Neurosteroids are reduced in diabetic neuropathy and may be associated with the development of neuropathic pain

Introduction: Peripheral and central sensitisation are implicated in the development of neuropathic pain. Hypersensitivity of pain pathway neurons has been described in animal models of diabetic neuropathy, which is postulated to be related to an imbalance between inhibitory and excitatory signals within the spinal cord. GABAergic neurons within the pain pathway are vital for the transmission of painful stimuli to higher centres. A developmental change in the rate of exponential decay of GABAergic synaptic events has been observed in other types of neurons and this may be associated with fluctuations in endogenous neurosteroid tone. Methods: The whole-cell patch-clamp technique was used on slices of neural tissue. Electrophysiological recordings were obtained from wild type mice between the ages of 6 and 80 days in the spinal cord, the nucleus reticularis of the thalamus and the cerebral cortex. Recordings were also obtained from mice with diabetic neuropathy (ob/ob and db/db) between the ages of 60 and 80 days. Behavioural experiments were performed to examine mechanical and thermal nociception. Results: Electrophysiological recordings from cortical pain pathway neurons from mature type-2 diabetic mice revealed that the endogenous neurosteroid tone is reduced compared to control. However, selected neurosteroid compounds had a more pronounced effect on the GABA A receptors of these diabetic mice. ob/ob mice exhibit mechanical hyperalgesia and allodynia, which was reduced by neurosteroids applied exogenously. Conclusions: The reduced endogenous neurosteroid tone in ob/ob mice may be linked to their hypersensitivity. Neurosteroids may exert analgesic effects in pathological pain states by attempting to restore the physiological GABAergic inhibitory tone.


Introduction
A loss of physiological inhibitory tone is associated with hypersensitivity to painful stimuli such as allodynia and hyperalgesia (Chen & Pan, 2002;Zeilhofer, 2008). The GABA A receptor (GABA A R) is the major inhibitory receptor in the mammalian nervous system and mediates inhibitory tone throughout the pain pathway ( Therefore, pharmacological agents that enhance GABA A R function could be useful to counteract lost inhibitory tone (Knabl et al., 2008;Munro et al., 2009). Neurosteroids such as allopregnanolone are potent allosteric modulators of this receptor (Callachan et al., 1987;Hosie et al., 2006; Figure 1). Indeed, an upregulation in the production of endogenous neurosteroids within the spinal cord in response to peripheral inflammation has been shown to have an analgesic effect. The analgesic effect could be suppressed by the administration of finasteride to inhibit the enzyme 5α-R, which converts progesterone to its more active metabolites (Poisbeau et al., 2005;Schlichter et al., 2006).
There is a burgeoning obesity and type-2 diabetes epidemic worldwide and the incidence is likely to become even greater (Danaei et al., 2011). Neurosteroids such as progesterone have been studied for their potentially protective effects for a number of different neuropathologies including stroke, brain and spinal injuries (Mensah-Nyagan et al., 2009;Stein, 2008). It is possible that fluctuations in endogenous neurosteroid levels may have a pathophysiological role in the development of diabetic neuropathic pain. In mice, diabetic neuropathy develops over weeks, which allows disease progression and the efficacy of interventions to be studied within a relatively a short timeframe (Cefalu, 2006;Kaplan & Wagner, 2006). The type-2 diabetic ob/ob mouse, which has an autosomal recessive nonsense mutation on chromosome 6 causing leptin deficiency develops morbid obesity due to its rapacious appetite and exhibits a predictable and spontaneous neuropathic phenotype (Drel et al., 2006;Latham et al., 2009;Lindstrom, 2007;Vareniuk et al., 2007). In contrast, the db/db mouse has an autosomal recessive mutation of the leptin receptor gene, which prevents leptin from activating its receptor (Chen et al., 1996;Chung et al., 1996).
Pain has sensory, emotional and cognitive components and therefore multiple areas within the cerebral cortex are involved in generating the experience of pain (Flor & Bushnell, 2005;Treede et al., 1999). Areas such as the insular, somatosensory, anterior cingulate and prefrontal cortices and the thalamus may be considered as a 'pain matrix.' The cerebral cortex is organised into parallel mini-columns of synaptically linked neurons that span part, or all of the six horizontal cortical layers. Mini-columns may be clustered together to constitute functional modules (Lubke & Feldmeyer, 2007;Mountcastle, 1997). GABAergic inhibitory interneurons are present in all layers of the cortex but are most abundant in layers 2/3 and lower in layer 4 (Meyer et al., 2011). By improving the understanding at the molecular level it may be possible to identify novel therapeutic targets for painful diabetic neuropathy.

Methods
Breeding and housing of mice All procedures were carried out in compliance with the University of Dundee code of practice following consideration by the University Research Ethics Committee (UREC) and in accordance with Schedule 1 of the Animals (Scientific Procedures) Act 1986 (UK). Home Office Project Licence numbers: PPL 60/4144 and PPL 60/4005. Wellcome Trust Grant number 090667. Wild Type (WT) mice aged less than 2 months were obtained from an in-house colony. C57/Bl6J WT mice aged 2 months were purchased from Charles River, UK while OlaHSD ob/ob mice, db/db mice and their respective strain-matched WT littermates, all aged Text box 1

What's already known about this topic?
• There is a worldwide obesity diabetes epidemic, causing a huge amount of morbidity including neuropathy and neuropathic pain.
• The mechanisms responsible for the development of neuropathy and neuropathic pain are not well understood.
• Peripheral and central sensitisation are implicated in the development of neuropathic pain with neuroplastic changes occurring at multiple levels of the pain pathway.
• Hypersensitivity of pain pathway neurons has been described in animal models of diabetic neuropathy, which is postulated to be related to an imbalance between inhibitory and excitatory signals within the spinal cord. GABAergic neurons within the pain pathway are vital for the transmission of painful stimuli to higher centres involved in the perception of pain.
• GABA A receptors are an important target for many drugs, and specific endogenous neurosteroids act as potent allosteric modulators of these receptors.
• A developmental change in the rate of exponential decay of GABAergic synaptic events has been observed in other types of neurons and this may be associated with fluctuations in endogenous neurosteroid tone.

