jpet.aspetjournals.org/content/e … 4.full.pdf
This is a great article…It’s fresh too(June 15th, 2010)
The article talks about the importance of Neurosteroid Androstanediol and how the brain processes. It also mentions how finasteride can cause hypogonadism and seizures due to the blockage of Androstandediol. Androstandediol is a very important neurosteroid that protects us from seizures. I hope this of use folks…
The Testosterone-Derived Neurosteroid Androstanediol is a
Positive Allosteric Modulator of GABAA Receptors
Department of Neuroscience and Experimental Therapeutics, College of Medicine, Texas A&M
Health Science Center (D.S.R. and K.J), College Station, Texas 77843-1114, USA.
ABSTRACT
Testosterone modulates seizure susceptibility, but the underlying mechanisms are obscure. Recently,
we demonstrated that testosterone affects seizure activity via its conversion to neurosteroids in the
brain. Androstanediol (5α-androstan-3α,17β-diol) is an endogenous neurosteroid synthesized from
testosterone. However, the molecular mechanism underlying the seizure protection activity of
androstanediol remains unclear. Here we show that androstanediol has positive allosteric activity as
a GABAA receptor modulator. In whole-cell recordings from acutely dissociated hippocampus CA1
pyramidal cells, androstanediol (but not its 3β-epimer) produced a concentration-dependent
enhancement of GABA-activated currents (EC50, 5 μM). At 1 μM, androstanediol produced a 50%
potentiation of GABA responses. In the absence of GABA, androstanediol has moderate direct
effects on GABAA receptor-mediated currents at high concentrations. Systemic doses of
androstanediol (5-100 mg/kg), but not its 3β-epimer, caused dose-dependent suppression of
behavioral and electrographic seizures in the mouse hippocampus kindling, which is a model of
temporal lobe epilepsy. The ED50 for antiseizure effects of androstanediol was 50 mg/kg, which did
not produce sedation/motor toxicity. At high (2×ED50) doses, androstanediol produced complete
seizure protection that lasted for up to 3 h after injection. The estimated plasma concentrations of
androstanediol producing 50% seizure protection in the kindling model (10.6μM) are within the range
of concentrations that modulate GABAA receptors. These studies suggest that androstanediol could
be a neurosteroid mediator of testosterone actions on neuronal excitability and seizure susceptibility,
via its activity as a GABAA receptor modulator, and may play a key role in men with epilepsy
especially during the age-related decline in androgen levels.
Drugs.
Stock solutions of androstanediol (Steraloids Inc., Newport, RI) and other drugs for injection
were made in 30% β-cyclodextrin in water, and additional dilutions were made using normal saline.
By itself, β-cyclodextrin at concentrations as high as 50% failed to affect kindled seizures. Drug
solutions were administered subcutaneously in a volume equaling 1% of the animal’s body weight.
Cyclodextrin and flutamide were procured from Sigma (St. Louis, MO).
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Results
Androstanediol Potentiates GABA Responses in Acutely Dissociated CA1 Neurons. To
investigate the physiological actions of androstanediol on GABAA receptor responses, we first
characterized the GABAA receptor-mediated Cl- currents in acutely dissociated CA1 neurons from
adult mice. An acutely dissociated CA1 pyramidal neuron had a clear primary apical dendrite with a
typical cell body (Fig. 2A). Application of GABA (0.1 – 1000 μM) for 5 s evoked brief inward currents
in a concentration dependent fashion (Fig. 2B). At 3 μM concentration, GABA evoked significant
currents with the average value of 479 ± 98 pA, which is ~10% of the maximum current at high GABA
application (EC10). The desensitization was minimal at 30 s interval between applications. Thus, we
chose 3 μM GABA for further experiments. Repeated application of 3 μM GABA induced similar and
consistent Cl– currents without desensitization when applied with 30 s interval (Fig. 2C). Like cells
from the enzymatic method, mechanically dissociated neurons showed similar GABA responses but
showed high background noise (not shown). Next, we sought to confirm the GABAA receptor
component of inhibition in CA1 neurons (Fig.2D). The GABA evoked currents were blocked by the
GABAA receptor competitive antagonist bicuculline and the GABAA receptor channel blocker
picrotoxin (Fig. 2E), indicating that these currents were mediated by the GABAA receptors.
We next assessed the ability of androstanediol to potentiate currents generated by 3 μM
GABA. Neurons were preapplied with androstanediol for 5 s and then GABA was coapplied with
androstanediol for 5 s. Androstanediol caused a concentration-dependent increase in peak-current
responses evoked by GABA and, at high concentrations, seemed to enhance desensitization
(Fig.3A). The effect of androstanediol was fully reversible but required long wash times at higher
concentrations (Fig.3A). At a concentration of 10 nM, the mean percentage of control value for
androstanediol was 109 ± 2 (n = 4). At higher concentrations, androstanediol caused up to a four-fold
potentiation of control responses (Fig.3B). The estimated concentration that produced a 50 percent
potentiation of the control GABA current was 1.0 μM. The concentration of androstanediol producing
half of the maximal enhancement of the GABA-evoked current (EC50) was 5 μM. To determine
whether the potentiating effect is structurally specific, we next compared the response to
androstanediol with that of its 3β-epimer (Fig.1). As shown in Fig.4, only the natural 3α-isomer was
active at concentration of 10 μM (2 × EC50). The 3β-epimer at 10 μM failed to increase GABAactivated
currents in acutely dissociated CA1 neurons.
