1. Volume 19, Issue 5, Pages 1103-1114 (November 1997)
Plasticity in Fast Synaptic Inhibition of Adult Oxytocin Neurons Caused by Switch in GABAA Receptor Subunit Expression 
A.B Brussaard, K.S Kits, R.E Baker, W.P.A Willems, J.W Leyting-Vermeulen, P Voorn, A.B Smit, R.J Bicknell, A.E Herbison 
Neuron 
Volume 19, Issue 5, Pages (November 1997)
DOI: /S (00)

2. Figure 1
Quantitative Cellular Analysis of GABAA Receptor α1 and α2 Subunit mRNA Expression in Magnocellular Neurons of the Dorsal Supraoptic Nucleus on P19, P20, and PPD1 Rats
(A) Mean (± SD) number of silver grains per magnocellular neuron in each experimental group (n = 6 animals per group) following hybridization for the α1 or α2 subunit. Double asterisks indicate p < 0.003, post-hoc Student-Newman-Keuls test.
(B) Histogram distribution analysis of cellular α1 subunit mRNA expression in the dorsal supraoptic nucleus of P19, P20, and PPD1 rats. Each bar shows the percentage of neurons (n = 120 pooled from six different animals) expressing different levels of silver grains per magnocellular neuron. Silver grain expression was grouped in units of ten; for example, “80” represents all neurons expressing 80–89 silver grains. Note the generalized leftward shift to lower levels of α1 subunit expression per cell as the day of parturition becomes imminent.
Neuron  , DOI: ( /S (00) )

3. Figure 2
Slower Decay of GABAA Receptor-Mediated Synaptic Currents in Dorsal SON Neurons of Adult Female Rats with Parturition
(A and B) Spontaneous IPSCs from dorsal SON neurons before (A) and after (B) parturition.
(C) Superimposed averages of 50 sIPSCs from three different recordings at P20 and PPD1, normalized to the maximal amplitude of IPSC.
(D) IPSC decay time constant histograms from representative SON recordings at P20 and PPD1 with lognormal functions fitted to the raw data.
(E) Amplitude histogram of sIPSCs at P20 and PPD1. Lognormal functions fitted to the raw amplitude data in each experiment yielded a mean and standard deviation for each experiment.
(F) Summary of the sIPSC properties recorded from all P20 and PPD1 rats. Note the absence of any effect upon sIPSC amplitude or interval, while a significant increase in sIPSC decay time constant was observed in PPD1 animals compared with P20 rats (n = 21 and 8, respectively; triple asterisks indicate p < 0.001, t test).
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4. Figure 3
Shift in 3α-OH-DHP Sensitivity of GABAA Receptor due to Parturition
(A) Superimposed averages of 50 spontaneous IPSCs recorded from dorsal SON neurons both before (P20) and after (PPD1) parturition, in the absence and presence of 1 μM 3α-OH-DHP concentrations.
(B) Lognormal curves fitted to the IPSC decay time constant histogram before (control) and after 3α-OH-DHP application (1 μM) at P20 and PPD1.
(C) Overall effect of 3α-OH-DHP at 1 μM and 10 μM on sIPSC decay in P20 (n = 5) and PPD1 (n = 7) animals. ANOVA F[5,23] = 4.271; p < 0.007; post-hoc comparison indicated: double asterisks, p < 0.05, and triple asterisks, p < 0.001, compared to the control decay.
(D) Scatter plots of the decay time constants (each dot represents the average sIPSC decay time constant of one neuron in the absence of neurosteroid) versus the relative effect at 1 μM or 10 μM of 3α-OH-DHP on the sIPSC decay. Linear regression lines fitted to these data showed, for both concentrations, the significant correlation in a Pearson correlation test at p < (Bartlett Chi-square statistics).
Neuron  , DOI: ( /S (00) )

5. Figure 4
Expression of α1 and α2 Subunit mRNA in Organotypical Neocortex Explants and Effect of Antisense Deletion of α2
(A) In situ hybridization showing α1 and α2 subunit mRNA expression at the cellular level in layer II-IV neocortex cells in slices cultures. Magnification, 250×.
(B) Detail of immunoblots of total protein from the neocortex explants probed with an α2 subunit–specific polyclonal antiserum (McKernan et al. 1991). Molecular markers indicate the extent of migration of a 52 kDa protein (line), the predicted size of the α2 protein. Identical amounts of total protein from untreated control (lane 1), α2 AS (lane 2), and α2 MM (lane 3) treated explants were loaded. The amount of α2 protein relative to control was determined by densitometric measurement of autoradiograms. This showed a 68% ± 8% reduction in α2 protein content in α2 AS treated explants compared to either control or α2 MM treated cultures (n = 4 per group).
Neuron  , DOI: ( /S (00) )