What does this study add?
• This paper explores potential underlying mechanisms and identifies potential therapeutic targets in order to promote translational work in this field.
• This is the first electrophysiological GABA A receptor characterisation of two models of type-2 diabetes mellitus and reports the discovery of a reduced endogenous GABA-ergic neurosteroid tone that may mediate painful neuropathic hypersensitivity.
• The paper then reports the anti-nociceptive impact of neurosteroids on live mice, thus complementing the electrophysiological data.
• These discoveries may ultimately lead to a new rational avenue of research aimed at understanding and treating painful diabetic neuropathy and the neuropathic mechanisms may be analogous in other neuropathic conditions such as chemotherapyinduced neuropathic pain.
2 months were purchased from Harlan UK and housed in the same in-house colony. All animals were male and were kept under an alternating 12hr light/dark cycle and had ad libitum access to food and water.
Brain slicing technique Brain tissue was also obtained following the cervical dislocation method (Schedule 1) and submerged in aCSF as above. For all nucleus reticularis (nRT) preparations the aCSF solution was the same as that described above for spinal cord but the sucrose concentration was increased to 234mM giving a total osmolarity Cholesterol is taken through the mitochondrial membrane by the translocator protein where it is converted to pregnenolone by the cytochrome P450 side chain cleavage enzyme. Pregnenolone undergoes stepwise enzymatic conversion to other neurosteroid compounds and ultimately allopregnanolone which modulates GABA A R function. Neurosteroids may act via paracrine or autocrine mechanisms. Postsynaptic GABA A Rs are activated by GABA that has been released from vesicles in the presynaptic nerve terminal. GABA induces a conformational change in the GABA A R, which 'opens' its central pore, allowing the passage of chloride ions. The negatively charged chloride ions typically induce hyperpolarisation of the neuronal membrane, which is associated with neuronal inhibition. Neurosteroids such as allopregnanolone enhance GABA A R function and therefore facilitate neural inhibition.
of ~340mOsm/l as this improved the condition of the slices. For cortical preparations in mice below 2 months of age the aCSF was the same as for the spinal cord. Neuronal viability deteriorates with increasing age and for mice above the age of 2 months, including all ob/ob and db/db, a different solution was required to optimise the condition of the slices as previously described by Maguire et al. (2014). This consisted of 140mM potassium gluconate, 10mM HEPES, 15mM sodium gluconate, 0.2mM EGTA, 4mM NaCl, 1mM ascorbic acid and 3mM pyruvic acid. Sodium hydroxide solution was then added to bring the pH up to 7.2. The brain was removed carefully from the skull and slices were obtained using a Vibratome series 1000 PLUS Sectioning System (Intracell, Royston, Hertfordshire, UK). Cortical slices were cut in the coronal plane and nRT slices in the horizontal plane. Slice thickness was greatest for the youngest mice and least for the oldest mice (250-350μM). Slices were transferred immediately to a storage chamber as previously described.

Electrophysiology
Subsequently, the slices were transferred to a recording chamber under an Olympus BX51WI fixed-stage upright microscope. An infrared differential interference contrast disc and a water immersion objective (× 40) were used for visualisation of neurons within the dorsal horn of the spinal cord, the nucleus reticularis of the thalamus and the somatosensory area of the cortex respectively. The chamber was perfused continuously with oxygenated artificial extracellular solution (aECS). Slices were held in position using a small grid. Whole-cell patch-clamp recordings were obtained using an Axopatch 200B amplifier and all recordings were made at a holding potential of -60mV and at a temperature of 35°C. The extracellular solution contained kynurenic acid (2mM), strychnine (0.5μM) and tetrodotoxin (0.5μM) to antagonise ionotropic glutamate, glycine receptors and voltage-gated sodium channels respectively. Patch electrodes were prepared from thick walled borosilicate glass capillaries (Garner Glass Co., Claremont, CA, USA). Such electrodes had an open-tip resistance of ~4MΩ when filled with intracellular solution containing: 135mM CsCl, 10mM HEPES, 10mM EGTA, 1mM CaCl 2 , 1mM MgCl 2 , 2mM Mg-ATP, 5mM QX-314, pH 7.2, titrated with CsOH. The measured osmolarity was 310mOsmol/l. Typically, the mean whole-cell capacitance was 5-15pF. Series resistance was compensated for by up to 80% and recordings were considered invalid where the series resistance changed by more than 20% or if it exceeded 15MΩ. A 2 kHz frequency filter was used for all recordings and analysis of each cell was performed offline.

Data analysis
The Strathclyde Electrophysiology Software, WinEDR and WinWCP, (Dr J Dempster, University of Strathclyde, Glasgow, UK) was used for the analysis of recordings. Only recordings that met specific quality criteria were included in the analysis. Using an algorithmic detection protocol, miniature inhibitory postsynaptic currents (mIPSCs) were detected with an amplitude threshold of at least -5pA and duration > 2ms. Each individual mIPSC was then inspected visually to ensure validity and exclude artifactual events. mIPSCs with a rise time of more than 1ms were excluded to prevent the inclusion of events originating from a distal source. At least 50 events were sought for each recording, and the average peak amplitude, rise time (10-90%), charge transfer (area under the curve) and T 70 (time required to decay by 70%), were analysed. The mIPSCs were digitally averaged by alignment at the midpoint of the rising phase. The mIPSC decay was fitted by monexponential (y(t) = Ae -t/τ ) and biexponential (y(t) = A fast e (-t/τfast) + A slow e (-t/τslow) ) functions to determine which one was more appropriate (A is amplitude, t is time and τ is the decay time constant). The standard deviations of the residuals of the monexponential and biexponential functions were measured and an F test applied. For the vast majority of mIPSCs analysed, the decay was best described by a biexponential function. Consequently, a mean weighted decay constant (τ w ) was calculated to determine the relative contribution of each decay component. τ w is a mathematical constant generated by considering the fast initial component of biexponential decay τ 1 and the later slower component τ 2 . The value of τ w is determined for the mean mIPSC for each cell by determining the relative proportion that τ 1 and τ 2 contribute to the biexponential decay. The following equation was used: τ w = τ fast P 1 + τ slow P 2 , where P 1 and P 2 represent the proportions of the synaptic current decay curve described by each component.

