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1Neuroscience Research Institute, Department of Psychology, Carleton University, Ottawa, Ontario, Canada; and 2Department of Neuropharmacology, Brain Research Institute, University of Bremen, Bremen, Germany
Submitted 2 December 2004; accepted in final form 25 May 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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1 subunit expression in the NC rat strains and the higher 2, 3, and 5 subunit expression in the SP strain. Using patch-clamp recording, we investigated how the neurosteroids tetrahydrodeoxcorticosterone (THDOC) and allopregnanolone at physiological and pharmacological concentrations may differentially affect the mIPSCs in the perirhinal cortex of brain slices isolated from SP and NC rats. We found that 100 nM THDOC prolonged the time course and increased the amplitude of both the mono- and biphasic mIPSCs in the SP rats, but these effects were smaller in the NC rats. By comparison, allopregnanolone (100 nM) had small effects in both the NC and SP rats. At 1.0 µM, THDOC enhanced mIPSCs in both strains, but this effect was not greater in the SP rat than it was at 100 nM. By contrast, allopregnanolone (500 nM) enhanced the time course of the mIPSCs in both strains but it reduced mIPSC amplitudes as well. THDOC (100 nM) was much more effective than 100 nM allopregnanolone in inducing a tonic current in SP and NC rats. These data show that neurosteroids modulate synaptic activity at synapses having different biophysical behaviors. As differing GABAA receptors are targeted by subsets of interneurons, these data suggest these neurosteroids may selectively modulate one inhibitory input over another. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Cooper et al. 1999; Harrison et al. 1987; Jorge et al. 2000), and evidence indicates that they may be endogenous acute and perhaps long-term modulators of GABAA receptor activity and synaptic inhibition (Bitran et al. 1995; Calixto et al. 1995; Fahey et al. 1995; Fernandez-Guasti and Picazo 1995; Frye 1995; Frye and Sturgis 1995; Hollis et al. 2004; Lambert et al. 1995; Leroy et al. 2004; Poisbeau et al. 1997; Poulter et al. 1997; Puia et al. 1990; Yu and Ticku 1995; Zhu and Vicini 1997). Additionally, several studies have reported differential sensitivity to neurosteroids in different neurons in discrete brain regions (Harney et al. 2003), in the same neurons in wild-type and GABA receptor subunit knockout mice, in the same neurons before and after pilocarpine-induced status epilepticus (Peng et al. 2004) or before and after progesterone withdrawal (Smith et al. 1998a). Neurosteroids are metabolites of steroid hormones that have modulatory effects on GABAA receptor function by a number of mechanisms, including enhancement of affinity and by alteration in the desensitization kinetics (Lambert et al. 2003).
One class of neurosteroids having both positive and negative modulatory effects on GABAA receptors are metabolites of pregnanolone. The best characterized is the positive modulator allopregnanolone, which is synthesized in numerous body tissues (Compagnone et al. 1995; Mickan and Zander 1979). Evidence suggests that allopregnanolone levels may rise and fall depending on the progesterone concentration. In particular the "crash" in progesterone plasma concentrations after parturition has been implicated in increased susceptibility to drug-induced seizures (Smith et al. 1998a,b). Tretrahydrodeoxycorticosterone (THDOC) is another neurosteroid of interest that is synthesized from deoxycorticosterone (DOC). DOC is released during stress under the control of corticotrophin releasing hormone and adrenocorticotropin hormone (Reddy 2003). Thus it has been suggested that psychological stressors may cause a modulation of GABAA receptor activity. Although an attractive hypothesis, in the presence of stress, plasma concentrations of THDOC rise to levels that would have only small effects on GABAA receptor activity (Reddy 2003). So, although one may surmise that these compounds are endogenous modulators of inhibition (synaptic or extrasynaptic), conclusive evidence that they fulfill an important physiological function so far has not been presented.
Nevertheless, other evidence suggests that pharmacological doses of THDOC may be effective anticonvulsants in young rats and for the treatment of infantile seizures in humans. In this way, administration of adrenocortical hormones may act by enhancing the levels of THDOC via the release of its precursor DOC (Edwards et al. 2002a,b).
The molecular pharmacology of neurosteroids is complex and may vary from one steroid to the next (for review, see Lambert et al. 2003). Some neurosteroids (e.g., allopregnanolone) seem to have little selectivity, whereas others like THDOC may show significant, albeit not fully appreciated selectivity. This selectivity may determine neurosteroid activity in synapses having different subunit compositions as well as between synaptic versus extrasynaptic sites.