6. Figure 5
Faster sIPSC Decay Following Antisense Deletion of the α2 Subunit
(A) Representative single (top) and averaged (from 50 events, lower trace) sIPSCs recorded from neurons in control (n = 9), α2 AS (n = 7), and α2 MM (n = 4) treated explants.
(B) sIPSCs from these cultures but here normalized for peak amplitude and with monoexponential curves fitted to their decays. Note the faster decay at α2 AS.
(C) Lognormal curves fitted to the sIPSC decay time constant histograms for each experimental.
(D) Scatter plots of average sIPSC amplitude versus the average sIPSC decay time constant per experiment. Overall, α2 AS significantly reduced the sIPSC amplitude (ANOVA F[2,17] = 6.3; p < 0.009; post-hoc comparison with the control and the α2 MM, respectively: p < and 0.05) as well as the sIPSC decay time constant variance (Fmax test: S2max/S2min = 8.78, p < 0.05).
Neuron  , DOI: ( /S (00) )

7. Figure 6
Allopregnanolone Sensitivity of GABAA Receptor-Mediated Synaptic Input Inhibits the Firing of Oxytocin Neurons before, but Not after, Parturition
(A) Whole-cell current clamp revealed the membrane properties characteristic for the oxytocin neurons: from a membrane potential of −50 mV (−0.1 ± 0.03 nA; n = 10), during hyperpolarizing command pulses, outward rectification was observed, followed by rebound depolarizations with bursts of spontaneous firing activity at the offset of such pulses. Upon release from prolonged hyperpolarization (>5 min using −0.1 to −0.2 nA), most of these cells (10 out of 13) displayed tonic firing activity with a firing frequency of 10.1 ± 0.1 Hz.
(B) Application of bicuculline (20 μM) revealed that the firing rate of oxytocin neurons is dependent upon a GABAA receptor-mediated tonic inhibition.
(C) Whole-cell voltage clamp of the same cells at −40 mV showed outward (upward plotted) GABAA receptor-mediated postsynaptic currents (Erev = −65 ± 3 mV; n = 5) with an average interevent interval of 83 ± 19 ms as well as (downward plotted) glutamatemediated synaptic events.
(D) Superimposed traces showing individual GABAA receptor-mediated synaptic events from the same recording.
(E) The frequency of tonic spiking of oxytocin neurons before parturition was reduced after application of 10 μM 3α-OH-DHP.
(F) Three minutes of 3α-OH-DHP application at P20 (n = 6) leads to a 46% ± 12% reduction in firing frequency (ANOVA: F[3,16] = 4.210; p < 0.023; post-hoc testing: asterisk indicates p < 0.01), whereas at PPD1 (n = 4), there was no effect.
(G) Ongoing continuous firing activity in these cells was prematurely terminated upon application of 3α-OH-DHP. At P20, 3α-OH-DHP (arrow, moment of application) stopped ongoing firing activity within 284 ± 15 s, resulting in a total duration of the discharge of 454 ± 22 s (n = 4), as compared to 1067 ± 229 s (n = 4) obtained under control conditions from the same cells (ANOVA: F[3,12] = 4.597; p < 0.05; post-hoc testing: double asterisks indicate p < 0.05). At PPD1, no effect on the total duration of the discharge upon 3α-OH-DHP application was observed (control versus discharge with 3α-OH-DHP, both n = 4).
Neuron  , DOI: ( /S (00) )

8. Figure 7
GABAA Receptor-Mediated Restraint of Oxytocin Secretion in the Late Pregnant Rat
Individual rats were bled at five time points over 25 min with IV administration of saline (closed circlesn = 6) or 2 mg/kg bicuculline (open squaresn = 6) given following the 10 min sample (arrow). Plasma oxytocin concentrations (given as percentage change from levels at 0 min) were raised by up to 200% following bicuculline (ANOVA; F = 8.943, p = with post-hoc Dunnett's test) but unchanged following saline (ANOVA; F = 0.059, p > 0.05).
Neuron  , DOI: ( /S (00) )