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S T Anterior Brain

Fig. 1. Persistent DHEA and PREG after removal of steroidogenic glands. Male Sprague-Dawley rats were killed at the age of 77 ± 3 d. Groups of five males either intact (CONTROL), or castrated and adrenalectomized (ORX/ADX) 15 d before killing or sham-operated controls (SHAM) were used. Decapitation was performed 2-3 h after lights were on. Individual brains were divided in the posterior part (cerebellum, pons, medulla oblongata) and anterior part (cortex cerebri and mesencephalon). Brain samples were processed for RIA of DHEA, DHEAS, PREG, and PREGS. Results are expressed in ng/g (mean ± SD, n = 5). Fifteen days after the removal of steroidogenic glands, the concentrations of neurosteroids in brain were quite similar in ADX and in SHAM groups (adapted from ref. 11).

Fig. 1. Persistent DHEA and PREG after removal of steroidogenic glands. Male Sprague-Dawley rats were killed at the age of 77 ± 3 d. Groups of five males either intact (CONTROL), or castrated and adrenalectomized (ORX/ADX) 15 d before killing or sham-operated controls (SHAM) were used. Decapitation was performed 2-3 h after lights were on. Individual brains were divided in the posterior part (cerebellum, pons, medulla oblongata) and anterior part (cortex cerebri and mesencephalon). Brain samples were processed for RIA of DHEA, DHEAS, PREG, and PREGS. Results are expressed in ng/g (mean ± SD, n = 5). Fifteen days after the removal of steroidogenic glands, the concentrations of neurosteroids in brain were quite similar in ADX and in SHAM groups (adapted from ref. 11).

concentrations were found in the hypothalamus and the striatum (3.5 ± 0.7 and 4.6 ± 0.9 ng/g, respectively). After ovulation, PROG concentrations in the hypothalamus were increased almost tenfold (27 ± 5.2 ng/g), and there was a significant positive correlation between PROG plasma levels and the concentration in the cerebral cortex in the postovulatory, but not in the preovulatory, rat, although the adrenal gland is a documented source of PROG in the female rat (15). Therefore, PROG was measured in the brain of male and female rats before and after combined ADX + gonadectomy (13,16). Residual brain PROG levels in the 1-2 ng/g range were measured in operated rats of both sexes, whereas plasma levels were undetectable, thus strongly suggesting that PREG is converted to significant amounts of PROG in the brain. Similar observations were made in the peripheral nervous system (17). This conclusion was also supported by the administration of trilostane, a 3^-hydroxy steroid dehydrogenase inhibitor, to operated males, which resulted in a large decrease of PROG and increase of PREG in the brain in accordance with a precursor-to-product relationship (18).

The biosynthesis and metabolism of PROG are reviewed in Chapter 2 in this book by Mellon and Compagnone (19) and Chapter 6 by Poletti et al. (20). PROG is mainly converted to its 5a-reduced metabolite 5a-DH PROG, which is in turn converted to

3a- and 3p-hydroxy-5a-pregnan-20-ones. The 3a, 5a-reduced metabolites of PROG (3a,5a-TH PROG) and of deoxycorticosterone (3a,5a-THDOC) have raised considerable interest because they are potent allosteric modulators of gamma-aminobutyric acid A (GABAa) receptors, with sedative, anxiolytic and anesthetic properties (review in refs. 21-24).

Radioimmunoassay and gas chromatographic-mass fragmentographic methods (26) have been developed for the measurement of allopregnolone and its precursors in the brain.

There is no consensus about the basal levels of 3a, 5a-TH PROG in the young adult male rat brain, whereas Corpechot et al. (13) report barely detectable levels, other reports using sham-operated and/or restrained males, indicate concentrations of 1-3 ng/g (16,27). In these 2 reports, adrenalectomy induced only a partial decrease of 3a, 5a-TH PROG in the brain. Allopregnanolone levels have also been measured in the brain of female rats and found to parallel PROG levels, i.e, to be strongly dependent on estrus cycle or pregnancy, when they reach approx 12 ng/g (13,22). Allopregnanolone was still detectable after combined adrenalectomy and ovariectomy. The intermediate compound between PROG and 3a, 5a-THPROG (5a-DH PROG) has also been measured in the rat brain, and found at concentrations approaching those of PROG (13) or of -3a-, 5a-TH PROG (16).

The concentrations of neurosteroids have also been measured in castrated adult male Swiss mice (28). The concentrations of DHEA, PREG, PREG S, PROG, 5a-DH PROG, and 3a, 5a-TH PROG were similar to those of intact male rats. However, injection of DHEA (80 ^g daily for 2 wk) to castrated mice markedly increased the concentrations of 5a-DH PROG (approx 5 ng/g) and 3a, 5a-TH PROG (approx 2 ng/g). This was attributed to an androgenic effect, as it was also observed after injection of testosterone (40 ng daily for 5d) and counteracted by coadministration of an antiandrogen (J. Young and P. Robel, unpublished results).

