3alpha-hydroxy-5alpha-pregnan-20-one in the midbrain ventral tegmental area mediates social, sexual, and affective behaviors

Progestins mediate the onset and duration of lordosis, the mating posture of female rodents, through actions in the hypothalamus and ventral tegmental area. In the hypothalamus, progesterone has traditional, "genomic" actions via intracellular progestin receptors. In the ventral tegmental area, 3alpha-hydroxy-5alpha-pregnan-20-one has "non-genomic" actions independent of progestin receptors to facilitate lordosis that involve GABA(A)/benzodiazepine receptors, NMDA type glutamate receptors, and/or dopamine receptors. 3alpha-Hydroxy-5alpha-pregnan-20-one levels also change with behavioral and/or environmental stimuli and may have a role in other reproductively-relevant behaviors, such as affiliation, exploration, and anxiety (socio-sexual behaviors). Data are reviewed that support the notion that: 1) effects of 3alpha-hydroxy-5alpha-pregnan-20-one in the midbrain ventral tegmental area facilitate lordosis and other reproductively-relevant behaviors. 2) 3alpha-Hydroxy-5alpha-pregnan-20-one, formed in the ventral tegmental area from metabolism of progestins, produced peripherally by endocrine glands, or centrally from biosynthesis in glial cells mediates socio-sexual behaviors. 3) 3alpha-Hydroxy-5alpha-pregnan-20-one's actions at GABA(A)/benzodiazepine receptors, NMDA type glutamate receptors, and dopamine receptors in the ventral tegmental area are important for lordosis; however, effects at these substrates on socio-sexual behaviors have not been elucidated. Given 3alpha-hydroxy-5alpha-pregnan-20-one's involvement in stress responses, its putative role as a homeostatic regulator and in the pathophysiology and treatment of neuropsychiatric disorders is discussed.     

Novelty-induced opioid analgesia in the terrestrial snail, Cepaea nemoralis

Exposure for 1-15 min to the surface of either a non-functional (22 degrees C) hot-plate or a polypropylene tube increased the thermal (38.5 degrees C) nociceptive thresholds of the terrestrial snail, Cepaea nemoralis. This "analgesic response," which was evident 10-15 sec after exposure to the new stimuli and lasted for 15-30 min, was blocked by the opiate antagonist naloxone. The analgesic response was not observed in snails previously made familiar with the test situation. Snails exposed to a functional (36.5 degrees C) stressful hot-plate surface to which they were aversive also displayed a naloxone-reversible analgesia. However, the level and duration of analgesia was markedly greater than that observed following exposure to the novel non-physically stressful stimuli and was not inhibited by prior familiarization with the stimuli. These observations demonstrate that novelty per se can activate endogenous opioid system(s) and induce an analgesic response in a snail. They also suggest a phylogenetic continuity in responses to novel stimuli and environmental conditions.     

Inhibitory effects of 60-Hz magnetic fields on opiate-induced "analgesia" in the land snail, Cepaea nemoralis, under natural conditions.

There is accumulating laboratory evidence that magnetic fields can affect a variety of opioid-mediated behavioral and physiological functions in both vertebrates and invertebrates. The present study examined the effects of various durations (0.50, 1.0 and 2.0 h) of exposure to a low intensity (1.0 gauss rms) 60-Hz magnetic field on opioid-mediated aversive thermal ("nociceptive") responses and morphine-induced "analgesia" in the land snail, Cepaea nemoralis, under natural environmental conditions. Exposure to the powerline-related 60-Hz magnetic fields significantly attenuated morphine-induced analgesia and the basal nociceptive responses of Cepaea, with the degree of attenuation being related to the duration of exposure to the magnetic fields. These results with Cepaea show that 60-Hz magnetic fields can affect opioid-mediated behavioral responses outside the laboratory under natural environmental conditions.     

Increased mortality in land snails (Cepaea nemoralis) exposed to powerline (60-Hz) magnetic fields and effects of the light-dark cycle

The effects of various durations (0.5, 2, 12, 48, or 120 h) of day- and night-time exposures to a 1.0 gauss (rms) 60-Hz magnetic field or sham field on mortality levels in the nocturnally-crepuscularly active land snail, Cepaea nemoralis, were examined. These snails were injected with morphine or saline vehicle and tested for reaction to an aversive thermal stimulus as part of another study. Mortality levels were monitored over a 2-week period following the initial exposure to the fields and were shown not to be differentially affected by the drug injection procedures. Mortality levels increased linearly as a function of increased length of exposure to the magnetic fields (P less than 0.001) but not when exposed to the sham fields. As well, night-time exposures resulted in greater mortality levels than day-time exposures (P less than 0.025). These results indicate that day-night rhythms are important in determining the magnitude of the magnetic field exposure effect. It is speculated that the magnetic fields may disrupt endogenous opioid- and calcium-modulated homeostatic mechanisms and augment stress effects, modifying a variety of systems including immunocompetence.     