In cortical neurons, we have shown that two kinds of miniature inhibitory postsynaptic current (mIPSC) behaviors are discernable in voltage clamp. One type having monoexponential deactivation kinetics is 
20–30% smaller in amplitude than the other type, which deactivates with biexponential kinetics (Hutcheon et al. 2000; McIntyre et al. 2002). We have also shown that seizure-prone rats (SP; fast kindling) overexpress immature forms of the GABAA receptors (2,3 and 5) (Poulter et al. 1999). This appears to have a functional impact because mIPSCs recorded from SP rats, particularly on their interneurons, have slowly decaying small-amplitude mIPSCs in comparison to normal outbred rats. Here, we have extended these studies where we have investigated the effects of neurosteroids on the unitary synaptic events in these two strains of rat. The aim was to determine if neurosteroids affect these two synaptic behaviors in a similar manner and whether a rat expressing different GABAA receptors would have unique synaptic responses to neurosteroid modulation.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Animals
The SP rat strains were originally developed at McMaster University from an outbred parent population consisting of a Long Evans hooded and Wistar cross. They were selectively bred originally from that parent population to be fast in their rates of amygdala kindling (Racine et al. 1999). Male SP rats used in the present project were taken randomly from 41st to 48th generations. Long Evans hooded rats were chosen as normal controls' (NCs) as they were one of the two original parent strains used to derive the fast kindling (seizure prone) strain. The NC rats were purchased from Charles River Canada.
Electrophysiology
Patch-clamp recordings were performed on brain slices isolated from adult rats (60–200 days old). To obtain viable slices, heavily anesthetized rats (sodium pentobarbital, 80 mg/kg ip) were perfused with an ice-cold Ringer solution in which sodium was replaced by choline (composition in mM: 110 choline Cl, 2.5 KCl, 1.2 NaH2PO4, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2, 2.4 Na pyruvate, 1.3 ascorbate, and 20 dextrose), thus cooling and neuroprotecting the brain in situ. This methodology has been previously reported (McIntyre et al. 2002). During electrical recordings, the submerged slices were bathed in a standard artificial cerebrospinal fluid (ACSF) solution consisting of (in mM) 120 NaCl, 2.5 KCl, 1.2 NaH2PO4, 25 NaHCO3, 2.0 CaCl2, and 1 MgCl2, pH 7.3–7.4 and osomolarity adjusted to 295–305 mosM.
Recordings were done using KCl patch electrodes having an internal composition (in mM) of 145 KCl, 10 NaCl, 2 CaCl2, 10 EGTA (yielding a free Ca2+ concentration of 100 nM), 2 MgATP, 10 dextrose, and 10 HEPES; the solution was 300–320 mosM, pH adjusted to 7.3–7.4. The input resistances of these electrodes were 3–8 M
, and their shanks were coated in bee's wax to reduce electrode capacitance. Recordings from neurons in layers 3 and 5 of the perirhinal cortex were made using an Axopatch 200B amplifier (Axon Instruments, Carver City, CA) or HEKA EPC-9 (HEKA, Mahone Bay, N.S., Canada). Series resistance compensation was performed in all recordings. Acceptable recordings were those where the initial access resistance was <20 M, that could be compensated by 60–90% (50–100 µs lag). The series resistance was monitored throughout the recordings, and if it rose irreversibly >20 M, the recording was terminated. Under these conditions and based on size of the currents monitored (20–200 pA), the remaining uncompensated series resistance caused a voltage error of <1%. Filtering errors were also negligible.
Neurons were visualized with differential interference contrast optics. In voltage-clamp mode, spontaneously occurring mIPSCs were collected in a "blocking ACSF" solution containing 200–500 nM tetrodotoxin (TTX; Alomone Labs, Tel Aviv, Israel), 10 µM dinitroquinoxaline 2,3 dione, and 20 µM 2-amino phosphonopentanoic acid (DNQX and APV, respectively; RBI). Neurosteroids compounds, both allopregnanolone (Sigma-Aldrich, Mississauga, Ontario, Canada) and tretrahydrodeoxycorticosterone (THDOC; Sigma-Aldrich), were dissolved in 100% ethanol at a concentration of 10 mM. Final dilution in ACSF was done on the day of the experiment, and drugs were applied by bath perfusion. Drug equilibrium was assumed after twice the length of time that was required for the blocking ACSF to suppress the sodium current (this usually occurred within 5–10 min). Thus all neurosteroid data were collected after a minimum of 10–20 min after the perfusion commenced. Collection of mIPSCs continued for up to another 20 min with no obvious change in mIPSC attributes. Washout of the drug required 45–90 min. As maintaining the recording for this period of time happened only rarely, complete reversibility was rarely achieved. The cumulative distributions of the washout data reflect this slow washout.
