Neurosteroid replacement therapy using the allopregnanolone-analogue ganaxolone following preterm birth in male guinea pigs.

BACKGROUND: Children born preterm, especially boys, are at increased risk of developing attention deficit hyperactivity disorder (ADHD) and learning difficulties. We propose that neurosteroid-replacement therapy with ganaxolone (GNX) following preterm birth may mitigate preterm-associated neurodevelopmental impairment. METHODS: Time-mated sows were delivered preterm (d62) or at term (d69). Male preterm pups were randomized to ganaxolone (Prem-GNX; 2.5 mg/kg subcutaneously twice daily until term equivalence), or preterm control (Prem-CON). Surviving male juvenile pups underwent behavioural testing at d25-corrected postnatal age (CPNA). Brain tissue was collected at CPNA28 and mature myelinating oligodendrocytes of the hippocampus and subcortical white matter were quantified by immunostaining of myelin basic protein (MBP). RESULTS: Ganaxolone treatment returned the hyperactive behavioural phenotype of preterm-born juvenile males to a term-born phenotype. Deficits in MBP immunostaining of the preterm hippocampus and subcortical white matter were also ameliorated in animals receiving ganaxolone. However, during the treatment period weight gain was poor, and pups were sedated, ultimately increasing the neonatal mortality rate. CONCLUSION: Ganaxolone improved neurobehavioural outcomes in males suggesting that neonatal treatment may be an option for reducing preterm-associated neurodevelopmental impairment. However, dosing studies are required to reduce the burden of unwanted side effects.

Progesterone is actively metabolized to 5α-pregnane-3,20-dione and 3ß-hydroxy-5α-pregnan-20-one by the marine mussel Mytilus galloprovincialis.

Progesterone (P4) and synthetic progestins enter the aquatic environment through wastewater treatment plant effluents and agricultural run-off, posing potential risks to aquatic organisms due to their biological activity. P4 is a precursor of a number of steroids in vertebrates, including estrogens and androgens. Mussels Mytilus galloprovincialis were exposed to P4 at the ng to low µg/L range (0.02-10µg/L) for 7 days with the aim of (a) assessing potential alterations on endogenous steroids as a consequence of exposure, and (b) describing the enzymatic pathways involved in P4 metabolism in mussels. No significant alteration of the levels of testosterone (T) and estradiol (E2) was observed in mantle/gonad tissue of exposed mussels, in spite of a 5.6-fold increase in immunoreactive T in those exposed to 10µg P4/L, which was attributed to cross-reactivity. P4 was actively metabolized to 5α-pregnane-3,20-dione (5α-DHP) and 3ß-hydroxy-5α-pregnan-20-one (3ß,20-one) in digestive gland, with no evidence for the synthesis of 17α-hydroxyprogesterone or androstenedione. The metabolism of P4 to 5α-DHP was not altered by exposure. Histological examination of the gonads suggested that exposure to 10µg/L P4 induced gamete maturation and release in mussels. Nonetheless, environmental concentrations of P4 are unlikely to have an endocrine action in mussels.

The neurosteroid allopregnanolone impairs object memory and contextual fear memory in male C57BL/6J mice.

Allopregnanolone (ALLO, or 3α-hydroxy-5α-pregnan-20-one) is a steroid metabolite of progesterone and a potent endogenous positive allosteric modulator of GABA-A receptors. Systemic ALLO has been reported to impair spatial, but not nonspatial learning in the Morris water maze (MWM) and contextual memory in rodents. These cognitive effects suggest an influence of ALLO on hippocampal-dependent memory, although the specific nature of the neurosteroid's effects on learning, memory or performance is unclear. The present studies aimed to determine: (i) the memory process(es) affected by systemic ALLO using a nonspatial object memory task; and (ii) whether ALLO affects object memory via an influence within the dorsal hippocampus. Male C57BL/6J mice received systemic ALLO either before or immediately after the sample session of a novel object recognition (NOR) task. Results demonstrated that systemic ALLO impaired the encoding and consolidation of object memory. A subsequent study revealed that bilateral microinfusion of ALLO into the CA1 region of dorsal hippocampus immediately following the NOR sample session also impaired object memory consolidation. In light of debate over the hippocampal-dependence of object recognition memory, we also tested systemic ALLO-treated mice on a contextual and cued fear-conditioning task. Systemic ALLO impaired the encoding of contextual memory when administered prior to the context pre-exposure session. Together, these results indicate that ALLO exhibits primary effects on memory encoding and consolidation, and extend previous findings by demonstrating a sensitivity of nonspatial memory to ALLO, likely by disrupting dorsal hippocampal function.