5 beta-pregnan-3 beta-ol-20-one, a specific antagonist at the neurosteroid site of the GABAA receptor-complex.

Studies were made on the potentiation of [3H]flunitrazepam binding to rat brain membranes by gamma-aminobutyric acid (GABA), pentobarbitone and pregnanolone (5 beta-pregnan-3 alpha-ol-20-one). Epipregnanolone, the 3 beta isomer of pregnanolone, inhibited competitively the potentiation by pregnanolone with a Ki of 10.5 microM without affecting that of GABA. The potentiation by pentobarbitone was slightly enhanced. Epipregnanolone alone showed only slight potentiation of benzodiazepine binding. These findings demonstrate that epipregnanolone is a specific antagonist of the neurosteroid site of the GABAA receptor and raise the possibility of a physiological role for 3 beta-hydroxysteroids in modulating this receptor.     

The effect of stress on the concentration of 3beta-hydroxypregn-5-en-20-one (pregnenolone) and pregn-4-ene-3,20-dione (progesterone) in the adrenal gland of the rat

1. In cats anaesthetized with pentobarbitone sodium the third ventricle was perfused with artificial c.s.f., the effluent was collected in 30 min samples and assayed for 5-hydroxytryptamine (5-HT) on the rat stomach-strip preparation. Rectal temperature was monitored continuously.2. On perfusion of artificial c.s.f. through the third ventricle, small amounts of 5-HT appeared in the effluent; the amounts decreased with successive samples.3. When tranylcypromine (Parnate), an inhibitor of amine oxidase, was added to the perfusion fluid and perfusion was continued, the 5-HT output increased. This increase was associated with shivering and a rise in temperature which was not, however, maintained.4. When tranylcypromine was injected intraperitoneally, during the fall in temperature produced by the pentobarbitone sodium anaesthesia, the 5-HT output also increased, shivering occurred and the fall in temperature was halted or reversed. The effect on temperature was maintained.5. When the cat was killed and perfusion was continued, the 5-HT output, already elevated by the tranylcypromine before death, increased even further in the next few samples.     

Intracellular chloride activity and the effects of acetylcholine in snail neurones

1. Cl(-)-sensitive micro-electrodes were used to measure intracellular Cl(-) in snail neurones. The electrodes consisted of a sharpened and chlorided silver wire mounted inside a glass micropipette.2. The electrodes appeared to record changes in internal Cl(-) accurately but in H cells the chloride equilibrium potential (E(Cl)) as measured by the Cl(-)-sensitive electrode was always less negative than E(ACh).3. In some H cells ACh caused a measurable increase in internal Cl(-) when the cell was at its resting potential. In voltage-clamped cells there was a close correlation between the change in internal Cl(-) and the extra clamp current caused by a brief application of ACh. This confirmed that ACh increases the cell's membrane permeability only to Cl(-) ions, and that E(ACh) was equal to E(Cl).4. There was good agreement between the measured change in internal Cl(-) and that calculated from the cell size and clamp charge only when it was assumed that a constant voltage offset was added to the potential of the Cl(-)-sensitive electrode while it was inside the nerve cell.5. Cl(-)-sensitive electrodes with AgCl as the sensitive material appear to be unsuitable for intracellular measurement of Cl(-), although they might be suitable for following changes in E(Cl).6. In certain D cells ACh also caused an increase in internal Cl(-) although it decreased the membrane potential. In the presence of hexamethonium, ACh caused a hyperpolarization and a smaller increase in internal chloride.7. It is concluded that the intracellular Cl(-) in both H and D cells is about 8.3 mM, giving an E(Cl) of about -58 mV.     