Recordings were made in layers 3–5 of the perirhinal cortex. The recordings were performed at room temperature (22–26°C) because at higher temperatures, mIPSCs arrived too quickly and were rarely separated from one another enough to permit useful fitting and analysis. All mIPSCs were acquired at a holding potential of –60 mV. Steroids were added to the blocking ASCF at the required concentration.
Cells were identified as interneuronal based on the criteria and methodology used in McIntyre et al. (2002). Cells from which recordings were made were filled with 0.5% biocytin (Sigma-Aldrich, Oakville, Ontario, Canada) or DiI (Molecular Probes, Eugene, OR). Biocytin was subsequently visualized by streptavidin conjugated to the fluorescent 6-(7-amino-4-ethylcoumarin-3-acetyl) amino hexonic acid molecule (Jackson ImmunoResearch, West Grove, PA). A series of digital photos in the z axis (1.5 µM apart) were taken with a Photometrics Star 1 camera (x250 magnification; 580 x 380 pixel resolution). The stack of images was subsequently deconvolved using a commercially available software package (Exhaustive Photon Reassignment; Scanalytics, McClean, VA). The entire cell morphology was visualized by projecting the deconvolved optical sections onto a single plane. Classification of the cells as pyramidal was based on the morphology of the soma, the existence and orientation of a dominant dendrite, and the projection of the axon.
Analysis of mIPSCs
To provide insight on how synaptic currents might temporally summate, 50–100 individual mIPSC events were chosen at random, aligned by their rising phases (<1.5 ms), and averaged together. The resulting signals, denoted by an appended subscripted "av" (hence mIPSCav), had their declining phases fitted with sums of exponentials. One-way within-group ANOVA was used to assess for drug effects in these analyses. The estimate of the change in charge transfer after drug application was calculated from the amplitude and time course of the mIPSCav in each cell.
To evaluate the differential effects of the drug on the mono- and biexponential mIPSCs that comprise the average response, the deactivation phases of the mIPSCs were individually fitted with exponential functions. We used Axon instrument pClamp 7.0 for fitting and an in house macro that runs in Excel for sorting attributes and generating distributions. Thus mono- and biexponential fits were performed on each mIPSC. The residual deviations were then compared to decide which of the fits to retain as we have described previously; therefore it will only be briefly described here. First, all mIPSCs that were collected in a recording were sorted to eliminate events with 10–90% rise times >1.5 ms, half-width durations <4 ms (i.e., events that were too brief to be considered genuine GABAergic synaptic transients) as well as those temporally nondiscrete events. After sorting, 80% of the collected events were rejected for further analysis. Our initial screen of these rejected events showed that 30% of the mIPSC had rise times that were >1.5 ms, 10% were false events (shifts in the baseline or noise), while another 40% could not be fitted because another mIPSC occurred within the fitting window.
As allopregnanolone and THDOC have been reported to effect GABAA receptor-mediated inhibition, both were used in this study to compare and contrast their activity in each strain. This study was divided into two parts. First, a relatively low concentration of steroid was used (100 nM) that may be considered to be in the high physiological range (Barbaccia 2004; Barbaccia et al. 2001; Cooper et al. 1999; Lambert et al. 2003; Paul and Purdy 1992). Second, higher concentrations (500 nM and 1.0 µM) were also used. These concentrations are not likely attained in the brain (unless local high concentrations occur) and may be considered pharmacological.
After categorization, in a comparison of the drug's effects, 90–150 mIPSCs were selected as a representative sample to include diversity with respect to amplitude and kinetics parameters for each event. These representative samples were otherwise randomly selected. The median values describing the amplitudes and deactivation kinetics of the selected mono- and biexponential mIPSCs from each cell were calculated and then compared with the median values from the corresponding mIPSCs in the presence of the neurosteroid in the same cell. The differences between the median mIPSC attributes (time constants, amplitudes etc.) from each recording were then averaged to calculate the response to the drug in the two strains of rat. As the average of these median differences is normally distributed, we evaluated the drug effects by a paired Student t-test. Significance of the differences was set at P < 0.05. Values reported in the text are the average median values (±SE) for a given set of recordings.
