Endogenous neurosteroids and synthetic neuroactive steroids are among the most potent and efficacious modulators of the brain’s main ligand-gated inhibitory neurotransmitter receptor, the γ-aminobutyric acid type A (GABA_A) receptor, producing wide-ranging effects on behavior, including motor relaxation and impairment (Baulieu et al., 2001; Belelli et al., 2006). The concentrations of neurosteroids in the brain are increased in stress and by drugs, such as alcohol and antidepressants (Barbaccia, 2004). Endogenous concentrations of neurosteroids fluctuate during the ovarian cycle and in pregnancy, inducing changes in GABA_A receptor subtype populations, particularly in those containing the δ subunits (Maguire et al., 2005; Uusi-Oukari and Korpi, 2010; Wu et al., 2013; Mackenzie and Maguire, 2014), which may result in impaired regulation of mood in some patients (Backstrom et al., 2011, 2014). Neurosteroid mechanisms have been implicated in acute liver failure and hepatic encephalopathy (Ahboucha et al., 2012). Furthermore, allopregnanolone, a metabolite of progesterone and one of the main endogenous GABAergic neurosteroids, promotes proliferation of neural progenitor cells in vitro via activation of GABA_A receptors and, after a single dose, enhances neurogenesis in the hippocampal subgranular zone and in the dopaminergic substantia nigra pars compacta of adult transgenic model mice for Alzheimer’s disease (Wang et al., 2010; Zhang et al., 2015). However, chronic treatment with allopregnanolone accelerates Alzheimer’s disease in several types of mouse models (Bengtsson et al., 2012, 2013). There appears to be a need to better understand the mechanisms underlying neurosteroid sensitivity.

The mechanisms of neurosteroid sensitivity remain unclear. Neurosteroid actions can be mediated by all subtypes of GABA_A receptors, with just α and β subunits giving full potency and efficacy (Hosie et al., 2006). The type of the α subunit mediates some of the variation noted in receptor properties, whereas the type of the β subunit has less influence. In recombinant receptors, inclusion of a γ2 subunit has little effect on binding or efficacy compared to binary αβ receptors, but the inclusion of the δ subunit strongly increases the GABA-potentiating effects of neurosteroid agonists (Belelli et al., 2002; Wohlfarth et al., 2002). Neurosteroids affect both synaptic (mainly γ2-GABA_A dependent) and extrasynaptic (mainly δ-GABA_A dependent) inhibition in brain slices, but, in line with the increased efficacy of neurosteroids on δ subunit-containing receptors, their behavioral and neurophysiological effects are strongly reduced when extrasynaptic GABAergic inhibition has been attenuated, e.g., in the GABA_A receptor δ subunit knockout mice (Mihalek et al., 1999; Spigelman et al., 2003; Stell et al., 2003). In the case of extrasynaptic GABA_A receptors containing the α4 and δ subunits, these mechanisms involve protein kinase C activation, and subsequent GABA_A α4 subunit phosphorylation, increased membrane insertion of these receptors, leading to enhanced tonic conductance by prolonged exposure to neurosteroids (Abramian et al., 2014). Thus, whereas neurosteroid agonists affect both synaptic and extrasynaptic receptors, their behavioral effects seem to primarily depend on extrasynaptic receptors. It is evident that more information is needed on the effects of GABA_A receptor subunit composition on neurosteroid sensitivity at the whole animal level.

In the present experiments, we genetically removed the γ2 subunit in discrete neuronal populations. We used male mice to avoid estrus cycle-associated neurosteroid fluctuations. Using a simple cerebellum-related behavioral task on rotating rods, the mouse models enabled us to ascertain how the γ2 subunit affects the sensitivity of the neuroactive steroid pregnanolone in cerebellar circuitry. The cerebellum was very suitable for these experiments, since the two main targets we used (Purkinje and granule cells) are known to express the γ2 subunit and a limited number of other subunits [Purkinje cells α1, β2/3 and γ2; granule cells α1/6, β2/3, δ and γ2 (Wisden et al., 1996)]. The contributions of extrasynaptic and synaptic GABA_A receptors in mediating pregnanolone effects were further validated by transgenic re-introduction of the γ2 subunit into Purkinje cells (PC-γ2-swap mice) (Wulff et al., 2007; Wisden et al., 2009).

