Production of 5alpha-reduced neurosteroids is developmentally regulated and shapes GABA(A) miniature IPSCs in lamina II of the spinal cord

Abstract

In lamina II of the spinal dorsal horn, synaptic inhibition mediated by ionotropic GABA(A) and glycine receptors contributes to the integration of peripheral nociceptive messages. Whole-cell patch-clamp recordings were performed from lamina II neurons in spinal cord slices to study the properties of miniature IPSCs (mIPSCs) mediated by activation of GABA(A) and glycine receptors in immature (<30 d) and adult rats. Blockade of neurosteroidogenesis by 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide (PK11195), an inhibitor of the peripheral benzodiazepine receptor (PBR), or finasteride, which blocks 5alpha-reductase, accelerated the decay kinetics of GABA(A) receptor-mediated mIPSCs in immature, but not in adult animals. Glycine receptor-mediated mIPSCs remained unaffected under these conditions. These results suggest the presence of a tonic production of 5alpha-reduced neurosteroids in young rats that confers slow decay kinetics to GABA(A) mIPSCs. At all of the ages, selective stimulation of PBR by diazepam in the presence of flumazenil prolonged GABA(A) mIPSCs in a PK11195- and finasteride-sensitive manner. This condition also increased the proportion of mixed GABA(A)/glycine mIPSCs in the immature animals and led to the reappearance of mixed GABA(A)/glycine mIPSCs in the adult. Our results might point to an original mechanism by which the strength of synaptic inhibition can be adjusted locally in the CNS during development and under physiological and/or pathological conditions by controlling the synthesis of endogenous 5alpha-reduced neurosteroids.

Figure 1.

Allopregnanolone and diazepam prolong GABA_AR mIPSCs without affecting GlyR mIPSCs. Pharmacologically isolated GlyR and GABA_AR mIPSCs were recorded at a holding potential of –60 mV from immature lamina II neurons (<P23) in the presence of 10μm bicuculline or 1 μm strychnine, respectively. A, Histograms illustrate the mean value ± SEM of the decay time constant for GlyR (left histogram; black bars) and GABA_A mIPSCs (right histogram; white bars) fitted with a monoexponential function under control conditions (CT) (n = 10) and after application of AP (100 nm; n = 5), DZP (1 μm; n = 7), or DZP in the presence of the CBR antagonist flumazenil (DZP+FLU) (10 μm). Asterisks indicate statistically significant difference (t test; p < 0.05) with respect to control. B, Traces representing averages of 10 isolated glycine (top traces) and GABA_A mIPSCs (bottom traces) recorded under control conditions and after application of DZP (1μm; left traces) or DZP plus 10μm FLU (DZP+FLU) (right traces). The results of the monoexponential fits are shown as gray lines superimposed on the original traces (black lines). τ indicates the value of the mean decay time constant of mIPSCs for each experimental condition.

Figure 2.

Flumazenil antagonizes the short-term effect of diazepam, whereas PK11195 antagonizes the delayed prolongation of GABA_AR mIPSCs. A, Pharmacologically isolated GABA_AR mIPSCs (strychnine; 1μm) were recorded at a holding potential of –60 mV. Effect of DZP (1μm) on mean decay time constant as function of time of application (in minutes) in immature (<P23) lamina II neurons under different conditions: absence of flumazenil (No FLU) (open circles), preincubation of slices with FLU (10 μm; filled circles), or with both flumazenil and the PBR antagonist PK11195 (FLU+PK11195) (10μm; filled triangles). Each point represents the mean±SEM value of the decay time constant of mIPSCs sampled over periods of 5 min during superfusion of DZP (horizontal gray bar), except in the case of FLU+PK11195, for which we sampled the values of mIPSC decay time constants after steady-state incubation for 60–90 min. B, Cumulative distribution of GABA_AR mIPSCs decay time constants pooled from five immature neurons (<P23) and recorded after different pharmacological treatments. The dotted line illustrates the distribution under control conditions. Left panel, Short-term application (<1 hr) of DZP shifted the distribution to the right (Kolmogorov–Smirnov test; p < 0.01), an effect blocked by flumazenil. Right panel, Long-term application of DZP and FLU (>1 hr) increased the duration of mIPSCs (rightward shift of the distribution; Kolmogorov–Smirnov test; p < 0.01). This effect was blocked by PK11195 (10μm).

Figure 3.