Also as it was possible that these compounds may potentiate the activity of ambient GABA or GABA spillover from the synaptic cleft and/or directly potentiate GABA receptors, the holding current was carefully monitored.
In sum, our analysis first focused on the alterations in the amplitude and the deactivation phases of the average synaptic time course. We then examined the individual responses (mono- and biphasic mIPSCs) that make up these averages to determine if categorized populations are differentially sensitive to steroids. We also kept track of the holding current during steroid application as this indicates the enhancement of a tonic inhibitory component. Thus the experiments described below examine how low and high concentrations of THDOC and allopregnanolone may alter the inhibitory synaptic and tonic inhibition in these two strains of rat.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Low concentration responses of THDOC and allopregnanolone
First we compared the activity of THDOC on the average time course of mIPSCs in NC and SP rats. Figure 1 shows that 100 nM THDOC prolonged the time course of the averaged mIPSC in NC rats (baseline 
f: 2.5 ± 0.5 ms and s: 23.2 ± 4.6 ms; THDOC: 4.4 ± 0.6 and 35.6 ± 5.2 ms, P < 0.03, n = 8). THDOC also enhanced the amplitude significantly by 13 pA (25%; baseline: 51.3 ± 4.8 pA; THODC: 64.2 ± 5.2 pA; P < 0.01, n = 8 see Fig. 1). The combination of the prolonged deactivation and enhanced amplitude increased the total charge transfer by 71 ± 12% (summarized in Table 1). A further analysis of these data where we segregated the events into the mono- and biphasic responses showed that in NC rats the changes in the average mIPSCav were accounted for by an enhancement of the slow time course of the biphasic population's deactivation. This value changed from an average median value of 36.0 ± 5.5 to 44.2 ± 6.3 ms (P < 0.01). The time course of the monophasic mIPSCs was, surprisingly, unchanged, but the amplitude of these events was increased by 12 pA (control: 34 ± 4.1 pA; 100 nM THDOC: 46.6 ± 0.4 pA; P < 0.02). The amplitudes of the biphasic mIPSCs were unchanged.
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18 ms (from 40.3 ± 3.3 to 58.6 ± 5.6 ms; P < 0.01, Fig. 2). The contribution of the fast deactivation to the overall decay significantly changed from 50 to 25% as well. Thus a larger proportion of current decayed more slowly. Like in the NC rats the amplitude of the mIPSCav also increased significantly by 12.8 pA (baseline; 35.2 ± 5.8 vs. 48.0 ± 4.6 pA; n = 9 for all observations). Overall, based on charge transfer, THDOC enhanced inhibition in the seizure prone rat by 182 ± 25% and therefore was about twice as effective in enhancing inhibition compared with NC rats (summarized in Table 1).
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26 ms (control: 52.2 ± 5.9 ms; THDOC: 77.8 ± 7.8 ms, P < 0.03). In contrast to NC rats, the biphasic mIPSCs were increased in amplitude by 15.5 pA (control: 37.1 ± 2.6 pA; THDOC: 52.6 ± 4.1 pA, P < 0.01). The cumulative distributions of all the attributes that were measured for all mIPSCs summarizes these analyses for all recordings.
Next we compared the activity of allopregnanolone. In general, allopregnanolone had only weak effects at 100 nM in the NCs. For the mIPSCav, only the fast time constant of the fast deactivation component was reduced by 1.5 ms (baseline: f: 3.8 ± 0.7 ms and s: 25.6 ± 4.2 ms; allopregnanolone f: 2.3 ± 0.6 ms and s: 29.6 ± 3.9 ms; n = 7, P < 0.04, Fig. 3, A and B). It also significantly enhanced the amplitude of the mIPSCav. Only statistically significant effects on the individual monophasic mIPSCs were identified where allopregnanolone enhanced their amplitude and prolonged the time constant (control: 13.6 ± 2.9 ms, allopregnanolone: 18.8 ± 2.3 ms; control: 40.4 ± 2.1 pA, allopregnanolone: 44.6 pA, P < 0.05). These effects had only a small impact on the total charge transfer that increased by 7% (summarized in Table 1).