A mouse line [Pv-Δγ2, (Wulff et al., 2009a)] with inactivated synaptic GABA_A receptor-mediated inhibition of Pv-expressing neurons exhibited a high sensitivity to the exogenous neuroactive steroid 5β-pregnan-3α-ol-20-one (pregnanolone; Figure 1). Naive Pv-Δγ2 mice exhibited ataxia, but they could learn to stay on a rotating rod that accelerated from 5 to 20 rpms (Figure 1A). However, when these mice were challenged by cumulative dosing of pregnanolone they quickly fell down from the rod, while the control littermates were only slightly affected (Figure 1B, drug treatment × genotype interaction F_3,53 = 5.33, P < 0.01). Electrophysiology of the Pv-expressing hippocampal interneurons in the genetically engineered mice indicated abolition of synaptic GABA_A receptor-mediated inhibitory postsynaptic currents (IPSCs) (Wulff et al., 2009a). Pv-expression is widespread in the brain, including the cerebellar PCs (Meyer et al., 2002; Leppa et al., 2011; Kaiser et al., 2015). Thus, it seemed possible that neurosteroids can induce strong neuronal effects especially in the absence of their target receptors from a set of synapses.

Since Pv-expressing neurons exist at a low and diffuse level in most areas of the central nervous system, including motor neurons of the brain stem and spinal cord, and because they have abundant connections between each other and to principal neurons throughout the brain, interpretation of the effect of global Pv-cell-specific elimination of synaptic inhibition is complicated. Thus, we next wanted to use a mouse model with a more restricted ablation of synaptic inhibition. We produced cerebellar granule cell-selective GABA_A receptor γ2 subunit knockout mice (Gr-Δγ2) by cross-breeding α6-Cre (Aller et al., 2003) and floxed γ2 mice (Schweizer et al., 2003). Autoradiography of cerebellar sections showed that in the granule cell layer, the Gr-Δγ2 knockouts lack the γ2 subunit-dependent high-affinity labeling of the benzodiazepine binding sites, but contain normal labeling of integral ionophore sites of GABA_A receptors (Figure 2A). This is consistent with the loss of γ2 subunit-containing receptors at synapses between GABAergic Golgi interneurons and granule cells. In spite of these deficits in synaptic receptors, the Gr-Δγ2 mice learnt normally to run on a rotarod and did not show obvious motor deficits (Figure 2B). Interestingly, these mice showed a significant increase in pregnanolone sensitivity as compared to littermate controls and heterozygous knockouts (Figure 2C; drug treatment × genotype interaction F_6,72 = 2.87, P < 0.05).

Our third mouse model focused on manipulating inhibitory synaptic input to cerebellar Purkinje cells. We performed motor tests in male PC-Δγ2 mice (Wulff et al., 2007). These mice lack the spontaneous IPSCs of Purkinje neurons (Wulff et al., 2007), but they exhibited only a minor transient motor impairment during the 1st day of rotarod training. The motor-impairing effect of pregnanolone on rotarod performance was clearly enhanced in PC-Δγ2 mice as compared to littermates (Figures 3A,B; drug treatment × genotype interaction, F_6,69 = 2.96, P = 0.05). Importantly, the re-introduction of wildtype γ2 subunits to PCs, as evidenced by autoradiographic (Figure 3C) and electrophysiological experiments (Wulff et al., 2007), fully abolished the increased pregnanolone sensitivity (Figure 3B; P > 0.05 for the difference between PC-γ2-swap and γ2I77lox lines). These results finally implicate PCs as targets of pregnanolone in the cerebellar cortex resulting in impaired motor coordination.

To test whether PC-Δγ2 mice show enhanced sensitivity in other than motor coordination tests, doses of pregnanolone that are not strongly motor impairing (7 and 10 mg/kg) were tested for possible anxiolytic/exploration-increasing effects in the elevated plus maze and light:dark exploration tests, but the results were essentially similar in γ2I77lox and PC-Δγ2 mice (Figure 4), thus showing no baseline differences in any measures, nor any significant mouse line or interaction effects in the ANOVA. Pregnanolone treatment induced an anxiolytic-like effect that was similar in both mouse lines. It significantly increased the time on the open arms (F_1,29 = 11.8, P = 0.002) and the proportion of open arm entries (F_1,29 = 12.2, P = 0.0015) and reduced the distance moved (F_1,29 = 7.6, P = 0.01) in the plus maze test, and it tended to increase the time in the lit compartment (F_1,31 = 3.44, P = 0.07), increased number of crossings between the compartments (F_1,31 = 7.07, P = 0.012) and increased distance traveled in the lit compartment (F_1,31 = 7.34, P = 0.010) in the light:dark test. Furthermore, PC-Δγ2 mice showed no reduction in locomotor activity in these tests as compared to controls.