Neuroprotective effect of the 3α5ß-pregnanolone glutamate treatment in the model of focal cerebral ischemia in immature rats.

The perinatal hypoxic-ischemic insult frequently leads to mortality, morbidity and plays a key role in the later pathological consequences. The ischemic insult causes a massive release of glutamate and subsequent excitotoxic damage. The neuroactive steroid 3α5ß-pregnanolone glutamate (PG) is a NMDA receptor antagonist acting via use-dependent mechanism and can be used as a neuroprotective agent that may alleviate glutamatergic excitotoxicity in the brain. First, a possible neurotoxic effect of the PG, a novel use-dependent NMDA antagonist, was studied in immature rats. In addition, to compare this effect with a well-described non-competitive NMDA antagonist, the MK-801 (positive control) was used. Animals at postnatal day 12 (P12) were injected intraperitoneally with PG in a doses 1 or 10mg/kg or with MK-801 in a dose 1mg/kg. Effect of PG treatment on the immature brain was evaluated on Fluoro Jade B (FJB) stained sections. Second, a neuroprotective effect of the PG was studied in the model of focal cerebral ischemia in P12. Focal cerebral ischemia was induced by the infusion of the endothelin-1 (ET-1) into the right dorsal hippocampus. PG at the doses 1 or 10mg/kg was administrated intraperitoneally 5min after the end of ET-1 infusion. To evaluate the neuroprotective effect after the PG treatment FJB staining was used. Our results demonstrate a lack of the neurotoxicity of the PG in intact P12. In the second part of the study in the model of the focal ischemia we detected significantly lower occurrence of FJB-positive cells in the afflicted hippocampus in PG treated groups, while animals without PG treatment exhibited massive neurodegeneration. The neuroprotective potential of the PG can serve in the development of therapeutic strategies for brain damage induced by the glutamate excitotoxicity.

A stress steroid triggers anxiety via increased expression of α4ßδ GABAA receptors in methamphetamine dependence.

Methamphetamine (METH) is an addictive stimulant drug. In addition to drug craving and lethargy, METH withdrawal is associated with stress-triggered anxiety. However, the cellular basis for this stress-triggered anxiety is not understood. The present results suggest that during METH withdrawal (24h) following chronic exposure (3mg/kg, i.p. for 3-5weeks) of adult, male mice, the effect of one neurosteroid released by stress, 3α,5α-THP (3α-OH-5α-pregnan-20-one), and its 3α,5ß isomer reverse to trigger anxiety assessed by the acoustic startle response (ASR), in contrast to their usual anti-anxiety effects. This novel effect of 3α,5ß-THP was due to increased (three-fold) hippocampal expression of α4ßδ GABAA receptors (GABARs) during METH withdrawal (24h-4weeks) because anxiogenic effects of 3α,5ß-THP were not seen in α4-/- mice. 3α,5ß-THP reduces current at these receptors when it is hyperpolarizing, as observed during METH withdrawal. As a result, 3α,5ß-THP (30nM) increased neuronal excitability, assessed with current clamp and cell-attached recordings in CA1hippocampus, one CNS site which regulates anxiety. α4ßδ GABARs were first increased 1h after METH exposure and recovered 6weeks after METH withdrawal. Similar increases in α4ßδ GABARs and anxiogenic effects of 3α,5ß-THP were noted in rats during METH withdrawal (24h). In contrast, the ASR was increased by chronic METH treatment in the absence of 3α,5ß-THP administration due to its stimulant effect. Although α4ßδ GABARs were increased by chronic METH treatment, the GABAergic current recorded from hippocampal neurons at this time was a depolarizing, shunting inhibition, which was potentiated by 3α,5ß-THP. This steroid reduced neuronal excitability and anxiety during chronic METH treatment, consistent with its typical effect. Flumazenil (10mg/kg, i.p., 3×) reduced α4ßδ expression and prevented the anxiogenic effect of 3α,5ß-THP after METH withdrawal. Our findings suggest a novel mechanism underlying stress-triggered anxiety after METH withdrawal mediated by α4ßδ GABARs.

Brief but chronic increase in allopregnanolone cause accelerated AD pathology differently in two mouse models.