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f: 4.6 ± 1.6 ms and s: 47.5.6 ± 4.2 ms; allopregnanolone f: 3.8 ± 0.7 ms and s: 32.6 ± 3.9 ms; n = 12; P < 0.03, Fig. 4A). The amplitudes of the mIPSCav, unlike in NC rats, significantly decreased by 7 pA (approximately –20%; control: 33.3 ± 3.1 pA; allopregnanolone: 26.2 ± 2.6 pA; P < 0.02, Fig. 4A). After sorting the events into their respective categories, we found that the alterations in averaged mIPSCs were due to a complex combination of effects on the mono- and biphasic mIPSC populations. First like that seen with THDOC, there was a similar increase in the amplitude of the biphasic population (control: 36.8 ± 4.5 pA; allopregnanolone: 62.4 ± 4.2 pA, P < 0.03, Fig. 4). By contrast this increase was mirrored by a decrease in the amplitude of the monophasic population (control: 25.0 ± 3.8 pA; allopregnanolone: 14.1pA ± 2.6 pA, P < 0.02). As the latter events compromise between 65 and 80% of all mIPSCs collected, this offsets the effect on the biexponential population, and the overall effect on the mIPSCav was therefore a sum of these opposing responses. With regard to time course, we found no change in the monophasic mIPSC's deactivation, but the slow time constant of the biphasic mIPSCs became faster by 22 ms (baseline: f: 6.1 ± 2.3 ms and s: 55 ± 4.8 ms; allopregnanolone f: 5.8 ± 2.6 ms and s 23.1 ± 4.8 ms, P < 0.03) accounting for the change on the IPSCav. The sum of these two effects was to reduce the total amount of inhibition by 33% (summarized in Table 1).
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As the lower concentrations of THDOC and allopregnanolone employed in the preceding text had only small effects on the NC, we wished to compare higher pharmacological doses. In NC rat, both allopregnanolone and THDOC had similar effects. We chose 1.0 µM THDOC as it has been used at this concentration previously in a number of studies (Lambert et al. 2003). However, an equivalent concentration of allopregnanolone produced very large increases in holding current that were accompanied by an apparent block of the mIPSCs in all tissues (see Effects on holding current). Thus we used 500 nM allopregnanolone, which did not block the mIPSCs in both NC and SP rats.
Figure 5 shows the effects of 1.0 µM THDOC in NC rats. In NC rats the time course of mIPSCav was lengthened by increasing the slow time constant by 74% (baseline: 24.5 ± 3.7 ms; THDOC: 43.0 ± 7.1 ms, P < 0.03, n = 8). THDOC had a small but statistically significant effect on the contribution of the fast deactivation increasing it by 5% (baseline: 36 ± 2%; THDOC: 41.6 ± 3%). THDOC also uniformly and reliably increased the amplitude of mIPSCav by 16 pA (baseline: 48.1 ± 5.2 pA; THDOC: 64.3 ± 6.9 pA, P < 0.03). Again, breaking the average down into its two components showed differential pharmacological effects. THDOC (1.0 µM) produced a very large (32.7 ms) prolongation of the deactivation in the monophasic responses (190%; baseline: 17.1 ± 3.7 ms; THDOC: 49.6 ± 7.4 ms, P < 0.001), while increasing amplitude by 10.2 pA (33%; baseline: 31.3 ± 2.7 pA; THDOC: 41.5 ± 5.4 pA, P < 0.02). THDOC also prolonged the fast phase of the biexponential mIPSCs by 2.4 ms (85%; baseline: 2.8 ± 0.4 ms; THDOC: 5.2 ± 0.8 ms, P < 0.02) and prolonged the second time constant by 7 ms (21%, baseline: 31.2 ± 1.2 ms; THDOC: 38.8 ± 4.7 ms, P < 0.03, n = 8 for all observations).