Neurosteroids Variations in the Rodent Brain

Changes in neurosteroid levels have been observed in several situations, such as ontogenesis, biological rhythms, heterosexual exposure, or stress. In most instances, the relative contributions of locally synthesized neurosteroids and blood-borne steroids has not been established.

Ontogenesis

In newborn rats of both sexes, PREG(S) (as the sum PREG + PREG S) and corticos-terone (B) are high at the time of delivery in both rat brain and plasma. Brain PREG decreases steadily during the first day of life (postnatal day 1, PN 1), then remains within the range of adult levels, whereas B decreases to insignificant levels between days PN 1 and PN 10 (29). Contrary to B, brain PREG was unchanged when 4-d-old rats were treated with ACTH or dexamethasone for 2 d. The concentrations of DHEA(S) (as the sum DHEA + DHEA S) were very stable between birth and PN 22, in the 1.8-3.4 ng/g range not different from the values found in adults (30).

Clrcadian and Infradian Rhythms

In adult male Sprague-Dawley rats, timing of the removal of the brain, as related to the light-dark cycle, demonstrated prominent circadian rhythms of 3^-hydroxy-A5-steroids in plasma and brain. When the data were represented by the cosinor method, the acrophases of PREG(S) in brain and of DHEA(S) in plasma significantly preceded the acrophase of B, suggesting partly separate coordinatory mechanisms (31). Fifteen days after ADX + ORX, a significant rhythm of brain DHEA(S) persisted with an acrophase at the beginning of the dark period and a similar trend was observed for PREG (32).

In female Holtzman rats, data were collected over 11 d. The variations of brain DHEA(S), DHEA L, and PREG L followed a complex pattern that could best be approximated by the concomitant fit of cosine functions with about 5- and 1-d periods (33).

Concentrations of PROG, of 5a-DH PROG, and of 3a,5a-TH PROG have been measured in the mouse brain throughout the estrous cycle. Plasma PROG concentrations were also measured for comparison. At each stage, circadian fluctuations were found in the concentrations of brain PROG and its metabolites (34). Such fluctuations were greater than those attributable to any particular stage of the estrous cycle. Over the entire cycle, a significant correlation was found between brain 5a-DH PROG or 3a,5a-TH PROG and PROG concentrations. Brain PREG S also underwent circadian variations during the estrous cycle that unexpectedly were in phase with plasma PROG but not brain PROG concentrations. Results suggested that circadian and ovarian influences on the concentrations of PROG and its 5a-reduced metabolites in female whole mouse brain were caused predominantly by changes in the supply of PROG from within the tissue, whatever the contribution of peripheral sources.

Heterosexual Exposure

When intact male rats were exposed to the scent of estrous females for 7 d, a significant decrease of PREG(S) concentrations occurred in the olfactory bulbs but not in any other brain structure. Moreover, DHEA(S) concentrations tended to increase in the hypothalamus (30) (Fig. 2). When male rats were exposed to the scent and view of female rats, PREG(S) decreased significantly in the olfactory bulb, whereas DHEA(S) increased significantly in the olfactory bulb and the retina, and PROG decreased in the hypothalamus, amygdala, and parietal cortex (35).

Stress

The transient increase of brain DHEA S that occurs in the brain of male rats 2 d after adrenalectomy or the corresponding sham operation has been related to the heavy stress following anesthesia and surgery (10). Several later reports have provided interesting informations about the effect of stress on the levels of neurosteroids in the rat brain. In rats habituated to the manipulation that precedes killing, the cerebral cortical concentration of PREG and PROG were about 2-fold lower than in naive animals (36). Rapid (25 min) and robust (4- to 20-fold) increases of 3a,5a-TH PROG, and 3a,5a-TH DOC were detected in the brain and plasma of naive male rats after exposure to ambient temperature swim stress (27). Adrenalectomy essentially obliterated the response to stress, with the notable exception of brain 3a,5a-TH PROG. Another type of acute stress, CO2 inhalation, elicited a marked increase in the concentrations of PREG, PROG, and DOC in the brain cortex and hippocampus of handling habituated rats, whereas DHEA levels were unchanged (36). Foot shock also increased the concentrations of PREG, PROG, and 3a,5a-TH PROG in both brain and plasma of intact rats (37).

Acute Ethanol Intoxication

Sprague-Dawley male rats (175 g) were given ethanol (16% in saline) by intraperitoneal injection to obtain a blood alcohol level of approx 50 mg/100 mL. Control rats were given an equal volume of saline. Ethanol injection resulted in a dramatic decrease

Fig. 2. PREG(S) decreases in the olfactory bulb of male rats exposed to the scent of females. PREG(S) (as the sum PREG + PREGS) and DHEA(S) were measured in the limbic system of male rats exposed to the scent of either males (M/M) or estrous females (M/F). OB, olfactory bulb; OT, olfactory tubercle; A, amydala; HYP, hypothalamus. The only significant differences were the decrease of PREG(S) in the OB and the increase of DHEA(S) in the HYP of M/F groups vs M/M ones (adapted from ref. 30).