Previously, we have shown that chronic treatment with allopregnanolone (ALLO) for three months impaired learning function in the Swe/PS1 mouse model. ALLO is a neurosteroid, produced in the CNS and a GABAA receptor agonist. ALLO modulates the general inhibitory system in the CNS by enhancing the effect of GABA. Chronic treatment with other GABAA receptor active compounds, such as benzodiazepines, ethanol and medroxy-progesterone acetate has been associated to cognitive decline and/or increased risk for dementia. In this study, we sufficed with a treatment period of one month for the Swe/PS1 mouse, and included another Alzheimer's disease mouse model; the Swe/Arc model. We found that one month of chronic treatment with elevated ALLO levels within physiological range impaired learning and memory function in the Swe/Arc female and male mice. Male Swe/PS1 mice also showed marginally impaired function, while the female mice did not. Furthermore, the chronic ALLO treatment caused increased levels of soluble Aß in the Swe/PS1 mouse model while the levels were unchanged in the Swe/Arc model. Therefore, both Swe/Arc and Swe/PS1 mice showed signs of accelerated disease progression. Still, further studies are required to determine the mechanisms behind the cognitive impairment and the increased Aß-levels caused by mildly elevated ALLO-levels.

Allopregnanolone infusion induced neurobehavioural alterations in a neonatal foal: is this a clue to the pathogenesis of neonatal maladjustment syndrome?

REASONS FOR PERFORMING THE STUDY: Increased plasma progestagen concentrations have been reported in foals with neonatal maladjustment syndrome (NMS). These steroids may cross the blood-brain barrier and have dampening effects in the central nervous system. OBJECTIVES: To evaluate if the infusion of a progesterone derivative (allopregnanolone) in a healthy neonatal foal would induce clinical signs compatible with NMS. METHODS: A healthy neonatal foal from a healthy mare with a normal gestation (length, no complications), birth and placenta was infused with allopregnanolone to observe its neurobehavioural effects. Heparinised blood samples were collected pre- and post infusion to determine various progestagen concentrations using liquid chromatography mass spectrometry. A second healthy neonatal foal was infused with ethanol and saline for comparison of clinical observations. RESULTS: Infusion of allopregnanolone resulted in obtundation, lack of affinity for the mare and decreased response to external stimuli. These effects were short-lasting and associated with measurable concentrations of progestagens. CONCLUSIONS AND POTENTIAL RELEVANCE: Infusion of a steroid metabolite to a healthy neonatal foal resulted in neurobehavioural alterations compatible with those observed in foals with NMS. These findings suggest that increased progestagen concentrations may be responsible for some of the behavioural changes observed in foals with NMS.

3beta-20beta-dihydroxy-5alpha-pregnane (UC1011) antagonism of the GABA potentiation and the learning impairment induced in rats by allopregnanolone.

Allopregnanolone is a progesterone metabolite and GABA-A receptor modulator with benzodiazepine like effects, including decreased learning and memory. In vitro 3beta-hydroxypregnane steroids antagonize allopregnanolone-induced effects, but no antagonism has been shown in vivo. Our purpose was to evaluate 3beta-20beta-dihydroxy-5alpha-pregnane (UC1011) as a blocker of allopregnanolone-induced effects in vivo and in vitro in rats. We tested adult male Wistar rats in the Morris water maze 8 min after daily injections (i.v.) of allopregnanolone 2 mg/kg (n = 21); allopregnanolone : UC1011 2 : 6 (n = 7), 2 : 8 (n = 7), 2 : 20 (n = 14) mg/kg; UC1011 20 mg/kg (n = 14); or vehicle (10% 2-hydroxypropyl-beta-cyclodextrin, n = 4). Studies of chloride ion uptake into cortical and hippocampal membrane preparations were performed. The latency to find the hidden platform was still high in the allopregnanolone-injected group on day 6. Day 3-6 rats injected with allopregnanolone and UC1011 (2 : 20 mg/kg) had lower latency (P < 0.05), compared to the allopregnanolone-injected group. The group that only received UC1011 learned the location of the platform as fast as the controls. There was no significant difference in swim speed between groups. The time spent swimming close to the pool wall was in the allopregnanolone : UC1011 group (2 : 20 mg/kg) significantly decreased (P < 0.05, day 3-6), compared to the allopregnanolone-injected group. The increased chloride ion uptake induced by increasing dosage of allopregnanolone in the presence of 10 micro m GABA was significantly decreased with UC1011 (P < 0.01), in both cortical and hippocampal homogenates. In conclusion, UC1011 can via antagonism at the GABA-A receptor reduce the negative allopregnanolone effect on learning in the water maze.