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f: 5.0 ± 1.2 ms; THDOC: 7.0 ± 1.6 ms, n = 9, P < 0.02), whereas the slower deactivation increased by 19 ms (45%; baseline: s 42.2 ± 3.6 ms; THDOC: 61.2 ± 8.4 ms, P < 0.02). The amplitude of IPSCav was also enhanced by 14 pA (45%; baseline: 38.2 ± 3.7 pA; THDOC: 52.6 ± 8.4 pA, P < 0.03, n = 15). Interestingly, the contribution of the fast deactivation was reduced from 44% of the decay to just 16%. Overall and similar to 100 nM, these effects sum to a very large increase in charge transfer of 140% (1,137 ± 257 vs. 2,800 ± 589 pC) per mIPSC. Concomitant changes in both the mono- and biphasic populations were found that account for these large effects on mISPCav. The monoexponential mIPSCs increased by 18 pA (61%; baseline: 29.2 ± 5.2 pA; THDOC: 47.7 ± 10.2 pA, P < 0.03, Fig. 6). The deactivation rate slowed by 18 ms (83%; baseline: 21.6 ± 3.2 ms; THDOC: 39.2 ± 5.2 ms, P < 0.02). Both deactivation phases of the biphasic population were increased by 2 and 52 ms, respectively, an increase of 33 and 100%, respectively (baseline: f: 6.1 ± 1.8 ms and s: 54.6 ± 3.4 ms, THDOC f: 8.3 ± 2.8 ms and s:106.3 ± 4.7 ms, P < 0.02). Finally, the amplitude of both the mono- and biphasic mIPSCs was enhanced by 18 and 13 pA, respectively (baseline: 28.5 ± 4.8 and 37.2 ± 2.1 pA; THDOC: 47.0 ± 5.0 and 50.3 ± 4.4 pA, P < 0.02). Statistically, there was no difference in the effects of THDOC at 100 nM and 1.0 µM, indicating the GABAA receptors in the SP rats were already maximally modulated at the lower concentration.
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f: 3.6 ± 1.7 ms and s: 26.3 ± 5.8 ms; allopregnanolone f: 3.1 ± 1.4 ms and s: 40.3 ± 9.2 ms, n = 15 P < 0.03), but the amplitudes of the mIPSCav were not increased (baseline: 45.7 ± 5.9 pA; allopregnanolone: 50.1 ± 7.9 pA, P < 0.10). In contrast to THDOC, allopregnanolone acted selectively on the monoexponential mIPSCs (87%; baseline: 16.0 ± 4.9 ms; allopregnanolone: 30.3 ± 8.0 ms, P < 0.03), but it had no effect on the biphasic population (baseline f: 3.3 ± 1.9 ms and s: 28.3 ± 4.8 ms; allopregnanolone f: 3.2 ± 1.4 ms and s: 35.1 ± 6.3 ms, Fig. 7).
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15 pA or 52%; baseline: 28.9 ± 3.9 pA; allopregnanolone: 43.9 ± 10.1 pA, P < 0.01). There was no significant increase in the monophasic mIPSCs time course (baseline: 21.6 ± 3.0 ms; allopregnanolone: 23.0 ± 3.4 ms). The biphasic mIPSCs were also statistically unaltered. Thus in sum, allopregnanolone in the SP rat increased the amplitude of the mIPSCs but had little impact on the timing of the synaptic events.
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Effect of THDOC and allopregnanolone on holding current
Both steroids, with varying efficacy, were able to alter the holding current in each strain. Neurosteroids can potentiate the baseline current by enhancing the activity of any ambient GABA that may be present and/or directly gate GABAA receptors on their own (Shu et al. 2004). Thus during our experiments, we carefully monitored holding current to see if these compounds could enhance it. As shown on Fig. 9A THDOC induced significant changes in the holding potential of NC rats that were dose dependent (P < 0.01). In the SP strain, THDOC induced a >100% increase in holding current at 100 nM with no further increase at the 1.0 µM concentration. This holding current was not potentiated by allopregnanolone in either strain at 100 nM, but it significantly increased the current in SP strains at 500 nM (Fig. 9B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), we found that the synaptic responses are pharmacologically distinct. These biophysical and pharmacological distinctions support the view that these synapses both within and between strains are comprised of differing GABAA receptors. The best evidence is that the between strain differences are due to the documented differences in subunit expression (Poulter et al. 1999), so at least some of these effects are undoubtedly related to expression patterns. One of the most striking findings was that the neurosteroids generally have bigger effects (both time course and amplitude) on the mIPSCs and tonic current in the SP rats that overexpress 2, 3, and 5 subunits. What is also clear is that allopregnanolone, even at a relatively high physiological concentration (100 nM), had only small effects. So while the physiological concentration of THDOC used here had potent effects, the physiological concentration of allopregnanolone did not. Thus the activity of allopregnanolone seems to depend on the presence of another GABAA receptor subtype or state (phosphorylation) that is not highly expressed in our preparations. Differential effects of THDOC within and between strain