Drug and solution preparation and administration
Salts used in the preparation of aECS and aCSF solutions were purchased from VWR (West Chester, Pennsylvania, USA). Strychnine (Sigma Chemicals, St. Louis, MO, USA), tetrodotoxin (Tocris, Bristol, UK) and bicuculline (Axxora, Nottingham, UK) and THIP (generous gift from B Ebert) were prepared as concentrated stock solutions in double-filtered water to be added to the aECS. Other compounds such as progesterone, ganaxolone, allopregnanolone, dihydroxy-progesterone, provera, indometacin, finasteride were purchased from Tocris or Sigma and prepared as concentrated stock solutions in dimethyl sulfoxide (DMSO). The cyclodextrins (CDs; Sigma) were dissolved directly into the aECS, or the intracellular solution. Brain and spinal cord slices were placed under the microscope into a transparent plastic chamber filled with aECS. The aECS was perfused through the chamber using a rate-adjustable gravity-based system consisting of hard plastic tubing that connected an oxygenated reservoir to the chamber. Simultaneously, a peristaltic pump system (Minipuls 3, Gilson, UK) drained the aECS from the opposite side of the chamber and recycled it back to the reservoir. The fluid-filled circuit also ran through a custom made heating system (G23, UCL, London, UK) controlled by a temperature monitoring system (School of Pharmacology, London, UK) that maintained the near physiological temperature of 35°C throughout.
Behavioural experiments Drug administration. The drug, or vehicle, was administered by intra-peritoneal injection using a fine 1ml syringe and experiments were made pre-and post-injection in a randomised and blinded manner.  Figure 2A,B). The τ W is approximately equivalent to the time taken for the mIPSC to decrease by 67% from the peak amplitude. The unfavourable signal-to-noise of individual mIPSCs precludes the accurate fitting of the τ W to individual mIPSCs. Therefore, this function is fitted to the mean mIPSC derived for each neuron.  (Table S2). Intracellular application of γ-CD via the recording pipette was found to be the optimal method of application (see Additional material) and is consistent with the hypothesis Pipette-applied γ-CD reduces decay time of GABA A R mIPSCs of layer 2/3 pyramidal cortical neurons at two stages of development The mIPSC decay (τ W ) of L2/3 cortical neurons at two stages of maturity (P9-10 and P60-75) was significantly decreased in the presence of intracellular γ-CD (P <0.05, Table S1). These data are in contrast to the lack of effect of γ-CD observed in nRT neurons at P9-10 and P17-24. However, the data for P9/10 L2/3 cortical neurons are consistent with data published previously (Brown et al., 2015). Different regions of the nervous system reach maturation at different ages and it is possible that this may account for the regional variations observed. Interestingly, the endogenous neurosteroid tone that previously appeared to be lost during maturation re-emerges in the adult mouse cortex, which may have a significant physiological role.
A comparison of synaptic GABAAR mIPSCs of cortical layer 2/3 pyramidal neurons in adult WT, ob/ob and db/db mice incorporating the use of cyclodextrin As the behavioural studies were to be conducted in adult mice, it was decided to make recordings from adult layer 2/3 cortex neurons for three reasons: 1) Viable recordings of nRT neurons of  To exclude the presence of a direct effect of leptin itself recordings were additionally made in the db/db, which is able to synthesise leptin but lacks the receptor. There was a modest but significant reduction in the mIPSC decay (τ W ) of cortical neurons between the diabetic mice and the corresponding WT littermates, but no significant difference in the τ W , between the three WT strains (P <0.05; Table S4, Figure 3A,C).
Recordings were made to determine that neither the strain, nor the lack of leptin (directly) was an important factor in the shortening of mIPSC τ W with γ-CD. In the presence of intracellular γ-CD, there was no significant difference in the mIPSC τ W of between all five types of mice (Table S5, Figure 3D, P > 0.05). These findings suggest that synaptic GABAAR function is very similar across all these strains of mice included in the study when the endogenous neurosteroid tone is removed by γ-CD. The results are also consistent with the hypothesis that there is a neurosteroid tone at P60-75, but that it is reduced in both mouse models of diabetic neuropathy. However, the data do not exclude the possibility that the sensitivity of L2/3 cortical GABA A Rs to neurosteroids may be reduced.
A comparison of the effect of neurosteroids on cortical layer 2/3 neurons in adult WT, ob/ob and db/db mice The lipophilic intravenous anaesthetics etomidate and propofol, which in common with neuroactive steroids enhance the function of GABA A Rs, require relatively prolonged incubation times to approach equilibrium in a brain slice preparation-over 1-2 hours (Benkwitz et al., 2007;Gredell et al., 2004). It is conceivable that the same is true for neurosteroids (Li et al., 2007) therefore recordings were made after incubation treatment with allopregnanolone and ganaxolone. In contrast to the relatively modest prolongation of mIPSCs described above with an acute steroid application protocol (τ W : control = 4.0 ± 0.3 ms, n = 7; allopregnanolone 1 μM = 4.5 ± 0.4 ms, n = 7; paired Student's t test, P <0.05), a 2-hour incubation of the brain slice preparation with nanomolar concentrations of allopregnanolone (100 -300 nM) produced a dramatic concentration-dependent increase of the WT GABAAR mIPSCs (Table S6, Figure 4A,C,D; P <0.05). These results indicate that allopregnanolone is a potent modulator of synaptic GABAARs in mature cortical neurons, but additionally demonstrate that the steroid effect is greatly underestimated when applied acutely. The large difference between acute bath application and the 2-hour incubation (see Additional material) is probably a consequence of the time required for the steroid to approach equilibrium within the brain slice. No such effect is observed in time-matched controls.
A 2-hour incubation of the ob/ob brain slice preparation with allopregnanolone produced a clear concentration-dependent prolongation of GABAAR mIPSC τ W (Table S6, Figure 4B-D; P <0.05). The effect of allopregnanolone was similar for WT, ob/ob and db/db mice (Table S6; P > 0.05). When the data were normalised to reflect the control mIPSC τ W , there was no significant difference in the effect of between the three types of mice ( Figure S1, P > 0.05). These results suggest that the sensitivity of cortical GABAARs to allopregnanolone in diabetic mice is similar to age-matched WT.
A 2-hour incubation with ganaxolone produced a significant concentration-dependent increase of the WT mIPSC τ W , although the magnitude of the effect was less than that induced by allopregnanolone (Table S7, Figure 4E,G,H; P <0.05). In the ob/ob and db/db brain slices incubated with ganaxolone the mIPSC τ W prolongation was comparatively greater than for equivalent WT neurons, but there was no significant difference between the ob/ob and db/db (Table S7; P > 0.05). These observations contrast with that of allopregnanolone. These findings suggest that there is a difference in the effect of ganaxolone incubation treatment (but not allopregnanolone) in the ob/ob and db/db mice compared to the WT mice. A 2-hour incubation with progesterone produced a relatively modest prolongation of mIPSC τ W in WT mice. The highest concentration of progesterone tested was only slightly more effective than the lowest concentration investigated here (Table S8, Figure 5A,C; P <0.05). These results suggest that the enzymatic function (5α-R and 3α-HSD) is intact and neurosteroids may be synthesised with brain slice incubation of the precursor.
Progesterone incubation treatment also produced a similarly modest prolongation of mIPSC τ W in the ob/ob and db/db (Table S8, Figure 5B,C; P <0.05). When the data for τ W were normalised. When the effects of 50 μM progesterone were expressed as a percentage of the representative control, the steroid had a greater impact on the diabetic mice (P < 0.05), but there was no intergroup difference between the ob/ob and db/db mice (P > 0.05; Table S8, Figure S1; Figure 5D).
Bath application of DHP (3μM) had no effect on the GABA A R mIPSCs in WT neurons (τ W : Control = 5 ± 0.3 ms, n = 4; 3 μM DHP = 4.7 ± 0.3 ms, n = 4, P > 0.05). These findings are consistent with previous work on VB neurons (Brown et al., 2015). The lack of effect after the acute application of DHP contrasts to the modest effects of acutely applied allopregnanolone and ganaxolone (see Additional material). Contrastingly, 2 hours of incubation with DHP (1 -3 μM) produced a significant, concentration-dependent prolongation of GABA A R mIPSCs in WT (P <0.05; Table S9, Figure 5E,G). These results indicate that mature WT L2/3 cortical neurons have intact 3α-HSD enzymatic function and are able to convert DHP into the active metabolite allopregnanolone. 3μM DHP incubation produced a more pronounced effect in the ob/ob and db/db mice compared to the WT (P <0.05, Table S9), indicating that not only is 3α-HSD enzymatic function preserved in mature mice with T2DM, but may be up-regulated.