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The efficacy and potency of androstanediol potentiation of GABA-mediated inhibitory currents
was compared with the prototype neurosteroid allopregnanolone. In acutely dissociated CA1
pyramidal neurons, allopregnanolone caused a concentration-dependent increase in peak-current
responses evoked by 3 μM GABA. At 10 nM, the mean percentage of control value for
allopregnanolone was 161 ± 17 (n = 4). At higher concentrations (0.1 to 10 μM), the maximal peak
amplitude obtained was markedly greater (200 to 550 percent potentiation of the control GABA), as is
the degree of fluctuation. The data could not be fit because no response plateau was achieved even
at high concentrations; the maximal (4 to 5-fold) potentiation at higher concentrations (1-10 μM) was
comparable to androstanediol (Fig.3B). Values for percentage of potentiation at 1 μM were 482 ± 73
and 49 ± 11 for allopregnanolone and androstanediol, respectively, indicating a 10-fold higher
potency of allopregnanolone as compared to androstanediol.
Direct Activation of GABAA Receptor Currents by Androstanediol in Acutely Dissociated CA1
Neurons.
At high concentrations, neurosteroids, in the absence of GABA, directly activate GABAA
receptor Cl- currents (Kokate et al., 1994; Reddy and Rogawski, 2002). To investigate the direct
actions of androstanediol on GABA-A receptor currents, we tested androstanediol at several higher
concentrations in acutely dissociated CA1 pyramidal neurons. As illustrated in Fig.5, perfusion of
hippocampal neurons with 1 to 100 μM androstanediol (in the absence of GABA) resulted in
activation of inward current responses that were very small compared to the response evoked by
GABA. The androstanediol response amplitudes were normalized to the mean amplitude of the
GABA response (Fig. 5A). Androstanediol caused a moderate concentration-dependent increase in
the peak inward currents (Fig. 5B). The EC50 value could not be derived because responses were
saturated at concentrations <10 μM. The direct effect of the neurosteroid was picrotoxin-sensitive.
The maximal amplitudes of direct androstanediol responses at the highest concentration (100 μM)
were approximately 25% of the 3 μM GABA response amplitude recorded in the same neuron,
indicating that androstanediol has marginal efficacy in directly activating GABAA receptors relative to
GABA.
Antiseizure Activity of Androstanediol in the Hippocampus Kindling Model in Mice.Neurosteroids with GABAA receptor modulatory activity have protective effects in the kindling model
of epilepsy (Reddy and Rogawski, 2002; 2010). Therefore, we selected the kindling model for
characterization of the antiseizure activity of androstanediol and for correlation of androstanediol’s
effects in vitro on GABA currents with its systemic seizure protection. To evaluate the systemic
activity of androstanediol in protecting against kindled seizures, first we prepared the fully-kindled
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13 mice with stage 5 seizures by stimulation of the right hippocampus once daily at 125% of their
afterdischarge threshold. The overall average value of the initial afterdischarge threshold was 58 ± 9
μA. There was a progressive increase in seizure stage, with all animals achieving consistent stage 5
seizures after 15 stimulations. Then, we utilized these fully-kindled mice for drug testing. The fully
kindled mice were treated with various doses of androstanediol 15 min prior to stimulation. As shown
in Fig. 6, androstanediol produced a dose-dependent suppression of behavioral seizure activity (Fig.
6A) and afterdischarge duration (Fig. 6B) with significant effects on both at 25, 50 and 100 mg/kg.
The percentage inhibition at the various doses is shown in Fig. 6C. At the highest dose tested,
behavioral seizures were nearly completely suppressed. The estimated ED50 value for suppression of
seizure stage and afterdischarge duration was 50 ± 4.5 mg/kg and 22 ± 3.4 mg/kg, respectively. The
potency in the kindling model was marginally less than that in the chemoconvulsant and 6-Hz models
(Table 1). Sample recordings of afterdischarge from fully kindled mice are shown in Fig. 7.
Androstanediol pretreatment markedly reduced the afterdischarge duration in a dose-dependent
fashion. The stereoselective effects of the 3-hydroxy epimers of androstanediol against kindling
seizures are shown in Fig. 8. Unlike the 3α-androstanediol which, at a dose of 100 mg/kg (2 × ED50),
conferred complete protection against seizures, the 3β-epimer at a dose of 100 mg/kg failed to
protect seizures in the kindling model.