Interaction between 3α-hydroxy-5α-pregnan-20-one and carbachol in the control of neuronal excitability in hippocampal slices of female rats in defined phases of the oestrus

The effects of 3α-hydroxy-5α-pregnan-20-one (allopregnanolone) and carbachol on CA1 and dentate gyrus action potentials were studied in hippocampus slices in premature, follicular and luteal phase rats. A 0.5 nL droplet of allopregnanolone (12.5 μmol L^?1), carbachol (5 μmol L^?1) or a mixed solution of 12.5 μmol L^?1 allopregnanolone and 5 μmol L^?1 carbachol was applied locally onto the stratum oriens-pyramidale or granular layer. The amplitude of CA1 population spike (POPSP) was reduced by allopregnanolone (?38 ± 3%) and carbachol (?21 ± 4%) in the luteal phase slices. The mixture of allopregnanolone and carbachol doubled this inhibition (?77 ± 6%). The inhibition caused by allopregnanolone and the mixture of allopregnanolone and carbachol in CA1 was significantly larger in the luteal phase than in the follicular phase (P = 0.02 and 0.0002). In the granular layer of the dentate gyrus, these inhibitions showed no significant difference between the phases. Neither in CA1 nor in the dentate gyrus did the carbachol inhibition differ between the phases. Perfusion with 5–10 μmol L^?1 carbachol caused an increasing inhibition of the POPSP during the first few minutes. Thereafter the inhibition gradually diminished and was replaced by a facilitation. The local allopregnanolone inhibition was enhanced by simultaneous carbachol perfusion. Picrotoxin (100 μmol L^?1) substantially reduced the allopregnanolone but not the carbachol inhibition. Atropine (10 μmol L^?1) blocked the carbachol response, but not the allopregnanolone inhibition. Perfusion with a mixed solution of picrotoxin and atropine reduced, but did not block, the inhibition caused by local application of allopregnanolone or by the mixture of allopregnanolone and carbachol. Our data suggest that neuroprogestine modulators of the GABA_A-receptor-mediated inhibition may play a significant role in the control of the cholinergic excitation in the hippocampus.     

Suppressive effects of dehydroepiandrosterone and 3-beta-methylandrost-5-en-17-one on attack towards lactating female intruders by castrated male mice

Three experiments were performed. In Experiment I, triads of adult castrated male mice were chronically administered with either oil-vehicle or 80 micrograms/day of dehydroepiandrosterone (D) or 3-beta-methylandrost-5-en-17-one (D-CH3). They were subsequently tested for their attack on a lactating intruder female introduced for 15 min in their home-cage, 2 hr after their last injection. Both D and D-CH3 significantly reduced male aggressive responses (cf. oil-injected category). In Experiment II the effects of D-CH3 dosed at 20, 40 or 80 micrograms/day were measured in the same testing situation. D-CH3 dose-dependently reduced the mice attack probability on lactating intruders with the highest dose being the most effective. Finally, to test in Experiment III that D-CH3 was not demonstrably androgenic, castrated males received daily injections of 20, 40 or 80 micrograms of this synthetic steroid for 4, 8, 16 or 32 days before tissue sampling. At all doses and whatever treatment duration, D-CH3 did not significantly increase the weights of the accessory sex organs.     

Salivary testosterone, cortisol, and progesterone: Two-week stability, interhormone correlations, and effects of time of day, menstrual cycle, and oral contraceptive use on steroid hormone levels

With salivary assessment of steroid hormones increasing, more work is needed to address fundamental properties of steroid hormone levels in humans. Using a test-retest design and radioimmunoassay assessment of salivary steroids, we tested the reliability of testosterone, cortisol, and progesterone levels across two weeks, as well as the effects of oral contraceptives, menstrual cycle phase, and time of day on steroid hormone levels. Testosterone and cortisol were found to be highly reliable in both sexes. Progesterone was found to be reliable after collapsing across sex. Oral contraceptive use was associated with lower levels of testosterone, but did not affect cortisol. Contrary to expectations, oral contraceptives also did not affect progesterone. Menstrual cycle was found to affect levels of progesterone, but not testosterone or cortisol. Time of day had an effect on cortisol, on progesterone only at one testing time, and no effect on testosterone. We explored the interhormone correlations among testosterone, progesterone, and cortisol. All three hormones were positively correlated with one another in men. In women, progesterone was positively correlated with testosterone and cortisol, but testosterone and cortisol were uncorrelated.     

Antinociceptive effects of hydromorphone,bupivacaine and biphalin released from PLGA polymer after intrathecal implantation in rats

Intraspinal drug delivery, based on the concept of controlling pain by delivering drug to a nociceptive target rich in opioid and other relevant receptors is increasingly used clinically. The therapeutic ratio for opioids or other centrally acting agents is potentially greater if they are administered intrathecally (i.t.) than outside the central nervous system (CNS). The present study was designed with the ultimate goal of formulating a controlled release system for intrathecal analgesia characterized by effectiveness, rapid onset and few side effects for chronic pain control. A biodegradable copolymer poly(L-lactide-co-glycolide) (PLGA) was used to prepare a rod-shaped drug delivery system containing hydromorphone (HM), bupivacaine (BP), both HM and BP, or biphalin (BI). In vitro drug release kinetics of these systems showed a zero-order release rate for HM and BP from PLGA (85:15) rods. Drug-loaded rods were implanted i.t. Control groups received only placebo implants. Measurement of analgesic efficacy was carried out with tail flick and paw-withdrawal tests. In vivo studies showed potent, prolonged analgesia in comparison to controls for all active treatments. Analgesic synergy was observed with HM and BP. With further refinements of drug release rate, these rods may offer a clinically relevant alternative for intrathecal analgesia.     