Selective inhibition of enzymes within the neurosteroid synthesis pathway in mature layer 2/3 cortical neurons from WT, ob/ob and db/db mice Finasteride itself has no direct effect on GABA A R mIPSCs, but pre-treatment with this 5α-R enzyme inhibitor prevents the conversion of progesterone into GABA A R-active neurosteroid (Sanna et al., 2004). Recordings were made after at least 2 hours of incubation with finasteride and progesterone. Finasteride alone had no effect on WT mIPSC τ W but it did prevent the effect of progesterone (Table S10; P <0.05). Finasteride alone also had no  effect on ob/ob mIPSC τ W of mice, but it did prevent the effect of progesterone (Table S10; P < 0.05) indicating that progesterone requires the 5α-R for it to be converted to its' active metabolites, (although do not prove neurosteroid synthesis by the neurons itself). Finasteride alone had no effect on mIPSC τ W , despite the suggested presence of a modest endogenous neurosteroid tone in mature WT mice. This apparent paradox may be explained by comparing how finasteride and γ-CD act. γ-CD will remove the endogenous neurosteroid present, whereas although finasteride should prevent new allopregnanolone synthesis, it will have little impact on that already present. Therefore, the apparent lack of an effect of finasteride may reflect the relatively slow turnover of pre-synthesised allopregnanolone and is consistent with the literature (Brown et al.,  2015).
The 3α-HSD enzyme inhibitor provera suppresses the effects of DHP, but not ganaxolone on GABA A R-mediated mIPSCs of mature cortical neurons Recordings were made in WT mice after at least 2 hours of incubation with provera and DHP, or provera and ganaxolone. Provera alone exerted a modest effect on mIPSC τ W but, in addition, it prevented the effect of DHP (Table S11, Figure S2 A,B; P <0.05).
Provera did not inhibit the effect of ganaxolone on τ W in WT mice (Table S11, Figure S2 C,D, P >0.05). These results confirm that DHP requires enzymatic conversion by 3α-HSD for it to induce a prolongation of τ W . In contrast, ganaxolone was unaffected by 3α-HSD inhibition with provera.
The 3α-HSD enzyme inhibitor indometacin suppresses the effects of DHP and ganaxolone, but not those of allopregnanolone on GABA A R-mediated mIPSCs of mature cortical neurons Indometacin alone had no effect on τ W , but it also prevented the effect of DHP (Table S12, Figure S2 E,F; P <0.05). By contrast, indometacin had no effect on the prolongation of mIPSC τ W by preincubated allopregnanolone (Table S13, Figure S2 G,H; P >0.05) confirming that allopregnanolone does not require 3α-HSD in order to modulate GABA A R mIPSCs. Indometacin did not increase the effect of allopregnanolone by potentially inhibiting its metabolism to an inactive form. These experiments with another 3α-HSD enzyme inhibitor confirm that DHP requires conversion to a more active form by 3α-HSD in order for it to induce prolongation of GABA A R mIPSC decay time. By contrast, indometacin exhibited concentration-dependent inhibition of ganaxolone incubation treatment on τ W (Table S13, Figure S2 I,J, P <0.05). These results with ganaxolone were unexpected given the lack of impact that indometacin had on the effectiveness of allopregnanolone. Indeed, inhibiting 3α-HSD with provera had no impact on ganaxolone, but prevented the effect of DHP on GABA A R mIPSC τ W . This raises the question as to whether ganaxolone is an allosteric modulator of the GABA A R and a precursor to a more active neurosteroid such as allopregnanolone. Alternatively, it raises the question of whether indometacin could be a silent competitive steroid antagonist at the GABA A R and prevent the action of ganaxolone by that mechanism (explored in next section).
The effect of pipette-applied neurosteroids on layer 2/3 cortical GABA A R mIPSCs in mature WT mice (P60-75) Acute allopregnanolone had only a modest effect on the GABA A Rmediated mIPSCs decay time (see Additional material), but was far more efficacious in this respect when brain tissue slices were incubated with steroid for > 2 hours, suggesting that the steroid is relatively slow to equilibrate within the tissue. It has been proposed that endogenous neurosteroids may be synthesised in the postsynaptic neuron and act in an autocrine manner to influence GABA-ergic transmission (  , 1986). Recordings were made with allopregnanolone, or ganaxolone present in the recording pipette to explore whether the steroid could modulate synaptic GABA A Rs when delivered to the intracellular compartment. These recordings were compared to separate control recordings (i.e. they were not paired).
The presence of allopregnanolone in the recording pipette significantly increased the WT mIPSC τ W in a concentration-dependent manner (Table S14, Figure S3 A,C; P < 0.05). Here, the higher concentration of allopregnanolone (10 μM) had a comparatively large concentration-dependent effect on GABA A R mIPSCs when presented acutely within the recording electrode. This finding indicates that neurosteroids are able to exert their effect via the intracellular compartment and is in agreement with the literature (Akk et al., 2005).
The concentrations of pipette-applied allopregnanolone are relatively high, but the time scale from application to recording is short (< 10 minutes) and dialysis rate of the intracellular contents may be an influential limiting factor. The presence of ganaxolone in the recording pipette also significantly increased the WT mIPSC τ W in a concentration-dependent manner (Table S15, Figure S3 E,G, P < 0.05). The effect of pipette-applied ganaxolone is consistent with the recordings described above with allopregnanolone, although ganaxolone had a less pronounced effect on GABA A R mIPSCs. Collectively these experiments illustrate that steroid penetration of brain slice tissue is a significant limiting factor, the intracellular application is an effective method of presenting neuroactive steroids to GABA A Rs (Table S14-Table S16, Figure 1, Figure S3 A-H) and that, in these neurons, allopregnanolone is a more effective modulator of the synaptic GABA A R than ganaxolone ( Figure S3 C,D,G,H).
Recordings were also made in ob/ob neurons with allopregnanolone, or ganaxolone, present in the recording pipette. The ob/ob mIPSC τ W was increased by the presence of allopregnanolone in the recording pipette (Table S14, Figure S3 B,C; P < 0.05). After normalisation, intracellular allopregnanolone 3 -10 μM increased τ W to similar percentages of the strain representative controls (Table S14, Figure S3 D, P > 0.05). These findings are consistent with the hypothesis that there is no difference in the sensitivity of the cortical GABA A R to neurosteroids between the ob/ob and WT mice.
The ob/ob mIPSC τ W was increased by the presence of ganaxolone in the recording pipette (Table S15, Figure S3 F,G, P < 0.05). Ganaxolone did not have a concentration-dependent effect on ob/ob cortical GABA A R mIPSCs when acutely delivered intracellularly and the magnitude of the effect was less than that produced by equivalent pipette concentrations of allopregnanolone (Table S14-Table S15, Figure S3 C,D,G,H,L, P > 0.05). There was no difference in the GABA A R mIPSC τ W between the ob/ob and the WT when treated with intracellular ganaxolone. This finding is consistent with the recordings described above with allopregnanolone, although intracellular ganaxolone had a less pronounced effect.
In order to explore whether indometacin could be a silent competitive antagonist at the GABA A R (and therefore prevent modulation by ganaxolone) recordings were made with indometacin ± ganaxolone presented in the pipette. The WT mIPSC τ W was unchanged by the presence of indometacin in the pipette and indometacin had no effect on the modulatory action of ganaxolone (Table S16, Figure S3 I,J, P >0.05).
Recordings were also made in ob/ob neurons with indometacin ± ganaxolone presented in the pipette. The mIPSC τ W in ob/ob mice was unaffected by the presence of indometacin in the pipette and indometacin had no effect on the modulatory action of ganaxolone (Table S16, P >0.05). It is possible that indometacin may inhibit the action of ganaxolone by preventing its conversion to a more active compound (such as allopregnanolone).
The WT and ob/ob mIPSC τ W was unaffected by the presence of DHP in the recording pipette (Table S17, P > 0.05). These findings contrast with the significant effects of intracellular allopregnanolone and ganaxolone and are consistent with the idea that DHP is a precursor compound (Figure 1, Figure S3 K,L). Considering these results it would be difficult to make the case for the alternative hypothesis that GABA A R sensitivity is increased in ob/ob and db/db mice.

