations (50 or 100 nm) of neurosteroids by 20 days. It should be noted that the true physiological decay times would be considerably faster than those measured here as, like most of the studies quoted, the measurements in this study were made at room temperature (22-25°C).
A difference in sensitivity to neurosteroids of different brain regions at different stages of development has been suggested from a variety of biochemical studies (e.g. Nguyen et al. 1995). For example, Wilson & Biscardi (1997) recently demonstrated an increase in GABA-induced Cl− uptake into ‘microsacs’ prepared from various brain regions including hippocampus and cerebellum in the presence of THDOC or allopregnanolone (100 nm-3 μm). In contrast to the present study, these authors reported that the microsacs prepared from the hippocampus were more sensitive than those from cerebellum. This apparent difference may reflect the fact that it was not possible, in their study, to differentiate between cell types within the preparations or indeed between synaptic and extrasynaptic receptors on individual cells and thus the result reflects an average of a range of receptor types. This may suggest that a particular subtype of neurones, not sampled in the present study, has a different profile of steroid sensitivity. We cannot completely discount the possibility that temperature plays a role in the difference seen, as the study by Wilson & Biscardi was carried out at 30°C.
Interestingly the concentration of neurosteroids and the enzymes which produce them also vary in different brain areas (Bixo et al. 1997). This regional variation suggests that, whether produced locally in glia or entering the brain from peripheral sources, the neurosteroids could have their effects in very specific areas, i.e. only where the appropriate receptor is present.
Potentiation of GABAergic transmission via an increase in the synaptic decay time constant, as observed here, is consistent with the previously observed electrophysiological effects of this and related compounds. Belelli et al. (1996) demonstrated that GABAA receptors expressed in oocytes were sensitive to the neurosteroid 5α-pregnan-3α-ol-20one (THP) and other related compounds, regardless of whether the α1, 2 or 3 subunit was included. This is one of the few groups to have used physiological concentrations of neurosteroid and they were able to demonstrate effects of THP at concentrations as low as 3 nm with an EC50 of 90 nm. Most other studies have only tested effects of high concentrations (≥ 1 μm). However in these high-concentration studies the neurosteroids and related compounds have consistently been shown to increase the open time of GABA receptor channels without changing the conductance of the channel (Harrison et al. 1987). Moreover after it was demonstrated by Jones & Westbrook (1996) that desensitisation of the GABAA receptor can buffer the protein in a bound state, so that the rate of final closing is slowed, Zhu & Vicini (1997) demonstrated that the presence of neurosteroids may slow the recovery of the receptor from the desensitised state and hence prolong currents by this mechanism.
It seems likely that the differences in kinetics seen in the absence of neurosteroids and the different efficacy of steroids on GABAergic synaptic currents, reported here, are due to the presence of different subunits in the GABAergic synapses recorded in different brain areas and at different stages of development. A wide range of subunit combinations show neurosteroid sensitivity, though some specific subunit combinations have been shown to increase the sensitivity of the GABAA receptor to neurosteroids (Shingai et al. 1991; for review Lambert et al. 1999). Hauser et al. (1995) demonstrated an increase in sensitivity if the α6 subunit was included. Although it has not been directly tested, this suggests a role for the α4 subunit, which shows a high degree of homology with α6. Moreover a recent study has demonstrated that withdrawal from progesterone in rats results in a decrease in decay time constant of GABAergic currents and is associated with an increase in α4 subunit RNA (Smith et al. 1998). A recent study also compared various recombinant receptors containing different α or γ subunits. They concluded that a lack of α subunits greatly decreased the efficacy of allopregnenolone for enhancing GABAergic currents, with α2 being more effective than α1 subunits and γ3 imparting both greater sensitivity and greater efficacy than γ1 or γ2 subunits (Maitra & Reynolds, 1999). Other studies have suggested involvement of various other subunits. Zhu et al. (1996) showed a strong inverse correlation between steroid sensitivity and δ subunit transfection in HEK293 cells, causing a complete loss of neurosteroid sensitivity. Moreover, interestingly, studying cerebellar granule cells in primary cultures, they showed a very similar result to that seen here in hippocampal granule cells in brain slices. They reported a decrease in the potentiation of GABAA-mediated currents by THDOC (100 nm-10 μm) in cells which had been in culture for 14 days compared to those after 4 days in culture (Zhu et al. 1996). They concluded from RT-PCR on these cells and comparison with sensitivities in transfected HEK cells that this change was due to the increased number of cells expressing the δ subunit in the 14 day cultures.