THDOC had a larger effect on the monoexponential mIPSCs than on the other population of mIPSCs in NC rats, whereas in the SP strain the effects of THDOC were profound on both populations but the nature of action was different (increased amplitude and effects on deactivation). Thus it seems evident that the receptors both within each strain and between these strains are physiologically and pharmacologically modified in a different manner. A few previous studies provide some possible explanations for these observations. The first and most obvious possibility is that the molecular structure (subunit expression) of the synapses is different. Extrapolating from the known global differences in subunit expression between these two strains is difficult, but some endpoints seem clear. First, the biphasic synaptic responses in the NC correlate quite well with the kinetics of recombinant 1
3
2 receptors (Haas and Macdonald 1999; McIntyre et al. 2002), suggesting that 100 nM THDOC is relatively inefficient at 1 subunit containing receptors. In the SP strain, by contrast, THDOC had very profound effects on the equivalent biphasic population the kinetics of which best match recombinant 5 receptor deactivation (Burgard et al. 1999). Thus THDOC may favor 5 subunit containing receptors. We have reported that the monophasic population is sensitive to the 1-selective positive modulator Zolpidem (Ing and Poulter 2003). Thus in SP rats, the relative paucity of 1 subunits may explain the relatively larger effect of THDOC on monophasic mIPSCs as well. In agreement with these interpretations, studies using recombinant receptors have shown neurosteroids have some selectivity based on subunit expression (Hauser et al. 1995, 1996; Maitra and Reynolds 1998, 1999; Srinivasan et al. 1999), including the finding that the potency of neurosteroids may be relatively smaller on 1-containing receptors (Maitra and Reynolds 1999). Finally, as we have found no differences in 
subunit expression between these strains, this does not account for differences in potency.
Second, phosphorylation of GABAA receptors has been reported as a determinant of neurosteroid activity (Brussaard and Koksma 2002, 2003; Brussaard et al. 2000; Fancsik et al. 2000; Tasker 2000). For example, phosphorylation has been found to potentiate THDOC activity on recombinant receptors (Leidenheimer and Chapell 1997) while preventing allopregnanolone activity in supraoptic nucleus (Brussaard et al. 2000) and magnocellular neurons of the hypothalamus (Fancsik et al. 2000) Thus our results may reflect differences in the phosphorylation state of the two synaptic sites in both the NC and SP strains. So our data may indicate, aside from differences in subunit expression that the phosphorylation of GABAA receptors in the SP strain may be higher than that in NC strain.
Likewise in the NC strain, there was 100% enhancement of tonic current while in the SP strain this current was enhanced by nearly 300%. This enhancement likely arises from potentiation of any ambient GABA that is present. In a few recordings, we have documented that there is an apparent tonic current in all strains that is blockable by gabazine or bicuculline (unpublished observations). Thus the activity of both steroids may be to potentiate this activity. As well they may directly gate the receptor as has been recently demonstrated (Shu et al. 2004; our unpublished observations). No matter what the exact mode of potentiation, the differential sensitivity may reflect differences in subunit expression in the extrasynaptic membrane between the strains. So, although it is likely that most subunits found in the synapses (1 and 2 for example) are also expressed in the extrasynaptic membrane, the reverse is likely not true. There is evidence that 3, and particularly 5, are primarily extrasynaptic (Brunig et al. 2002; Caraiscos et al. 2004; Hutcheon et al. 2000). These data again are consistent with the interpretation that THDOC may have higher activity on 5 (and perhaps 3) subunits. At higher concentrations, the effects of THDOC in the two strains are quantitatively similar, thus these effects are not easily explainable due to differences in receptor density.
THDOC enhanced the mIPSC amplitude at both concentrations used. In NC rats, only the monophasic mIPSCs were enhanced, but both population's amplitudes were increased in the SP rats. The best explanation of this effect is that THDOC enhances the affinity of GABA at these receptors, thus increasing Popen similar to the effect of Zolpidem at room temperature (Perrais and Ropert 1999). This indicates that the SP strain has relatively lower affinity receptors that may not saturate in either of the two types of synapses. Again based on our previous data, these observations fit well with the high expression of 3 subunits in the SP rat, compared with the NC strain (Poulter et al. 1999). A number of studies employing recombinant receptors have shown that 3-containing receptors confer the lowest affinity to GABA (Ducic et al. 1995; Gingrich et al. 1995; Verdoorn 1994).