ob/ob mice have impaired sensorimotor coordination in comparison to WT mice
The ob/ob mice were able to remain on the rotarod for a significantly shorter time than were WT mice (WT = 216 ± 14 s, n = 15; ob/ob = 21 ± 5 s, n = 15; Mann-Whitney Rank Sum test, P < 0.001; Figure 6A). The impaired rotarod performance of the ob/ob mouse is consistent with a previous report (Mayers et al., 2009). It is not possible to have obese WT mice as controls because those obese mice would be likely to have similar neurological consequences as the ob/ob mice.
ob/ob mice exhibit prolonged tail withdrawal from noxious heat in comparison to WT mice The tail flick test was used to characterise the response of the WT and the ob/ob mouse to three distinct temperatures. At 48°C and 50°C ob/ob mice exhibited significantly longer tail withdrawal latencies compared to WT mice (Table S18, Figure 6B, P < 0.05). By contrast, there was no significant difference with a water temperature of 46°C (Table S18, Figure 6B,

Ganaxolone increases tail withdrawal latency in WT mice. Ganaxolone impairs thermal nociception in WT mice
The tail flick test is a useful tool for assessing thermal nociception. Ganaxolone was considered a priori considered to be a metabolically stable synthetic analogue of allopregnanolone, the molecular structure of which differs only by having an extra methyl group that prevents oxidation to an inactive form and has previously been used in clinical trials (Carter et al., 1997). Ganaxolone required to be solubilised with hydroxypropyl β-CD prior to intraperitoneal injection (Besheer et al., 2010; Carter et al., 1997; Reddy & Rogawski, 2010). There were no significant differences in the baseline withdrawal latencies in response to a noxious thermal stimulus (50°C) between the four groups of mice used to examine the effects of ganaxolone (Table S19, Figure 6C, P > 0.05). Ganaxolone induced a dose-dependent prolongation of tail withdrawal latency in WT mice after 30 minutes and this effect lasted more than 90 minutes (Table S19, Figure 6C, P < 0.05). These data suggest that ganaxolone exhibits a dose-dependent analgesic effect in a test of thermal nociception in WT mice. This finding is consistent with the intrathecal administration of ganaxolone (Asiedu et al., 2012) and also other reports of the analgesic effects of similar neurosteroids in rats in the setting of post-chemotherapy neuropathy (Meyer et al., 2010; Meyer et al., 2011).

