Postnatal handling increases glucocorticoid receptor expression in the rat hippocampus, thus altering the regulation of hypothalamic synthesis of corticotropin-releasing hormone and the hypothalamic-pituitary-adrenal response to stress. The effect on glucocorticoid receptor gene expression represents one mechanism by which the early environment can exert a long-term effect on neural development. The handling effect on hippocampal glucocorticoid receptor expression is dependent on peripheral thyroid hormone release and the activation of ascending serotonergic pathways. In primary hippocampal cell cultures, serotonin (5-HT) increases glucocorticoid receptor expression, and this effect appears to be mediated by increased cAMP levels. In the current studies we examined the in vivo effects of handling on hippocampal cAMP-protein kinase A (PKA) activity. In 7-d-old rat pups, we found that (1) postnatal handling increased adenylyl cyclase activity and hippocampal cAMP levels, (2) the effect of handling on cAMP levels was completely blocked by treatment with either propylthiouracil (PTU), a thyroid hormone synthesis inhibitor, or the 5-HT receptor antagonist, ketanserin, and (3) handling also increased hippocampal PKA activity. We then examined the effects of handling on cAMP-inducible transcription factors. Handling rapidly increased levels of the mRNAs for nerve growth factor-inducible factor A (NGFI-A) (zif268, krox24) and activator protein-2 (AP-2) as well as for NGFI-A and AP-2 immunoreactivity throughout the hippocampus. Finally, we found that the effects of handling on NGFI-A and AP-2 expression were significantly reduced by concurrent treatment with either PTU or ketanserin, effects that paralleled those on cAMP formation. NGFI-A and AP-2 have been implicated in the regulation of glucocorticoid receptor expression during development. Thus, these findings suggest that postnatal handling might alter glucocorticoid receptor gene expression via cAMP-PKA pathways involving the activation of NGFI-A and AP-2.

In aging humans and rodents, inter-individual differences in cognitive function have been ascribed to variations in long-term glucocorticoid exposure. 11␤-Hydroxysteroid dehydrogenase type 1 (11␤-HSD1) regenerates the active glucocorticoid cortisol from circulating inert cortisone, thus amplifying intracellular glucocorticoid levels in some tissues. We show that 11␤-HSD1, but not 11␤-HSD2, mRNA is expressed in the human hippocampus, frontal cortex, and cerebellum. In two randomized, double-blind, placebocontrolled crossover studies, administration of the 11␤-HSD inhibitor carbenoxolone (100 mg three times per day) improved verbal fluency (P < 0.01) after 4 weeks in 10 healthy elderly men (aged 55-75 y) and improved verbal memory (P < 0.01) after 6 weeks in 12 patients with type 2 diabetes (52-70 y). Although carbenoxolone has been reported to enhance hepatic insulin sensitivity in short-term studies, there were no changes in glycemic control or serum lipid profile, nor was plasma cortisol altered. 11␤-HSD1 inhibition may be a new approach to prevent͞ameliorate cognitive decline.

tion-mass spectrometry (LC-ESI-MS) analysis. We hereby report the exact identity of 16 oxysterols and downstream metabolites, including cholestenoic acids, found in human CSF (Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI68506DS1). The most abundant of these metabolites (19.48-0.40 ng/ml; Supplemental Figure 1) were 7α-hydroxy-3-oxocholest-4-en-26-oic acid (7αH,3O-CA), 3β-hydroxycholest-5-en-26-oic acid (3β-HCA), and 2 newly identified metabolites in CSF, 3β,7α-diHCA and 3β,7β-dihydroxycholest-5-en-26-oic acid (3β,7β-diHCA). Precursors of these acids, including 26-HC and newly identified 7α,26-dihydroxycholesterol (7α,26-diHC; cholest-5-ene-3β,7α,26-triol) and 7α,26-dihydroxycholest-4-en-3-one (7α,26-diHCO), were also found, but at lower levels (0.15-0.03 ng/ml). Our results thus identified 4 novel oxysterol metabolites in human CSF that were downstream of 26-HC ( Figure 1A). 26-HC is metabolized via 7α,26-diHC and 7α,26-diHCO, or via 3β-HCA and 3β,7α-diHCA, to 7αH,3O-CA. While 26-HC can cross the blood-brain barrier (BBB) and enter the brain from the circulation (25), 7αH,3O-CA traverses the BBB and is exported from the brain (26). Very low levels of 24S-hydroxycholesterol (24S-HC; cholest-5-ene-3β,24S-diol), 25-hydroxycholesterol (25-HC; cholest-5-ene-3β,25-diol), and newly identified 7α,25-dihydroxycholesterol (7α,25-diHC; cholest-5-ene-3β,7α,25-triol) and 7α,25-dihydroxycholest-4-en-3-one (7α,25-diHCO) were also found in CSF (0.08-0.03 ng/ml).Reduced levels of 7α-hydroxylated cholestenoic acids in CSF and plasma/serum of human patients with SPG5. SPG5 presents with upper motor neuron signs and results from mutations in CYP7B1, encoding the oxysterol 7α-hydroxylase responsible for 7α-hydroxylation of side-chain oxidized sterols that is required for extrahepatic synthesis of 7αH,3O-CA and its precursor, 3β,7α-diHCA ( Figure 1A and ref. 18). In order to examine the pathogenic role of such mutations, we sought to identify alterations in oxysterol and cholestenoic acid profiles in CSF and plasma from these patients and then examine the biological activities of the altered metabolites. We first studied the CSF from 3 patients with SPG5

11␤-hydroxysteroid dehydrogenase type 1 (11␤-HSD-1) intracellularly regenerates active corticosterone from circulating inert 11-dehydrocorticosterone (11-DHC) in specific tissues. The hippocampus is a brain structure particularly vulnerable to glucocorticoid neurotoxicity with aging. In intact hippocampal cells in culture, 11␤-HSD-1 acts as a functional 11␤-reductase reactivating inert 11-DHC to corticosterone, thereby potentiating kainate neurotoxicity. We examined the functional significance of 11␤-HSD-1 in the central nervous system by using knockout mice. Aged wild-type mice developed elevated plasma corticosterone levels that correlated with learning deficits in the watermaze. In contrast, despite elevated plasma corticosterone levels throughout life, this glucocorticoid-associated learning deficit was ameliorated in aged 11␤-HSD-1 knockout mice, implicating lower intraneuronal corticosterone levels through lack of 11-DHC reactivation. Indeed, aged knockout mice showed significantly lower hippocampal tissue corticosterone levels than wild-type controls. These findings demonstrate that tissue corticosterone levels do not merely reflect plasma levels and appear to play a more important role in hippocampal functions than circulating blood levels. The data emphasize the crucial importance of local enzymes in determining intracellular glucocorticoid activity. Selective 11␤-HSD-1 inhibitors may protect against hippocampal function decline with age.