Enhanced anticonvulsant activity of ganaxolone after neurosteroid withdrawal in a rat model of catamenial epilepsy.

Perimenstrual catamenial epilepsy, the exacerbation of seizures in association with menstruation, may in part be due to withdrawal of the progesterone metabolite allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one), an endogenous anticonvulsant neurosteroid that is a positive allosteric modulator of gamma-aminobutyric acid(A) receptors. Neurosteroid replacement is a potential approach to therapy, but natural neurosteroids have poor bioavailability and may be converted to metabolites with undesired progestational activity. The synthetic neuroactive steroid ganaxolone (3alpha-hydroxy-3beta-methyl-5alpha-pregnane-20-one) is an orally active analog of allopregnanolone that is not converted to the hormonally active 3-keto form. To assess the potential of ganaxolone in the treatment of catamenial seizure exacerbations, a state of persistently high serum progesterone (pseudopregnancy) was induced in 26-day-old female rats with gonadotropins, and neurosteroids were withdrawn on postnatal day 39 with finasteride, a 5alpha-reductase inhibitor that blocks the conversion of progesterone to allopregnanolone. Finasteride treatment during pseudopregnancy results in a reduction in the threshold for pentylenetetrazol seizures. During this state of enhanced seizure susceptibility, there was a 3-fold increase in the anticonvulsant potency of ganaxolone (control ED(50) = 3.5 mg/kg; withdrawn = 1.2 mg/kg) without a change in the potency for induction of motor toxicity in the rotarod test. The plasma concentrations of ganaxolone did not differ significantly in control and withdrawn animals; the estimated plasma concentrations of ganaxolone producing 50% seizure protection were approximately 500 and approximately 225 ng/ml in control and withdrawn rats, respectively. Unlike ganaxolone, neurosteroid withdrawal was associated with a decrease in the anticonvulsant potency of diazepam (control ED(50) = 1.9 mg/kg; withdrawn = 4.1 mg/kg) and valproate (control ED(50) = 279 mg/kg; withdrawn = 460 mg/kg). The enhanced anticonvulsant potency of ganaxolone after neurosteroid withdrawal supports the use of ganaxolone as a specific treatment for perimenstrual catamenial epilepsy.

The neurosteroid 3 alpha-hydroxy-5 alpha-pregnan-20-one induces catalepsy in mice.

Intracerebroventricular injection of the neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alphaTHP) induced catalepsy in mice, within less than 10 min of its administration, which lasted for more than 3 h. This catalepsy was reversed by i.p. administration of picrotoxin, bicuculline, bromocriptine and levodopa-plus-carbidopa combination. A low i.p. dose of 3alpha,5alphaTHP (1.5 mg/kg) did not induce catalepsy. However, significant catalepsy was noted when 3alpha,5alphaTHP (1.5 mg/kg) was administered by the i.p. route in the mice pretreated with subcataleptic doses of haloperidol, muscimol or aminooxyacetic acid. The results, for the first time, demonstrate the cataleptogenic potential of 3alpha,5alphaTHP and suggest that neurosteroids may affect central dopaminergic transmission through their direct action on GABA(A) receptors.

The neurosteroid 3 alpha-hydroxy-5 alpha-pregnan-20-one induces cytoarchitectural regression in cultured fetal hippocampal neurons.