Purkinje cells express especially the α1 subunit-containing GABA_A receptors (Wisden et al., 1996). Therefore, we used recombinant GABA_A receptors to assess whether different α1 subunit-containing receptor subtypes are affected by the presence of pregnanolone. In the absence of GABA, among α1β3, α1β3γ2, and α1β3δ receptors only the δ subunit-containing receptor α1β3δ responded to increasing concentrations of pregnanolone with increased binding of [³H]EBOB (Figure 5). At 1 μM GABA, pregnanolone reduced [³H]EBOB binding to α1β3 and α1β3γ2 receptors (Figure 5), but had no effects on α1β3δ. At the highest GABA concentration tested (10 μM), high nanomolar pregnanolone reduced the binding to α1β3δ receptors but had no more effects on the already low binding to α1β3 and α1β3γ2 receptors. These data suggest that also in a biochemical assay of recombinant GABA_A receptor function, the δ subunit-containing receptors respond strongly to the presence of pregnanolone. More importantly, the α1β3 receptors that do not have the address to go to synapses are as strongly affected as α1β3γ2 receptors by pregnanolone.

Neurosteroids are potent modulators of brain GABA_A receptors and their significance to various behavioral states is under active investigation. The level of endogenous neurosteroids is thought to influence neuronal excitability, e.g., in catamenial epilepsy, premenstrual syndrome and migraine as well as depression (van Broekhoven and Verkes, 2003; Wu et al., 2013; Backstrom et al., 2014). Neurosteroid mechanisms seem to be important also in liver failure and hepatic encephalopathy (Ahboucha et al., 2012) and in Alzheimer’s disease models (Wang et al., 2010; Bengtsson et al., 2012, 2013; Zhang et al., 2015). Using several GABA_A receptor genetically engineered mouse models we show here that the neuroactive steroid agonist pregnanolone can strongly modulate learned motor performance when the balance of non-synaptic (extrasynaptic) and synaptic GABA_A receptors was altered in a set of neuronal populations with inactivated γ2 subunits. Previous findings on α4 and δ subunit-containing receptors in hippocampal areas indicate that neurosteroid agonists powerfully facilitate tonic inhibition mediated by GABA_A receptors (Carver et al., 2014). These results, together with the reduced sensitivity of neurosteroids in GABA_A receptor δ knockout mice (Mihalek et al., 1999; Stell et al., 2003), strongly indicate that the extrasynaptic GABA_A receptors are the preferential targets of neurosteroids. Importantly, in the present study these preferential effects could be detected in mice with impaired synaptic inhibition only in a single population of neurons after inactivation of the γ2 subunit that is essential for synaptic targeting of GABA_A receptors (Crestani et al., 1999), without any general enhancement of δ subunit-containing GABA_A receptors. It should anyway be kept in mind that compensatory changes may take place after inactivation of γ2 subunits, although the δ subunit is not known to be expressed in the cerebellar PCs (Wisden et al., 1996) and global γ2 inactivation cannot be efficiently compensated in mice (Gunther et al., 1995).

The modulation of binding of the GABA_A receptor ionophore ligand [³H]EBOB was different in the δ subunit-containing receptors than in binary αβ or γ2 subunit-containing receptors (Figure 5), again indicating a possibility that δ subunit-containing GABA_A receptors have features that make neurosteroid effects in them more potent or efficient. Further study is needed to understand the molecular mechanisms how the general neurosteroid binding sites on α subunits (Hosie et al., 2009) are transducing the signal to the ionophore structure in the δ subunit-containing receptors differently from δ subunit-absent receptors.

The alteration of balance between synaptic and extrasynaptic inhibition might be also operative in the regulation of other neuronal pathways that mediate more subtle behaviors such as emotions and irritability, that are known to be associated with altered endogenous neurosteroid metabolism and GABAergic inhibition (Backstrom et al., 2003). In the present study the anxiolytic and exploration-related effects of pregnanolone were not affected by the γ2 inactivation in the cerebellar PCs (Figure 4). The tests should be extended in further studies by using more appropriate neuronal populations for emotional regulation, e.g., in the forebrain.

Learned motor performance depends on cerebellar circuitry. In a recent study, the conditioned eyeblink reflex was pinpointed to deep cerebellar nuclei, particularly the anterior interpositus nuclei, which receive input from cerebellar cortical PCs (Ito, 1984; Heiney et al., 2014). PCs have multiple roles in movement precision, including predictive activity and instructive signaling downstream to the deep cerebellar nuclei. Optogenetic activation of PCs-inhibiting interneurons in the molecular layer transiently decreases the constitutive inhibitory output of PCs to the deep cerebellar nuclei and consequently produces precisely timed movement activation in awake mice (Heiney et al., 2014). Our present findings of enhanced motor impairment by pregnanolone acting on non-synaptic GABA_A receptors in various cerebellar cortical neuron populations likely resulted from non-timed prolonged disinhibition of the deep cerebellar nuclei.