The α4 and δ subunits may be particularly relevant here. During postnatal development the dentate gyrus has been shown to express α1, α3 and α4, as well as β1 and γ2, until around day 12. Around day 12 there is a decline in α4 and β1 subunits and the δ subunit becomes detectable for the first time (Laurie et al. 1992). This correlates well with the relative insensitivity in the granule cells of the 20 day group shown in the present study and the variation in sensitivity seen in different cells of the 10 day group (P9-P13) where some synapses may have already switched and others may be still in the neonatal form. In contrast to the hippocampus, cerebellar Purkinje cells show no change in subunits expressed during development and do not express a δ subunit. This is again in agreement with the present study where the Purkinje cell synapses remained sensitive at both stages of development.
While the parallels which can be drawn from the literature concerning subunit expression and neurosteroid sensitivity are highly suggestive, other mechanisms are also possible. In particular cAMP-dependent phosphorylation of GABAA receptors has been shown to decrease whole-cell responses to GABA application and to change the desensitisation kinetics, which could also be compatible with the present result (e.g. Moss et al. 1992). All intracellular solutions contained MgATP (2 mm) but no other phosphate-regenerating solutions. Although no run-down was observed in the experiments, it is possible that different synapses could have localised supplies of phosphatases and/or kinases which can cause local regulation of GABAA receptors or that these factors can affect different subtypes of the receptor differentially. However, we consider the difference in subunit combination a more likely explanation.
Note that the decay kinetics of the control currents reported here for HG20 cells are slightly different from those reported previously with very close to identical recording conditions (Edwards et al. 1990). This is probably due to two main factors. (1) From experience of using Wistar rats for similar experiments in Germany, UK and Australia, there seem to be genuine differences between GABAergic synaptic currents (F. A. Edwards, unpublished observation). This may be a true factor of breeding, suggesting that differences occur within one strain as generations are bred in isolation from each other. Alternatively the exact breeding and holding conditions, though similar in different animal houses, may be sufficiently different to cause acute or chronic differences in neuromodulators which affect GABA channels or the exact proportions of particular subunits expressed. (2) The apparently slower fast exponential may be an artefact of the selection procedure. In the earlier study (Edwards et al. 1990) the important measured factor was the exact amplitude of individual currents. Recordings were thus highly selected, only being continued on cells where rise times were very fast (< 1 ms) and background noise extremely low. In the present study such selection was not necessary as the main factor affected appears to be the slow decay time of the currents. Thus this study represents a wider sample of the population, with all cells being accepted for recording. Currents were afterwards selected for the somewhat less stringent criterion of a < 2 ms 10-90 % rise, which only excluded currents where artefactual noise or overlap of consecutive currents impeded the measurement. Hence a certain degree of dendritic or series resistance filtering may have slowed the recorded fast component, which would be much more sensitive than the slow component, in which we were primarily interested in this study. Note that it is unlikely that differences in dendritic filtering between ages was a determining feature in the developmental effects seen as the rise times between the two age groups was unchanged in control conditions for hippocampal cells. Moreover changes in dendritic spread would be expected to be considerably greater in Purkinje cells where in fact no change was seen in the effects of THDOC with development.
It is interesting to note that, in the first postnatal week, GABAergic transmission is excitatory in many cells of both the hippocampus and cerebellum (Ben-Ari et al. 1989) due to lack of expression of the chloride transporter KCC2 (Rivera et al. 1999). This results in a relatively high intracellular chloride concentration in neurones of young animals, thus changing the reversal potential of chloride currents such as GABAergic IPSCs. In hippocampal slices from newborn animals, GABA-activated currents result in depolarising potentials which change to hyperpolarising inhibitory potentials during the P9-P12 period. Thus again the P9-P13 period of the 10 day groups in our present study is shown to be a transition period in which the adult pattern of inhibitory transmission is being established in the hippocampus. The effect of the steroid would therefore presumably be to enhance excitability in young animals, and in the dentate gyrus the inhibitory currents mediated by GABAA receptors seem to be largely insensitive to the effects of neurosteroids. In contrast, in the cerebellum, the neurosteroid sensitivity remains so that these intrinsic compounds would have an opposite effect on modulation of excitability in new born versus adult animals in Purkinje cells. It is perhaps relevant that the Purkinje cells, which were the most sensitive cell type tested, are themselves GABAergic and it would be interesting in future studies to investigate whether there is any systematic difference between the sensitivity of excitatory and inhibitory neurones to neurosteroids at physiological concentrations.
In summary, we have shown that the neurosteroid THDOC is likely to have important modulatory effects at concentrations which occur naturally in the brain, in some but not all GABAergic synapses of the central nervous system. The differential sensitivity of different cell types to the neurosteroids is of particular interest in terms of development of potential therapeutic compounds which may be able to be targeted to specific brain areas without causing a general effect across the whole brain.