The neurosteroid 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha, 5 alpha-THP) acts as a potent allosteric modulator and a direct activator of the GABA-chloride channel complex. This neurosteroid has also been found to protect against seizures that arise from blockade of the GABA-chloride channel complex. Because 3 alpha,5 alpha-THP protects against excitotoxin-induced seizure activity and because seizure activity has been found to be associated with aberrant hippocampal nerve cell growth, the rapid effect of the neurosteroid 3 alpha,5 alpha-THP upon nerve cell growth was investigated using videomicroscopy of hippocampal neurons in culture. Within 40 min of exposure 3 alpha,5 alpha-THP induced a significant decrease in the area and length of neurites. A concomitant decrement in the number and length of filopodia decorating neuritic extensions also occurred within the 40 min of 3 alpha,5 alpha-THP exposure. Both rapid and slow retrograde movement of intracellular organelles was observed in 3 alpha,5 alpha-THP-treated neurons. 3 alpha,5 alpha-THP-induced regression of neuritic extensions occurred only in nerve cells that had not yet established contact with other nerve or glial cells in culture. Established structural connections between neurons or glia did not erode during 3 alpha,5 alpha-THP exposure. Neither the inactive stereoisomer 3 beta-hydroxy-5 beta-pregnan-20-one nor progesterone had a significant effect upon any of the morphological parameters assessed. In approximately 25% of the cells in which 3 alpha,5 alpha-THP had induced regression, subsequent exposure to 17 beta-estradiol induced profuse filopodial growth within 60 sec of exposure. In cultures similar in age to those used in the morphological studies, 3 alpha,5 alpha-THP induced a significant increase in 36Cl- uptake within 10 sec. The magnitude of 36Cl- uptake was comparable to that induced by exposure to 100 microM GABA. In older, more mature cultures in which the nerve cells had established structural connections, 3 alpha,5 alpha-THP protected cells from picrotoxin-induced nerve cell death. These results demonstrate that 3 alpha,5 alpha-THP can induce regression of neuronal morphology within a relatively rapid time frame. 3 alpha,5 alpha-THP induction of 36Cl- uptake within 10 sec suggests that activation of neurosteroid-regulated chloride channels is an initial step in the biochemical mechanism underlying the retraction induced by this progesterone metabolite steroid. In select instances, 17 beta-estradiol induced an extremely rapid reversal of the filopodial regression produced by 3 alpha,5 alpha-THP.(ABSTRACT TRUNCATED AT 400 WORDS)

Anticonvulsant profile of the progesterone metabolite 5 alpha-pregnan-3 alpha-ol-20-one.

5 alpha-Pregnan-3 alpha-ol-20-one (3 alpha-OH-DHP) is a naturally occurring metabolite of progesterone that can modulate brain excitability through a specific steroid recognition site on the GABA/benzodiazepine receptor-chloride ionophore complex. The anticonvulsant properties of 3 alpha-OH-DHP were determined using standardized anticonvulsant screening tests in mice. This steroid was found to be effective against metrazol-, (+)-bicuculline- and picrotoxin-induced seizures. The steroid has maximum potency against (+)-bicuculline-induced convulsions and no activity against maximal electroshock and strychnine-induced seizures. These findings support the hypothesis that therapeutically useful anticonvulsant steroids active at the putative steroid recognition site associated with the GABA/benzodiazepine receptor-chloride ionophore complex can be identified.

Anticonvulsive properties of pregnanolone emulsion compared with althesin and thiopentone in mice.

The anticonvulsive properties of pregnanolone (as an emulsion) were evaluated in mice and compared with similar properties of Althesin and thiopentone. Pregnanolone emulsion was found to antagonize convulsions induced by the GABA antagonists pentetrazole, bicuculline and picrotoxin and by the specific glycine receptor antagonist, strychnine. The drug was effective in all four convulsive tests at subanaesthetic doses with maximal activity appearing within less than 1 min. The anticonvulsive therapeutic indices of pregnanolone emulsion were superior when compared with the therapeutic indices of Althesin and thiopentone in all four tests. Pregnanolone emulsion might be a useful alternative drug in the management of convulsive states resistant to conventional therapy.

Pregnanolone emulsion. A new steroid preparation for intravenous anaesthesia: an experimental study in mice.

The anaesthetic activity of pregnanolone (a metabolite of progesterone) in emulsion formulation administered intravenously to male mice was compared with that of Althesin. The loss of righting reflex for 15 seconds was used to estimate the anaesthetic effect. The mean anaesthetic dose (AD50) for the pregnanolone emulsion was 5.25 mg/kg and for Althesin, 2.8 mg/kg. The mean lethal dose (LD50) was 44 mg/kg for pregnanolone and 54 mg/kg for Althesin. The sleeping time after pregnanolone was 2.5-3.5 times longer than after Althesin in dosages above 7.5 mg/kg. No signs of pain or local reaction from injection were observed for either of the drugs. The onset of action was fast for both drugs, with only minor signs of excitation, and recovery was rapid and without excitation. The results indicate that the anaesthetic properties of pregnanolone emulsion are very similar to those of Althesin. Further studies will show whether it can fill the major vacuum left in anaesthetic practice after the withdrawal of Althesin.