In Pv-Δγ2 mice the predicted absence of synaptic inhibition with maintained extrasynaptic inhibition in widespread brain regions resulted in a pronounced increase in pregnanolone sensitivity in the rotarod test. Importantly, cerebellar molecular layer interneurons, such as stellate and basket cells are Pv-positive, as are the PCs as well (Meyer et al., 2002; Kaiser et al., 2015), and the inhibitory feed-forward control the molecular layer interneurons exert on PCs via synaptic GABA_A receptors is perforce altered in Pv-Δγ2 mice (Wulff et al., 2009b). When pregnanolone is administered, it affects the cerebellar function in these knockout mice more strongly than in wild-type mice. Considering that the synaptic inhibitory influence of molecular layer interneurons on PCs is already lacking, the effect of pregnanolone on remaining extrasynaptic receptors in both PCs and stellate/basket cells produces a network inhibitory effect that impairs the learned motor behavior of Pv-Δγ2 mice.

Pregnanolone produced a robustly increased effect on learned motor performance also in Gr-Δγ2 mice. Despite the missing synaptic inhibition from Golgi interneurons to granule cells (correlating with the absence of α1/6βγ2 subunit-dependent [³H]Ro 15-4513 binding, Figure 2A), these mice learned motor tasks as well as wild-type mice, suggesting compensations. Normally cerebellar granule cells have an excitatory effect on Purkinje cells via parallel fiber axons. When Gr-Δγ2 mice are administered pregnanolone, this excitation is possibly reduced to a greater extent and/or the phasic changes in PC firing are blunted compared to wild-type mice due to the prevalence of only extrasynaptic receptors in the granule cells. The resulting decrease in timed PC activity is a likely explanation for the increased impairing effect of pregnanolone on motor performance. This cerebellar granule-cell specific mouse model revealed that ablation of phasic inhibition only in a single population of cerebellar neurons, although in the most abundant one of the brain, is enough to bring about enhanced neurosteroid sensitivity.

Purkinje cell-Δγ2 mice were previously found to have disrupted inhibitory and excitatory input timing from PCs and mossy fiber collaterals (Wulff et al., 2009b). The lack of the GABA_A γ2 subunit disrupts the coordination of PC activity through molecular layer interneurons. In the present study powerfully prolonged tonic inhibition of PC activity by pregnanolone acting on extrasynaptic GABA_A receptors would disrupt the learning and performance of motor tasks more in PC-Δγ2 than in wild-type mice due to the sole availability of presumably α1β2 extrasynaptic receptors. Compensatory mechanisms, such as up-regulation of δ subunit expression, cannot be excluded, though. In PC-γ2-swap mice the situation was normalized due to the restoration of synaptic GABAergic inhibition in the molecular layer. As compared to the normal γ2F77 wild-type mice, the rescue of γ2-dependent benzodiazepine site binding in PC-γ2-swap was only 23% (Figure 3C), but this comparison may underestimate the rescue, since it does not take into account the binding to molecular layer interneurons missing at the γ2I77 background. Anyway, this mouse model with the rescue experiment pinpointed that the presence of γ2 subunits and associated synaptic inhibition strongly blunted the effect of pregnanolone on motor performance, underscoring the importance of phasic, directly neurotransmission-linked inhibition. Each PC receives a robust, convergent inhibition from about seven GABAergic, gap-junctionally connected interneurons in the molecular layer (Hausser and Clark, 1997; Park et al., 2012; Kim et al., 2014). The abolition of this phasic function should at least affect the timing of inhibition and thereby the regulation of firing of different PCs and at the end the motor performance. In the absence of γ2 subunits, pregnanolone presumably induced prolonged inhibition of PC firing via extrasynaptic receptors. Alternative mechanisms could involve cerebellar glutamatergic N-methyl-D-aspartate receptors that might undergo adaptive changes in neurons with deficient synaptic inhibition [synaptic scaling; cf. (Moykkynen et al., 2007)], especially as some neurosteroids blunt cerebellar responses to glutamate via potentiating GABA_A receptor functions or by blocking glutamate receptors (Cauli et al., 2011; Bali and Jaggi, 2014; Vyklicky et al., 2015).

Taken as a whole, these results from four different genetically engineered mouse lines with disruptions in the synaptic connections of the cerebellar network point to a delicate balance of inhibitory and excitatory input toward PCs. A possible interpretation of our results is that reduced inhibition from PCs toward the deep cerebellar nuclei, resulting from either decreased excitation (Gr-Δγ2 mice) or increased sustained inhibition of PCs (Pv-Δγ2 and PC-Δγ2 mice), caused the increased effect of pregnanolone on the rotarod test performance. These mouse models may form the basis for studies on molecular mechanisms of neurosteroid actions and for detailed understanding of how the cerebellar cortex regulates motor performance.

EL, A-ML, PW, BL, WW, and EK designed the experiments on mouse models and autoradiography. HL designed and carried out the recombinant receptor study. EL, A-ML, MA, and OV carried out the experiments. All authors wrote and approved the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.