Effects of allopregnanolone
In NC rats, there were only weak differential effects between each mIPSC population, and these responses were very similar to those shown by others (Poisbeau et al. 1997). In SP rats, our data show allopregnanolone had some selective effects that might differentiate one GABAA receptor-mediated response over another. This suggests that in another part of the brain, where either the phosphorylation state or subunit expression is similar to SP rat, allopregnanolone can differentially alter the attributes of one inhibitory synapse over another. In the SP rats, and like THDOC, allopregnanolone augmented mIPSC amplitudes, suggesting it enhanced affinity at a subset of synapses. However, allopregnanolone has been shown to have only weak subunit selectivity showing only a weak preference for 1- and 3-subunit-containing receptors (Belelli et al. 2002). Steroid sensitivity has also been shown to be dependent on or 
subunits (Lambert et al. 2003; Spigelman et al. 2003; Stell et al. 2003; Wei et al. 2003), but we have not detected a difference in subunit expression in these two strains (unpublished observations), whereas the subunit expression has not been assayed. Others have reported subunit selective activity for allopregnanolone. Allopregnanolone seems to potentiate subsaturating doses of GABA on recombinant 1-subunit-containing receptors about five times better than either 2- or 3-subunit-containing receptors (Shingai et al. 1991). By contrast, Brussaard and Koksma (2003) have shown that in knockout 1 mice allopregnanolone sensitivity is unchanged, and the loss of allopregnanolone sensitivity is related to the alteration in the balance of the phosphatase and kinase activity in the SON neurons. These data indicate that subunit expression is not a major determinant of allopregnanolone activity and that the known differences in subunit expression in our strains do not explain this steroid's pharmacology.
The finding that allopregnanolone reduced mIPSC amplitude also demonstrates a different (and or more complex) mechanism of action in comparison to THDOC. The prolongation of the deactivation may be explained by an enhancement of affinity, but amplitude reduction suggests enhanced desensitization that would tonically reduce Popen. The sum of these opposing effects therefore was estimated by calculating the total charge transfer from each averaged mIPSC (summarized in Table 1). Surprisingly, we found that the phasic inhibition may be decreased by as much as 35% in the SP rat, but in the NC, there was only a small increase. This implies that the allopregnanolone changes the timing/amplitude profile of inhibition, i.e., fast relatively large amplitude mIPSCs are converted to smaller long-lasting mIPSCs. Because these changes were usually not accompanied by an increase in tonic inhibition (except 500 nM in the SP strain), this suggests the balance between phasic and tonic inhibition is altered by allopregnanolone. Parenthetically we should add that THDOC increased both phasic and tonic inhibition and thus its effects would not alter (at least overtly) this balance.
Physiological implications of the differential activity
It is well documented that immunocytochemically and morphologically distinct interneurons innervate differing types of cells (DeFelipe 1997; Hof et al. 1999; Luth et al. 1993). Even subcellular regions expressing differing GABAA receptors can be uniquely targeted. The best example of this are parvalbumin positive interneurons in the hippocampus. Here these interneurons innervate the axon initial segment that selectively recruits 2 subunit containing receptors into the synaptic sites located in this region (Fritschy and Brunig 2003). In our preparation, the equivalent information is unknown and to what degree these synapses may or may not be uniquely targeted by various interneuron subtypes is not known. Nevertheless, our data show that the neurosteroid modulation of inhibitory currents acts in nonuniform manner at the cellular level. Our data raise the possibility that the modulation by these compounds could involve the preferential potentiation of one interneuronal input over another.
These data were surprising to us from another viewpoint. We had originally hypothesized that neurosteroids may be relatively ineffective in the SP rat compared with the NC strain and that a lack of sensitivity may contribute to the seizure prone phenotype. On the surface, this appears to be untrue. But as all these recordings are from nonpyramidal cells, the enhanced sensitivity of the THDOC may contribute to relative augmentation of disinhibition in the SP strain that may promote abnormal synchrony. At this point, however, it remains to be determined what the overall impact of this differential sensitivity is in these strains of rat.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. O. Poulter, Associate Professor, Neuroscience Research Institute, Department of Psychology, Carleton University, 1125 Colonel By Dr., Ottawa Ontario, K1S 5B6 Canada (E-mail: michael_poulter{at}carleton.ca)
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