Fig. 2. PREG(S) decreases in the olfactory bulb of male rats exposed to the scent of females. PREG(S) (as the sum PREG + PREGS) and DHEA(S) were measured in the limbic system of male rats exposed to the scent of either males (M/M) or estrous females (M/F). OB, olfactory bulb; OT, olfactory tubercle; A, amydala; HYP, hypothalamus. The only significant differences were the decrease of PREG(S) in the OB and the increase of DHEA(S) in the HYP of M/F groups vs M/M ones (adapted from ref. 30).

of DHEA(S) concentrations in the brain. This decrease occurred rapidly, DHEA(S) had completely disappeared after 30 min and reappeared progressively to reach control values after 4 h. All brain areas investigated were similarly affected. PREG and PREG S did not change (23).

To gain some insight into the mechanisms involved in the depletion of brain DHEA, 2.5 mg of the steroid were injected intramuscularly in sesame oil, 4 h before killing, to control or ethanol-treated rats. DHEA was cleared much more rapidly from the brain under the influence of ethanol, thus suggesting that ethanol induces a rapid metabolic conversion of DHEA and DHEA S. It was previously reported that ethanol markedly increases the metabolic conversion of DHEA to Androst-5ene-3p, 17^-diol in humans (38).

Other Mammalian Species

Monkeys

In primates, contrary to rodents, the adrenal secretes large amounts of DHEA S, which is the most abundant steroid in plasma. DHEA(S), PREG(S), and PREG L concentrations have been measured in the brain of Macaca fascicularis without or with suppression of adrenocortical steroid secretion by dexamethasone (DEX) (39). Two control adult spayed females and two DEX-treated females (4 mg DEX daily for 3 d) were studied. The effectiveness of adrenal suppression was indicated by the decrease of plasma

Cortisol to undetectable levels 20 h after the last injection of DEX. Concentrations of DHEA(S) in plasma were also much smaller after DEX treatment (91.5-93.5 ng/mL before DEX and 4.5-19.3 ng/mL after DEX, respectively). DHEA(S) concentrations in the brain were about threefold smaller than in plasma. Adrenal suppression resulted in about twofold smaller brain concentration, but the fraction of DHEA(S) remaining in the brain after adrenal suppression was much larger than the corresponding fraction in plasma, suggesting that the accumulation of DHEA(S) in monkey brain is, at least in part, independent of peripheral endocrine glands, as previously shown in the rat.

PREG(S) concentrations were severalfold larger in brain than in plasma but about twofold smaller than those of DHEA(S); and did not seem to be influenced by adrenal suppression.

The impermeability of the blood-brain barrier to DHEA S had been previously investigated in the Rhesus monkey (39). Two brains were perfused in vivo either with [14C]DHEA and [3H]DHEA S, or with [3H]DHEA. Plasma from the jugular vein and the brain were analyzed for free and conjugated metabolites. Much more free than sulfoconjugated DHEA was withdrawn from the blood. Slight conversion of [ 14C]DHEA to [14C]DHEA S and of [3H]DHEA S to [3H]DHEA occurred in the brain.

Humans

The concentrations of A5-3 p -hydroxysteroids have been measured in specific regions of the human brain (41,42). The tissue samples were obtained by routine craniotomy from cadavers that had been stored at 4°C until autopsy within 24 h.

The overall mean of PREG was 120.7 nmol/kg or 38.2 ng/g, the overall mean of PROG was 10.1 nmol/kg or 3.2 ng/g, and the overall mean of DHEA was 19.6 nmol/kg or 5.6 ng/g (41). The concentrations of the free steroids generally exceeded those of the sulfate esters (41), and were much higher than those of the sex steroid hormones and in the same range as those reported in the rat.

The ratios of PREG and DHEA concentrations in the brain to the corresponding plasma concentration typically found in aged subjects were 74 and 6.5, respectively. Hence it is tempting to speculate that the human brain is capable of de novo biosynthesis of steroids. With the immunoperoxidase technique, P450scc, adrenodoxin and adreno-doxin reductase have been detected in the human brain (43). The human brain possesses DHEA sulfotransferase and sulfatase activities (40).

Postmortem concentrations of PROG, 5a-DH PROG, and 3a,5a-TH PROG were measured in the brain and serum of fertile women in the luteal phase and postmenopausal women (44). There were regional differences in brain concentrations of all three steroids and in their relative amounts. Theses concentrations were significantly higher in the womens' luteal phase compared to their postmenopausal control subjects, thus showing that the levels of those neurosteroids were at least in part related to ovarian steroid (PROG) production.

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