High doses of ganaxolone impair rotarod performance of WT mice
Only the highest dose of ganaxolone significantly impaired the performance of WT mice on the rotarod. There was no significant difference in the baseline rotarod performance of the four groups in the study (Table S20, Figure 6D, P > 0.05). However, at 15, 30 and 60 minutes post-injection the highest dose of ganaxolone (30 mg/kg) significantly impaired performance of WT mice on the rotarod (Table S20, Figure 6D, P < 0.05). The effect of ganaxolone (30 mg/kg) was no longer apparent at 120 minutes post-injection (Table S20, Figure 6D, P > 0.05). Taken in conjunction with the tail flick data, these data suggest that ganaxolone exhibits an analgesic effect at a dose of 10 mg/kg but only impairs rotarod performance in WT mice at higher doses such as 30 mg/kg. In addition, the dose-dependent effect on rotarod performance is consistent with the literature (Carter et al., 1997).  (Meyer et al., 2011). The ob/ob mice responded significantly more frequently than the WT mice to the 0.16 g, 0.4 g and 0.6 g filaments (Table S21, Figure 6E, P < 0.05). By contrast, there was no significant difference in the frequency of responses to the 1 g vF filament (Table S21, Figure 6E, Ganaxolone reduces mechanical hypersensitivity in ob/ob mice and reduces mechanical nociceptive pain in both WT and ob/ob mice vF filaments were used for the comparison of mechanical sensitivity of WT and ob/ob mice before and after intraperitoneal drug administration. The β-CD vehicle had no impact on the response to any of the vF filaments in WT or ob/ob mice (Table S22, P > 0.05). Ganaxolone (10 mg/kg) had no impact on the response to the 0.16 g, 0.4 g or 0.6 g vF filaments in the WT mouse (Table S23, Figure 6F, P > 0.05) but ganaxolone did reduce the response of Figure 6. ob/ob mice have impaired sensorimotor coordination, exhibit hypoalgesia to noxious heat and mechanical hypersensitivity in comparison to WT mice. Ganaxolone impairs thermal nociception in WT mice at doses low enough to preserve rotarod performance. Ganaxolone reduces mechanical hypersensitivity in ob/ob mice and reduces mechanical nociceptive pain in both WT and ob/ob mice. (A) Histogram illustrating the dramatic impairment of sensorimotor function exhibited by mature ob/ob mice. The ob/ob mice were able to remain on the accelerating rotarod for a significantly shorter time than were WT mice (n = 15; Mann-Whitney Rank Sum test P < 0.001).
(B) Histogram illustrating that mature ob/ob mice have a delayed response to thermal noxious stimuli in comparison to age-matched WT mice. (P < 0.05). There was no significant difference at the less noxious temperature of 46°C (n = 20 per group, Mann-Whitney Rank Sum test P > 0.05). A maximum withdrawal latency of 15 seconds was enforced to minimise the possibility of tissue damage. (C) Histogram illustrating that ob/ob mice exhibit mechanical hypersensitivity in comparison to WT mice. The ob/ob mice responded significantly more frequently than the WT mice to the 0.16 g, 0.4 g and 0.6 g filaments (P < 0.05). By contrast, there was no significant difference in the frequency of responses to the 1 g vF filament (Mann-Whitney Rank Sum test P > 0.05). These results confirm the presence of mechanical hypersensitivity in the ob/ob mouse. (D) Histogram illustrating that ganaxolone induced a dose-dependent prolongation of tail withdrawal latency in WT mice after 30 minutes (P < 0.05). The solubilising vehicle β-CD had no effect (Kruskall Wallis One-Way ANOVA on ranks P > 0.05). (E) Histogram showing that at 15, 30 and 60 minutes post-injection the highest dose of ganaxolone (30mg/kg) significantly impaired performance of WT mice on the rotarod (P < 0.05). The effect of ganaxolone (30 mg/kg) was no longer apparent at 120 minutes post-injection (P > 0.05). The solubilising vehicle β-CD had no effect on rotarod performance (Kruskall Wallis One-Way ANOVA on ranks P > 0.05). (F) Histogram illustrating that ganaxolone had no impact on the response to the 0.16 g, 0.4 g or 0.6 g vF filaments in WT mice (P > 0.05) but ganaxolone did reduce the response of WT mice to the 1.0 g vF filament (n = 10 per group, Wilcoxon signed rank test (before & after) P < 0.05). These data suggest that ganaxolone is analgesic for mechanical nociceptive pain in WT mice. WT mice do not exhibit hypersensitivity under normal conditions; therefore it is not unexpected that these drugs had no effect on the response to the smaller vF filaments. (G) Histogram illustrating that ganaxolone had no impact on the response to the 0.16 g vF filament the ob/ob mouse (P > 0.05). In contrast, ganaxolone reduced the response of the ob/ob mouse to the 0.4 g, 0.6 g and 1 g vF filaments (n = 10 per group, Wilcoxon signed rank test (before & after) P < 0.05). Ganaxolone reduced the response of the ob/ob mouse to the 0.4 g and the 0.6g vF filaments, which is consistent with the idea that this neuroactive steroid may reduce mechanical hypersensitivity. As described earlier, the 1.0 g vF filament induces a withdrawal response on ~85% of occasions in the WT and ~93% in the ob/ob mouse and may therefore be considered as an unambiguous test of mechanical nociceptive pain. Taken as a whole, these results show that ganaxolone was effective for mechanical nociceptive pain in both strains of mice.
WT mice to the 1.0 g vF filament (Table S23, Figure 6F, P < 0.05). These data suggest that ganaxolone is analgesic for mechanical nociceptive pain in WT mice. By definition, WT mice do not exhibit hypersensitivity or allodynia under normal conditions; therefore it is perhaps unsurprising that these drugs did not impact on the response to the smaller vF filaments.
Ganaxolone had no impact on the response rate to the 0.16 g vF filament in ob/ob mice (Table S24, Figure 6G, P > 0.05), but in contrast, ganaxolone reduced the response rate of ob/ob mice to the 0.4 g, 0.6 g and 1 g vF filaments (Table S24, Figure 6G, P < 0.05). ob/ob mice have exaggerated baseline response rates to the 0.16 g, 0.4 g and 0.6 g vF filaments in comparison to WT mice ( Figure 6E). Ganaxolone reduced the response rates of ob/ob mice to the 0.4 g and 0.6 g vF filaments, which is consistent with the idea that these neurosteroids may reduce mechanical hypersensitivity. As described earlier, the 1.0 g vF filament induces a withdrawal response on ~85% of occasions in WT mice and ~93% in ob/ob mice ( Figure 6E) and may therefore be considered as an unambiguous test of mechanical nociceptive pain. Taken as a whole, these results show that ganaxolone was effective for mechanical nociceptive pain in both strains of mice.