Interaction between 17β-oestradiol and 3α-hydroxy-5α-pregnane-20-one in the control of neuronal excitability in slices from the CA1 hippocampus in vitro of guinea-pigs and rats

The effect of 17β-oestradiol and 3α-hydroxy-5α-pregnane-20-one (allopregnanolone) on the action potentials in the Schaffer collateral pathway was investigated in hippocampus CA1. Slices from male and female guinea-pigs and female rats were used. In the rat three groups were studied: (a) untreated prepubertal rats at day 25 after partus; (b) rats injected on day 26 with 10 IU of equine serum gonadotropin studied on day 28, when in the pro-oestrus follicular phase; and (c) on day 32 when in the luteal phase. The allopregnanolone (12.6 μ.M, 0.5 nL) was applied locally in stratum oriens-pyramidale. The 17β-oestradiol (0.7 nM) was perfused (4 mL min^-1) or applied locally. The amplitude of the population spike in stratum pyramidale was increased by oestradiol in guinea-pigs of both sexes and in all the three groups of rats. Allopregnanolone decreased the amplitude of the population spike in the guinea-pigs and in the luteal phase rats. The effect appeared within seconds after the application of the drugs. The allopregnanolone inhibition of the population spike was increased by perfusion with oestradiol in the guinea-pigs and in the luteal phase rats. This effect appeared within 7 min, and improved with increasing length of the perfusion (7–71 min). It remained for 55 min after return to perfusion with artificial cerebrospinal fluid. In prepubertal and follicular phase rats the allopregnanolone inhibition was seen only after perfusion with oestradiol for more than 15 min. The results show that 17β-oestradiol increases the allopregnanolone inhibition and that this inhibition is most efficient during the luteal phase of the rat.     

Antinociceptive effects of the 5-HT2 antagonist ritanserin in rats: evidence for an activation of descending monoaminergic pathways in the spinal cord

The antinociceptive properties of the 5-HT2 antagonist ritanserin have been investigated in the writhing test using rats implanted with chronic lumbar catheters. The antinociceptive action of 15 mg/kg ritanserin applied subcutaneously (s.c.) was powerfully inhibited by the intrathecal (i.t.) application of ritanserin (50 nmol), methysergide (15 nmol), yohimbine (20 nmol), alpha-flupenthixol (20 nmol) or naloxone (15 nmol), but not atropine (30 nmol), at doses which themselves produced no change in nociceptive threshold. It is concluded that ritanserin acts supraspinally to activate pain-modulating descending serotonergic, noradrenergic, dopaminergic and possibly opioidergic pathways, while spinopetal cholinergic pathways do not seem to be involved. Hyperalgesic effects of s.c. ritanserin following the i.t. application of methysergide or yohimbine were interpreted in terms of the co-release of an excitatory transmitter, possibly substance P, from descending serotonergic and noradrenergic nerve fibres. The supraspinal mechanism by which ritanserin activates spinopetal pathways and its dependence on 5-HT2 receptors have not yet been established.     

Antinociceptive effects and synergistic interaction with morphine of intrathecal metabotropic glutamate receptor 2/3 antagonist in the formalin test of rats

Spinal metabotropic glutamate receptors (mGluRs) have been known to be involved in the modulation of nociception. While the antinociceptive effects of the mGluR1/5 have been demonstrated, the role of mGluR2/3 for nociception is less clear. This study investigated the effects of an intrathecal mGluR2/3 agonist, APDC, and a mGluR2/3 antagonist, LY341495, for inflammatory and acute pain in the formalin test and thermal stimulation test. We also examined their interaction with intrathecal morphine for the antinociceptive effect. APDC had little effect on the formalin-induced nociception. In contrast, LY341495 caused a dose-dependent suppression of the phase 2 flinching response to the formalin stimulus without affecting phase 1 flinching response. Furthermore, the suppression of pain behavior by LY341495 during phase 2 was reduced significantly by pretreatment with APDC. LY341495 and morphine also showed synergistic drug interaction for antinociception during phase 2 in the formalin test.