PK11195 and finasteride accelerate GABA_AR mIPSCs decay kinetics in neurons from immature animals (<P23). Pharmacologically isolated GABA_AR mIPSCs were recorded in the presence of strychnine (1μm) at a holding potential of –60 mV. The graphs represent the cumulative distributions of GABA_AR mIPSCs decay time constants pooled from two immature neurons recorded under control conditions (dotted line), in the presence of the CBR antagonist FLU (10μm) or the PBR antagonist PK11195 (PK) (10μm)(A), or after long-term incubation (>6 hr) with finasteride (50μm), an inhibitor of 5α-reductase (B). The reduction in mIPSC decay time constant values observed in the presence of finasteride or PK11195 was statistically significant (Kolmogorov–Smirnov test; p < 0.01). The insets represent averages of 10 traces of individual mIPSCs recorded under each condition. The deactivation phase of GABA_AR mIPSCs was fitted with a monoexponential function shown as the superimposed gray lines. τ represents the value of the decay time constant determined by the fitting procedure. C, Whole-cell GABA_A receptor-mediated currents evoked by exogenous 100-msec-lasting pressure applications of 100 μm isoguvacine in the vicinity of the recorded cell during steady-state perfusion of either PK11195 (10 μm) or flumazenil (10 μm).

Figure 4.

Diazepam and allopregnanolone increase GABA_A mIPSCs and convert some GlyR mIPSCs into mixed GABA_A/GlyR mIPSCs. Synaptic mIPSCs were recorded at holding potential of –60 mV in the absence of any ionotropic inhibitory amino acid receptor antagonists in immature neurons (<P23). A, The histograms represent the mean (±SEM) decay time constant values of the fast glycine receptor-mediated component (τ₁) (black columns) and the slow GABA_A receptor-mediated component (τ₂) (white columns) of mixed GABA_A/GlyR mIPSCs. The trace in inset is a typical example of a mixed GABA_A/GlyR mIPSC best fitted with a biexponential function. Compared with the fit obtained in control conditions (dotted black line), superfusion with 100 nm AP prolonged the slowest decay time constant (i.e., the GABA_A component). Asterisks indicate statistically significant differences (t test; p < 0.05) with respect to control. B, The top panel shows the evolution of the frequency of total population of mIPSCs (filled circles). The bottom panel represents the evolution of the relative frequency of occurrence of each mIPSC category (open triangles, GlyR mIPSCs; open squares, GABA_AR mIPSCs; open circles, mixed GABA_A/GlyR mIPSCs) among the total population as a function of time of AP perfusion in a representative immature neuron (<P23). Each point corresponds to a 2 min sampling period. Horizontal gray bars denote application of AP and bicuculline. The total frequency of mIPSCs remained stable during AP application but decreased in the presence of bicuculline (Bicu) (10 μm). GABA_AR mIPSCs frequency was stable during AP application, whereas the proportion of GlyR mIPSCs decreased in parallel with the increase of mixed GABA_A/GlyR mIPSCs. In presence of bicuculline, only GlyR mIPSCs were detected, and their frequency corresponded to the sum of the frequencies of GlyR and mixed mIPSCs recorded before the blockade of GABA_AR receptors.

Figure 5.

Pharmacological treatments aimed at increasing or inhibiting neurosteroidogenesis affect mixed GABA_A/glycine mIPSC synaptic transmission. Neurons were recorded in the absence of any inhibitory amino acid receptor antagonists at a holding potential of –60 mV. Histograms give the percentage of mixed GABA_A/GlyR mIPSCs recorded among the overall population of mIPSCs in immature (<P23) (left histogram) and adult neurons (>P30) (right histogram). AP (100 nm) and DZP (1μm) were applied by superfusion. Other treatments consisted of short-time (<1 hr) and/or prolonged (>1 hr) incubation of slices with FLU (10μm), FIN (50μm), PK11195 (PK) (10μm), diazepam plus flumazenil (DZP+FLU), and diazepam plus flumazenil plus PK11195 (DZP+FLU+PK). A significant increase in the proportion of mixed GABA_A/glycine mIPSCs was noted in the presence of AP, DZP, and DZP+FLU (>1 hr) in immature slices. Conversely, a large reduction was noted with PK11195 or finasteride, two inhibitors of neurosteroidogenesis. After 30 d of postnatal life (>P30), no mixed events were detected. However, such mixed events reappeared in the presence of AP, DZP, or DZP+FLU (>3 hr). Incubation with finasteride (>6 hr) never revealed mixed mIPSCs. Asterisks represent statistically significant differences (t test; p < 0.05).

Figure 6.

Developmental changes in synaptic inhibition expressed as changes in decay time constant and fraction of total GABA_A, glycine, and mixed mIPSCs. Evolution of the proportion (left panels) and mean decay time constant (right panels) of monoexponential GABA_AR mIPSCs (A1), GlyR mIPSCs (A2), and biexponential mixed GABA_A/GlyR mIPSCs (B) in lamina II neurons of spinal cord slices from 6-d-old (P6), 15-d-old (P15), and adult (>P30) rats. Open symbols represent the values obtained from neurons recorded under control condition (i.e., without any pharmacological treatment). Filled symbols apply to slices that have been preincubated for at least 6 hr with finasteride (50μm). Asterisks indicate statistically significant differences (t test; p < 0.05) with respect to control.