Discussion
The decay time of GABA A R mIPSCs decreases with development at three levels of the pain pathway. GABA A Rs from pain pathway neurons are sensitive to modulation by neurosteroids, and this was explored using γ-CD applied within the recording pipette suggesting that neurosteroids may be synthesised within neurons themselves. The endogenous neurosteroid tone that previously appeared to be lost with maturation re-emerges in the adult mouse cortex, which is suggestive of a significant physiological role. There are no published reports of the electrophysiological characterisation of synaptic GABA A R function for T2DM. It was unknown whether neurosteroid tone would be altered in mice with diabetic neuropathy. In inflammatory pain, neurosteroidogenesis is upregulated to mediate a form of endogenous analgesia by enhancing GABAergic neural inhibition (Poisbeau et al., 2005) GABA A R mIPSCs from both ob/ob and db/db mice had significantly shorter decay times than the three WT strains, making it unlikely that leptin is involved. A decrease in the sensitivity of the GABA A R to allosteric modulators such as neurosteroids would be a possible explanation for the shorter GABA A R mIPSCs. However, reduced GABA A R sensitivity is inconsistent with the exaggerated effect of ganaxolone and DHP in ob/ob and the similar effect of allopregnanolone in both WT and diabetic cortical neurons. Pipette-applied allopregnanolone or ganaxolone induced the same concentration-dependent increase in mIPSC decay time, in ob/ob and WT, in keeping with Akk et al. (2005). A possible explanation may be that in T2DM mice there is a reduction in the endogenous neurosteroid tone, which is caused by a common pathological insult.
Reduced neurosteroid tone could result in diminished GABAergic inhibition and subsequently a hypersensitive phenotype. Theoretically, other endogenous compounds that modulate the GABA A R such as endocannabinoids may be affected by a common mechanism in T2DM; however the selective effects of the neurosteroid scavenger γ-CD (in comparison to the lack of effect with α-CD and β-CD) make this less likely. γ-CD reduced the GABA A R mIPSC decay time to similar baseline values for all five lines of mice. These findings are consistent with the hypothesis that mature WT mice possess an endogenous autocrine neurosteroid tone that may fine-tune GABA A R function under physiological conditions but is reduced in ob/ob and db/db mice (in which underlying GABA A R function and sensitivity are otherwise unchanged). Thus, in neuropathic pain associated with diabetes, neurosteroid levels may be reduced, which contrasts to inflammatory pain, where neurosteroid levels may be upregulated (Poisbeau et al., 2005).
With reference to neurosteroidogenesis in the diabetic mice, incubation treatment with progesterone produced a modest prolongation of GABA A R mIPSC decay time in ob/ob mice that was similar to WT controls and was not concentration-dependent from 1-50 μM. Additional recordings made at the highest progesterone concentration in db/db mice were no different to those of the WT or ob/ob. This suggests that enzymatic function responsible for the endogenous synthesis of allopregnanolone is intact in ob/ob and db/db. Indeed, due to the shorter baseline decay time of mIPSCs of ob/ob and db/db mice, progesterone actually had a proportionately greater effect in the diabetic mice than in WT controls. In keeping with what had been observed in the WT, incubation treatment with finasteride (5α-R inhibitor) also had no effect on ob/ob mIPSCs directly, but was able to block the effect of progesterone in ob/ob mice.
Incubation treatment with DHP (the progesterone metabolite) produced a significant, concentration-dependent prolongation of GABA A R mIPSC decay time that was exaggerated in diabetic mice compared to WT. This finding suggests that not only is 3α-HSD enzymatic function preserved in mature T2DM mice, but it may actually be upregulated. A potential increase in 3α-HSD activity could be a partial compensation for a reduced baseline endogenous neurosteroid tone in diabetic mice. In keeping with what had been observed in WT mice, pipette-applied DHP had no effect on the ob/ob mIPSCs. The lack of effect of DHP is consistent with DHP's role as a metabolic precursor to the active compound allopregnanolone.
Ganaxolone incubation treatment also had a greater impact in ob/ob mice compared with WT, despite the compound being considered previously to be more metabolically stable (Carter et al., 1997). This finding permits the possibility that ganaxolone may potentially be demethylated to allopregnanolone in order to exert maximal effect on cortical GABA A Rs. The idea of ganaxolone as both an agonist and precursor to allopregnanolone would also be consistent with the relatively larger effect of ganaxolone incubation in ob/ob mice, which may exhibit an upregulation of key enzymes such as CYP2C and 3α-HSD. The inhibitory effect of indometacin on ganaxolone incubation treatment is also consistent with the notion of ganaxolone as an active precursor of allopregnanolone. Indeed, indometacin had no impact on ob/ob cortical neurons when present in the pipette or after 2 hours of incubation treatment. When indometacin and ganaxolone were co-applied within the pipette, indometacin had no impact on the ability of ganaxolone to prolong mIPSCs.  ). Therefore, one possible explanation for this is that indometacin could compete with ganaxolone for the demethylating effects of CYP2C9, however, it must be noted that indometacin may have other non-specific effects.
The underlying mechanism responsible for the reduced neurosteroid tone in the diabetic mice is uncertain, but may be due to mitochondrial dysfunction ( The mechanical hypersensitivity of ob/ob mice and the increased response to ganaxolone reflected the observed electrophysiological findings. Ob/ob mice developed obesity and T2DM on a normal diet, which led to painful neuropathy by the age of P60-75. At this age they exhibited sensorimotor impairment, thermal hypoalgesia, cold allodynia and mechanical allodynia. In WT mice, the neuroactive steroid ganaxolone impaired sensorimotor function at 30 mg/kg, but the lower dose of 10 mg/kg did not, and was analgesic for thermal and mechanical nociceptive pain. The effect of ganaxolone on thermal nociception and sensorimotor impairment could not be tested on ob/ob mice due to their preexisting deficits. However, ganaxolone significantly reduced mechanical allodynia in ob/ob mice. These results suggest that GABA A R-modulatory neurosteroidal drugs such as ganaxolone may have analgesic properties for nociceptive pain and also neuropathic pain by restoration of the depressed endogenous neurosteroid tone. Please refer to Methods section for details of standard software used for data analysis.

Author contributions
Dr Stephen Humble is responsible for this all work, including planning the experiments, performing the experiments and writing the paper. Prof Hales, Lambert and Dr Belelli assisted Dr Humble with regards to planning many of the experiments. However, after discussion it was decided that their contributions merited being listed in the acknowledgements section rather than as co-authors.

Competing interests
No competing interests were disclosed.

Grant information
This research was supported by the Wellcome Trust (Grant No. 090667).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript Figure S1. Relative differences in the potencies of certain neurosteroidal compounds. Histogram summarising the relative differences in the potencies of certain neurosteroidal compounds (allopregnanolone, ganaxolone, DHP and progesterone) in three strains of mice (WT, ob/ob and db/db) after ~2 hours brain-slice exposure. When the mIPSC τ w were normalised for comparison, allopregnanolone and ganaxolone, which modulate the GABA A R directly, have the greatest potency, while the neurosteroid precursors DHP and progesterone have the least potency. There is no significant difference in the effect of allopregnanolone on the three strains of mice (n = 6-9, P > 0.05). In contrast, the precursor DHP had a greater impact on the diabetic mice n = 8-10; One-way RM ANOVA *P < 0.05. Post hoc Newman Keul's test revealed significant differences between the WT and both types of diabetic mice, *P < 0.05, but there was no intergroup difference between the ob/ob and db/db mice, P > 0.05). The precursor progesterone, also had a greater impact on the diabetic mice, n = 6-12; One-way RM ANOVA P < 0.05. Post hoc Newman Keul's test revealed significant differences between the WT and both types of diabetic mice, P < 0.05, but there was no intergroup difference between the ob/ob and db/db mice, P > 0.05). Ctrl = control; Allo = allopregnanolone; Ganax = ganaxolone; DHP = dihydroxyprogesterone; Prog = progesterone.

Supplementary material
Page 17 of 26 Figure S2. The 3α-HSD enzyme inhibitor provera suppresses the effects of DHP, but not ganaxolone on GABA A R-mediated mIPSCs of mature cortical neurons. The 3α-HSD enzyme inhibitor indometacin suppresses the effects of DHP and ganaxolone, but not those of allopregnanolone on GABA A R-mediated mIPSCs of mature cortical neurons. (A) Superimposed exemplar averaged GABA A R mIPSCs from a representative control cortical neuron and from equivalent neurons after ~2 hour pre-incubation of the brain slice with provera (1 μM), DHP (3 μM), or both. To facilitate comparison of their kinetics, the amplitude of the mIPSCs are normalised to that of the control averaged mIPSC (B) Histogram illustrating that provera prevents the effect of DHP pre-incubation to prolong GABA A R-mediated mIPSCs (τ w in ms; one-way ANOVA P <0.05. Post hoc Newman Keul's test revealed differences for DHP (3 μM) with or without provera (1 μM), *P <0.05, n = 7-9). (C) Superimposed exemplar averaged GABA A R mIPSCs from a representative control cortical neuron and from equivalent neurons after ~2 hour pre-incubation of the brain slice with provera (1 μM), ganaxolone (300 nM), or both. (D) Histogram illustrating that provera did not prevent the effect of ganaxolone on the duration of GABA A R mIPSCs (τ w in ms; one-way ANOVA P <0.05. Post hoc Newman Keul's test revealed no difference for the effects of ganaxolone (300 nM) with, or without provera (1 μM), P >0.05, n = 7-10). (E) Superimposed exemplar averaged GABA A R mIPSCs acquired from a representative control cortical neuron and from equivalent neurons after ~2 hours pre-incubation of the brain slice with indometacin (30 μM), DHP (3 μM), or both. (F) Histogram illustrating that indometacin prevents the effect of DHP on the duration of the GABA A R mIPSCs (τ w in ms, one-way ANOVA P <0.05. Post hoc Newman Keul's test revealed differences for DHP 3 μM with or without indometacin 100 μM, *P <0.05, n = 6-9). (G) Superimposed exemplar averaged GABA A R mIPSCs acquired from a representative control neuron and from equivalent neurons after an ~2 hour pre-incubation of the brain slice with indometacin (100 μM), allopregnanolone (100 nM), or both. (H) Histogram illustrating that indometacin does not prevent the effect of allopregnanolone to prolong the duration of the GABA A R-mediated mIPSC (τ w in ms, one-way ANOVA P >0.05. Post hoc Newman Keul's test revealed no differences for allopregnanolone with or without indometacin, P >0.05, n =7-9). (I) Superimposed exemplar averaged GABA A R mIPSCs acquired from a representative control neuron and from equivalent neurons after ~2 hour incubation of the brain slice with indometacin (100 μM), ganaxolone (300 nM), or both. (J) Histogram illustrating that indometacin in a concentration-dependant manner (30 -100 μM) prevents the effect of ganaxolone to prolong the GABA A R-mediated mIPSC (τ w in ms; one-way ANOVA P < 0.05. Post hoc Newman Keul's test revealed differences for ganaxolone 300 nM with or without indometacin 30 -100 μM, **P < 0.05, n = 8-10. Ctrl = control; Allo = allopregnanolone; Ganax = ganaxolone; DHP = dihydroxyprogesterone; Indo = indometacin.
Does the lack of finasteride on WT and ob/ob mice indicate that there is no endogenous basal neurosteroid-induced tone? The half-life of neurosteroid should be stated so that it may be considered with regard to being relevant.
It is interesting that galaxalone might be an allosteric modulator of GABA(A)Rs (as the other possibility of indomethacin as an antagonist has been excluded).
The rotarod test seems unfair when comparing fat mice to WT, but is valid in determining the detrimental effects of high doses of neurosteroid.
The effects of ganaxolone on vF is encouraging to indicate its potential relief from mechanical pain.
The possibility of differential binding sites to neurosteroids that could be differentially expressed is worthy of mention.
With the evidence presented here, I understand that the effects of Progesterone and DHP are greater in diabetic models, which would support an upregulation of the neurosteroid synthesis pathway as some compensatory mechanism. However, the evidence for the loss of neurosteroid tone remains elusive and probably upstream of the production of progesterone. This would presumably imply a deficit in mitochondrial cholesterol uptake, metabolism to pregnenolone, its efflux into the cytosol, or conversion to progesterone (from here on, the pathway to allopregnanolone is normal or enhanced). This needs to be spelled out more clearly.
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
I have provided the author with general advice on GABA(A) receptors in the past, Competing Interests: but have not contributed to this study.
Author Response 17 Feb 2017 , Imperial College London, UK

Stephen Humble
Authors Comments in response to the reviewer Thank you very much indeed for talking the time to review the paper 1. The author agrees that development does not fluctuate. Neurosteroid tone starts out very high in the most immature neurons then decreases as with maturity. At around P20 the neurosteroid tone is almost negligible, but a modest neurosteroid tone has reappeared in mature neurones by P60 (Humble 2013, Brown 2012). The presence of the neurosteroid tone may be revealed with the use of cyclodextrin. Separately the overall τ of the mIPCs also decreases independently of the neurosteroid tone with progressive maturity (Humble 2013, Brown 2012).
2. Loss of neurosteroid tone in development thought to be due to reduced production. But separately there is also changes in the expression of different receptor subtypes (e.g. alpha-2 as For reasons of brevity the issue of differential binding sites to neurosteroids was not explored significantly within the paper, but this was covered in substantial detail within my thesis (Humble, 2013). It is summarised in the Conclusions section of the thesis: 'Pipette-applied neurosteroids allopregnanolone and ganaxolone (but not DHP) were also able to prolong the decay time of cortical GABA R mIPSCs in a differential manner. Allopregnanolone induced a greater effect than ganaxolone on GABA R mIPSC decay time, but there was no difference in the response to the respective drugs between the WT and mice. These ob/ob findings suggest that neurosteroids are able to modulate the GABA R from the intracellular compartment and that the sensitivity of the GABA R is the same for WT and mice. In ob/ob addition, when indometacin was co-applied in the pipette with ganaxolone it had no impact on the efficacy of ganaxolone. This observation is inconsistent with the notion that indometacin competes with ganaxolone for its binding of the GABA R.' (Humble, 2013) 10. This is an excellent point and is the subject of a Brief Communication that I have written on the subject of Mitochondrial dysfunction and neurosteroid synthesis in diabetic neuropathy that will be published shortly. I would be happy to refer to this further in the discussion.
None to declare Competing Interests: