Critical Effects on Akt Signaling in Adult Zebrafish Brain Following Alterations in Light Exposure.

Evidence from human and animal studies indicate that disrupted light cycles leads to alterations of the sleep state, poor cognition, and the risk of developing neuroinflammatory and generalized health disorders. Zebrafish exhibit a diurnal circadian rhythm and are an increasingly popular model in studies of neurophysiology and neuropathophysiology. Here, we investigate the effect of alterations in light cycle on the adult zebrafish brain: we measured the effect of altered, unpredictable light exposure in adult zebrafish telencephalon, homologous to mammalian hippocampus, and the optic tectum, a significant visual processing center with extensive telencephalon connections. The expression of heat shock protein-70 (HSP70), an important cell stress mediator, was significantly decreased in optic tectum of adult zebrafish brain following four days of altered light exposure. Further, pSer473-Akt (protein kinase B) was significantly reduced in telencephalon following light cycle alteration, and pSer9-GSK3ß (glycogen synthase kinase-3ß) was significantly reduced in both the telencephalon and optic tectum of light-altered fish. Animals exposed to five minutes of environmental enrichment showed significant increase in pSer473Akt, which was significantly attenuated by four days of altered light exposure. These data show for the first time that unpredictable light exposure alters HSP70 expression and dysregulates Akt-GSK3ß signaling in the adult zebrafish brain.

Na+/K+-ATPase is involved in the regulation of food intake in rainbow trout but apparently not through brain glucosensing mechanisms.

To assess the hypothesis that Na+/K+-ATPase (NKA) is involved in the central regulation of food intake in fish, we observed in a first experiment with rainbow trout (Oncorhynchus mykiss) that intracerebroventricular (ICV) treatment with ouabain decreased food intake. We hypothesized that this effect relates to modulation of glucosensing mechanisms in brain areas (hypothalamus, hindbrain, and telencephalon) involved in food intake control. Therefore, we evaluated in a second experiment, the effect of ICV administration of ouabain, in the absence or in the presence of glucose, on NKA activity, mRNA abundance of different NKA subunits, parameters related to glucosensing, transcription factors, and appetite-related neuropeptides in brain areas involved in the control of food intake. NKA activity and mRNA abundance of nkaα1a and nkaα1c in brain were inhibited by ouabain treatment and partially by glucose. The anorectic effect of ouabain is opposed to the orexigenic effect reported in mammals. The difference might relate to the activity of glucosensing as well as downstream mechanisms involved in food intake regulation. Ouabain inhibited glucosensing mechanisms, which were activated by glucose in hypothalamus and telencephalon. Transcription factors and neuropeptides displayed responses comparable to those elicited by glucose when ouabain was administered alone, but not when glucose and ouabain were administered simultaneously. Ouabain might therefore affect other processes, besides glucosensing mechanisms, generating changes in membrane potential and/or intracellular pathways finally modulating transcription factors and neuropeptide mRNA abundance leading to modified food intake.

Distinct expression patterns for type II topoisomerases IIA and IIB in the early foetal human telencephalon.

TOP2A and TOP2B are type II topoisomerase enzymes that have important but distinct roles in DNA replication and RNA transcription. Recently, TOP2B has been implicated in the transcription of long genes in particular that play crucial roles in neural development and are susceptible to mutations contributing to neurodevelopmental conditions such as autism and schizophrenia. This study maps their expression in the early foetal human telencephalon between 9 and 12 post-conceptional weeks. TOP2A immunoreactivity was restricted to cell nuclei of the proliferative layers of the cortex and ganglionic eminences (GE), including the ventricular zone and subventricular zone (SVZ) closely matching expression of the proliferation marker KI67. Comparison with sections immunolabelled for NKX2.1, a medial GE (MGE) marker, and PAX6, a cortical progenitor cell and lateral GE (LGE) marker, revealed that TOP2A-expressing cells were more abundant in MGE than the LGE. In the cortex, TOP2B is expressed in cell nuclei in both proliferative (SVZ) and post-mitotic compartments (intermediate zone and cortical plate) as revealed by comparison with immunostaining for PAX6 and the post-mitotic neuron marker TBR1. However, co-expression with KI67 was rare. In the GE, TOP2B was also expressed by proliferative and post-mitotic compartments. In situ hybridisation studies confirmed these patterns of expression, except that TOP2A mRNA is restricted to cells in the G2/M phase of division. Thus, during early development, TOP2A is likely to have a role in cell proliferation, whereas TOP2B is expressed in post-mitotic cells and may be important in controlling expression of long genes even at this early stage.

Acute and specific modulation of presynaptic aromatization in the vertebrate brain.

Estrogens affect a diversity of peripheral and central physiological endpoints. Traditionally, estrogens were thought to be peripherally derived transcription regulators (i.e. slow acting). More recently, we have learned that estrogens are also synthesized in neuronal cell bodies and synaptic terminals and have potent membrane effects, which modulate brain function. However, the mechanisms that control local steroid concentrations in a temporal and spatial resolution compatible with their acute actions are poorly understood. Here, using differential centrifugation followed by enzymatic assay, we provide evidence that estrogen synthesis within synaptosomes can be modulated more dramatically by phosphorylating conditions, relative to microsomes. This is the first demonstration of a rapid mechanism that may alter steroid concentrations within the synapse and may represent a potential mechanism for the acute control of neurophysiology and behavior.

Evidence of anoxia-induced channel arrest in the brain of the goldfish (Carassius auratus).

The common goldfish (Carassius auratus) is extremely anoxia tolerant and here we provide evidence that "channel arrest" in the brain of these fish contributes to ATP conservation during periods of anoxia. Whole-cell patch-clamp recordings of slices taken from the telencephalon indicated that the N-methyl-d-aspartate (NMDA) receptor, an ionotropic glutamate receptor and Ca(2+)-channel, underwent a 40-50% reduction in activity during 40 min of acute anoxia. This is the first direct evidence of channel arrest in an anoxia-tolerant fish. Because goldfish produce ethanol as a byproduct of anaerobic metabolism we then conducted experiments to determine if the observed reduction in NMDA receptor current amplitude was due to inhibition by ethanol. NMDA receptor currents were not inhibited by ethanol (10 mmol L(-1)), suggesting that channel arrest of the receptor involved other mechanisms. Longer-term (48 h) in vivo exposure of goldfish to anoxic conditions (less than 1% dissolved O(2)) provided indirect evidence that a reduction in Na(+)/K(+)-ATPase activity also contributed to ATP conservation in the brain but not the gills. Anoxia under these conditions was characterized by a decrease in brain Na(+)/K(+)-ATPase activity of 30-40% by 24 h. Despite 90% reductions in the rates of ventilation, no change was observed in gill Na(+)/K(+)-ATPase activity during the 48-h anoxia exposure, suggesting that branchial ion permeability was unaffected. We conclude that rapid "channel arrest" of NMDA receptors likely prevents excitotoxicity in the brain of the goldfish, and that a more slowly developing decrease in Na(+)/K(+)-ATPase activity also contributes to the profound metabolic depression seen in these animals during oxygen starvation.

Enhanced CREB and DARPP-32 phosphorylation in the nucleus accumbens and CREB, ERK, and GluR1 phosphorylation in the dorsal hippocampus is associated with cocaine-conditioned place preference behavior.

Environment-induced relapse is a major concern in drug addiction because of the strong associations formed between drug reward and environment. Cocaine-conditioned place preference is an ideal experimental tool to examine adaptations in the molecular pathways that are activated upon re-exposure to an environment previously paired with drug reward. To better understand the mechanism of cocaine-conditioned place preference we have used western blot analysis to examine changes in phosphorylation of cAMP-response element binding protein (CREB), dopamine- and cyclic AMP-regulated phosphoprotein 32 (DARPP-32), extracellular signal-regulated kinase (ERK) and GluR1, key molecular substrates altered by cocaine, in the nucleus accumbens (NAc) and dorsal hippocampus (DHC) of C57BL/6 mice. Our studies revealed that re-exposing mice to an environment in which they were previously given cocaine resulted in increased levels of Ser133 phospho-CREB and Thr34 phospho-DARPP-32 with a corresponding decrease in Thr75 phospho-DARPP-32 in the NAc. In DHC there were increased levels of phospho-CREB, Thr183/Tyr185 phospho-ERK, and Ser845 phospho-GluR1. These data suggest that the formation of contextual drug reward associations involves recruitment of the DHC-NAc circuit with activation of the DARPP-32/CREB pathway in the NAc and the ERK/CREB pathway in the DHC.

Decrease of dehydrogenase activity of cerebral glyceraldehyde-3-phosphate dehydrogenase in different animal models of Alzheimer's disease.

Recently, a relationship between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the beta-amyloid precursor protein (betaAPP) in relationship with the pathogenesis of Alzheimer's disease (AD) has been suggested. Therefore, we studied the specific activity of GAPDH in the different animal models of AD: transgenic mice (Tg2576) and rats treated with beta-amyloid, or thiorphan, or lipopolysaccharides (LPS) and interferon gamma (INFgamma). We observed that GAPDH activity was significantly decreased in the brain samples from TG mice. The injection of beta-amyloid, or thiorphan, an inhibitor of neprilysin involved in beta-amyloid catabolism, in rat brains resulted in a pronounced reduction of the enzyme activity. The infusion of LPS and IFNgamma, which can influence the progression of the AD, significantly reduced the enzyme activity.

Tumor-targeted enzyme/prodrug therapy mediates long-term disease-free survival of mice bearing disseminated neuroblastoma.

Neural stem cells and progenitor cells migrate selectively to tumor loci in vivo. We exploited the tumor-tropic properties of HB1.F3.C1 cells, an immortalized cell line derived from human fetal telencephalon, to deliver the cDNA encoding a secreted form of rabbit carboxylesterase (rCE) to disseminated neuroblastoma tumors in mice. This enzyme activates the prodrug CPT-11 more efficiently than do human enzymes. Mice bearing multiple tumors were treated with rCE-expressing HB1.F3.C1 cells and schedules of administration of CPT-11 that produced levels of active drug (SN-38) tolerated by patients. Both HB1.F3.C1 cells and CPT-11 were given i.v. None of the untreated mice and 30% of mice that received only CPT-11 survived long term. In contrast, 90% of mice treated with rCE-expressing HB1.F3.C1 cells and 15 mg/kg CPT-11 survived for 1 year without detectable tumors. Plasma carboxylesterase activity and SN-38 levels in mice receiving both rCE-expressing HB1.F3.C1 cells (HB1.F3.C1/AdCMVrCE) and CPT-11 were comparable with those in mice receiving CPT-11 only. These data support the hypothesis that the antitumor effect of the described neural stem/progenitor cell-directed enzyme prodrug therapy (NDEPT) is mediated by production of high concentrations of active drug selectively at tumor sites, thereby maximizing the antitumor effect of CPT-11. NDEPT approaches merit further investigation as effective, targeted therapy for metastatic tumors. We propose that the described approach may have greatest use for eradicating minimum residual disease.

Effect of NMDA on staurosporine-induced activation of caspase-3 and LDH release in mouse neocortical and hippocampal cells.

To achieve a better understanding of developmentally regulated NMDA- and staurosporine-induced apoptotic processes, we investigated the concerted action of these agents on caspase-3 activity and LDH release in neocortical and hippocampal cell cultures at different stages in vitro (DIV). Hoechst 33342 and MAP-2 stainings were additionally employed to visualize apoptotic changes and cell damage. The vulnerability of neocortical cells to NMDA was more prominent at later culture stages, whereas hippocampal neurons were more susceptible to NMDA treatment at earlier stages. A persistent activation of caspase-3 by staurosporine was found at all experimental stages. Despite of certain differences in susceptibility to NMDA and staurosporine, both tissues responded to regulatory action of NMDA towards staurosporine-activated caspase-3 in a similar way. Combined treatment with NMDA and staurosporine resulted in a substantial increase in caspase-3 activity in neocortical and hippocampal neurons on 2 DIV. Additive effects were also observed in neocortical cultures on 12 DIV. In contrast, NMDA substantially inhibited staurosporine-induced caspase-3 activity on 7 DIV in neocortical and hippocampal cultures. Additionally, pro-apoptotic effects of 17beta-estradiol were attenuated by NMDA on 7 DIV. Changes in vulnerability to NMDA- and staurosporine-mediated activation of caspase-3 were not strictly related to LDH release. Our data revealed that NMDA can both enhance and inhibit the staurosporine-induced neuronal cell apoptosis. The pro-apoptotic effect of NMDA was exhibited at early and late culture stages, whereas the anti-apoptotic effect was transient occurring on 7 DIV only.

Sex differences in cytochrome P450 1B1, an estrogen-metabolizing enzyme, in the rhesus monkey telencephalon.

The metabolic enzyme CYP1B1 is a recently cloned member of the cytochrome P450 superfamily, expressed widely throughout primate tissue, including the CNS. Although CYP1B1 protein is known to metabolize estradiol to catecholestrogens in the uterus, its localization and function in brain have not yet been described. To better understand CYP1B1 distribution, we have combined in situ hybridization (ISH) for its mRNA with immunohistochemistry (IHC) for the CYP1B1 protein in selected brain regions of male and female adult rhesus monkeys (Macaca mulatta). Blocks of formalin-fixed tissue obtained from the frontal cortex, hippocampus, thalamus, and amygdala were processed and embedded in paraffin. They were then sectioned and stained as described for human tissue [Muskhelishvili, L., Thompson, P.A., Kusewitt, D.F., Wang, C., Kadlubar, F.F., 2001. In situ hybridization and immunohistochemical analysis of cytochrome P450 1B1 expression in human normal tissues. J. Histochem. Cytochem. 49, 229-236]. Results indicated widespread distribution of CYP1B1 mRNA in both male and female monkey frontal cortex, hippocampus, thalamus, and amygdala. In contrast, although CYP1B1 protein was co-localized with its mRNA in the female brains, it was primarily restricted to hippocampal pyramidal neurons in the male brains. These results suggest that CYP1B1 may subserve widespread metabolic functions in the female primate brain but have more restricted actions within the hippocampal pyramidal neurons of the male.

Developmental lag in superoxide dismutases relative to other antioxidant enzymes in premyelinated human telencephalic white matter.

Periventricular leukomalacia (PVL) involves free radical injury to developing oligodendrocytes (OLs), resulting from ischemia/reperfusion, particularly between 24 and 32 gestational weeks. Using immunocytochemistry and Western blots, we tested the hypothesis that this vulnerability to free radical toxicity results, in part, from developmental lack of superoxide dismutases (SOD)-1 and -2, catalase, and glutathione peroxidase (GPx) in the telencephalic white matter of the human fetus. During the period of greatest PVL risk and through term (> or = 37 weeks), expression of both SODs (for conversion of O2- to H2O2) significantly lagged behind that of catalase and GPx (for breakdown of H2O2), which, in contrast, superseded adult levels by 30 gestational weeks. Our data indicate that a developmental "mismatch" in the sequential antioxidant enzyme cascade likely contributes to the vulnerability to free radical toxicity of the immature cerebral white matter, which is "unprepared" for the transition from a hypoxic intrauterine to an oxygen-rich postnatal environment. All enzymes, localized to astrocytes and OLs, had higher-than-adult expression at 2 to 5 postnatal months (peak of myelin sheath synthesis), suggesting an adaptive mechanism to protect against lipid peroxidation during myelin sheath (lipid) synthesis. The previously unrecognized dissociation between the expression of the SODs and that of catalase and GPx in the fetal period has potential implications for future antioxidant therapy in PVL.

Estrogen contributes to seasonal plasticity of the adult avian song control system.

Songbirds show dramatic neural plasticity as adults, including large-scale anatomical changes in discrete brain regions ("song control nuclei") controlling the production of singing behavior. The volumes of several song control nuclei are much larger in the breeding season than in the nonbreeding season, and these seasonal neural changes are regulated by plasma testosterone (T) levels. In many cases, the effects of T on the central nervous system are mediated by neural conversion to estradiol (E(2)) by the enzyme aromatase. The forebrain of male songbirds expresses very high levels of aromatase, in some cases adjacent to song control nuclei. We examined the effects of aromatase inhibition and estrogen treatment on song nuclei size using wild male songbirds in both the breeding and nonbreeding seasons. In breeding males, aromatase inhibition caused the volume of a telencephalic song control nucleus (HVC) to decrease, and this effect was partially rescued by concurrent estrogen replacement. In nonbreeding males, estradiol treatment caused HVC to grow to maximal spring size within 2 weeks. Overall, these data suggest that aromatization of T is an important mediator of song control system plasticity, and that estradiol has neurotrophic effects in adult male songbirds. This study demonstrates that estrogen can affect adult neural plasticity on a gross anatomical scale and is the first examination of estrogen effects on the brain of a wild animal.

Seungnoidan increases cerebrocortical ATP and acetylcholine contents in ovariectomized rats.

This study investigated the effects of Seungnoidan (SND), which has been widely used as a remedy for cerebroneuronal diseases in Korean folk medicine, on the cerebrocortical adenosine triphosphate (ATP) and acetylcholine (ACh) contents in ovariectomized (OVX) rats. Female Sprague-Dawley rats were ovariectomized and maintained for 12 weeks to deplete ovarian steroid hormones, followed by oral administration of SND at 500 mg/kg/day for 14 weeks. SND markedly attenuated the high rate of body weight increase in OVX rats, and also reduced the decline of cerebral weight caused by ovariectomy (p < 0.05). Superfusion of SND at 50 mg/kg significantly increased the rate of cerebral blood fl ow, but did not change the mean arterial blood pressure. Deprivation of ovarian steroid hormones significantly decreased the cerebral ATP, choline and ACh contents, and these reductions were reduced by treatment of OVX rats with SND (p < 0.01). Additionally, SND also significantly elevated the cerebral choline acetyltransferase activities reduced by OVX (p < 0.01). Taken together, these results suggest that the pharmacological properties of SND may be implicated in the improvement of metabolic pathways of cerebral energy and cholinergic neurotransmitter function induced by deprivation of ovarian steroid hormones, and SND may be a promising herbal remedy for treatment of cerebral dysfunctions including dementia.

Telomerase activity in the subventricular zone of adult mice.

The subventricular zone (SVZ) is the most active site for the production of new neurons in the adult mouse brain. Neural stem cells in the adult SVZ give rise to neuroblasts that travel via the rostral migratory stream (RMS) to the olfactory bulb, where they differentiate into interneurons. The enzyme telomerase has been identified in other population of stem cells and is necessary for the synthesis of telomeric DNA to prevent chromosomal shortening, end-to-end fusions, and apoptosis during successive rounds of cell division. However, previous studies have failed to detect telomerase in the adult mammalian brain. Here we demonstrate that telomerase is expressed by all brain regions shortly after birth, but becomes restricted to the SVZ and olfactory bulb in the adult mouse brain. Cultures of neural precursor cells or of migratory neuroblasts purified from the SVZ were each found to possess telomerase activity. After elimination of migrating neuroblasts and immature precursor cells in vivo by treatment with cytosine-beta-D-arabinofuranoside (Ara-C), telomerase activity was still detectable in the remaining SVZ, which includes a population of neural stem cells. Following withdrawal of Ara-C, telomerase activity subsequently increased with a time course that parallels regeneration of the SVZ network and RMS. Finally, intracranial surgery alone, which has previously been shown to increase the number of cells in the SVZ, produced higher telomerase levels in the SVZ. We conclude that telomerase is active in neural precursor cells of the adult mouse and suggest that its regulation is an important parameter for cellular proliferation to occur in the mammalian brain.

In vitro effects of catecholamines and catecholestrogens on brain tyrosine hydroxylase activity and kinetics in the female catfish Heteropneustes fossilis.

Effects of catecholamines and catecholestrogens on tyrosine hydroxylase (TH) activity and kinetics were investigated in the telencephalon and hypothalamus of female Heteropneustes fossilis in gonad quiescent (resting) and recrudescent (preparatory) phases. Dopamine, noradrenaline and adrenaline and the catecholestrogen, 2-hydroxyestradiol-17 beta inhibited TH activity in a concentration-dependent manner in both resting and preparatory phases, with a higher effect in the resting phase. Two- methoxyestradiol-17 beta did not alter TH activity in any season. The catecholamines inhibited TH in a competitive manner increasing apparent K(m) values significantly without altering the apparent V(max). Two-hydroxyestradiol-17 beta inhibited significantly the enzyme in a noncompetitive manner and decreased apparent V(max) without altering apparent K(m) values. The apparent K(i) is higher for dopamine than noradrenaline or adrenaline. The apparent K(i) for 2-hydroxyestradiol-17 beta is not significantly different from that of noradrenaline. The present results suggest an interaction between oestradiol-17beta (E2) and catecholamine metabolism at the level of tyrosine hydroxylation and E2 effects on catecholamines may be mediated through its 2-hydroxylation.

Effects of estrogens on choline-acetyltransferase immunoreactivity and GAP-43 mRNA in the forebrain of young and aging male rats.

1. Previous work demonstrated that estradiol (E2) treatment prevented the abnormal response to stress and the reduction of glucocorticoid receptors (GR) in hippocampus from aging male rats. The mechanisms originating these effects were unknown. 2. In the present work, we investigated the E2 effects on the cholinergic, growth-associated protein (GAP-43) expressing neurons of the medial septum (MS) and vertical limb of diagonal band of Broca (VDB). These areas project to the hippocampus, and may be involved in the mentioned E2 effects in aging animals. Therefore, the response to E2 of choline-acetyltransferase (ChAT) in neurons and cell processes and GAP-43 mRNA as a marker of neurite outgrowth was studied in young and old male rats. 3. Young (3-4 months) and old (18-20 months) male Sprague-Dawley rats remained untreated or were implanted s.c. with a 14 mg pellet of E2 benzoate during 6 weeks. We used immoucytochemistry to determine ChAT and isotopic in situ hybridization to analyze GAP-43 mRNA expression. 4. Aging males showed a reduction in the number and length of ChAT-immunoreactive cell processes, but not in the number of positive neurons in MS and VDB. E2 reverted both parameters in old rats to levels of young animals. Regarding basal levels of GAP-43 mRNA, they were similar in old and young animals, but E2 treatment up-regulated GAP-43 mRNA expression in MS and VDB of old animals only. 5. Our data suggest that prolonged E2 treatment may affect hippocampal function of aging male rats by regulating in part the plasticity of cholinergic, GAP-43 expressing neurones of the basal forebrain. Without discarding a direct E2 effect on the limbic tissue, effects on the cholinergic system may have a pronounced impact on the neuroendocrine and stress responses of the aging hippocampus.

Effects of ovariectomy and oestradiol-17beta replacement on brain tyrosine hydroxylase in the catfish Heteropneustes fossilis: changes in in vivo activity and kinetic parameters.

In Heteropneustes fossilis, ovariectomy inhibited in vivo brain (hypothalamus-pituitary, telencephalon and medulla oblongata) tyrosine hydroxylase (TH) activity with significant effects in weeks 2, 3, 4 and 5 of the gonadal resting phase and in weeks 3, 4 and 5 of the prespawning phase (P<0.05, Tukey's test). Oestradiol-17beta (OE(2)) replacement in 3-week ovariectomised fish produced biphasic responses in both seasons; the low dosages of 0.05 and 0.5 micro g/g body weight (BW) elevated TH activity, whereas the high dosages of 1.0 and 2.0 micro g/g BW decreased it. The magnitude of the inhibition was higher in the resting phase than in the prespawning phase. The inhibitory effect of ovariectomy may be produced by elevating the apparent K(m) values (decreased affinity) of the enzyme for both L-tyrosine (substrate) and dimethyltetrahydropteridine (cofactor) and consequently decreasing the V(max). Significant changes (P<0.05) in both these parameters were noticed but showed minor differences with regard to the length of ovariectomy, season or brain regions. The biphasic effects of OE(2) replacement on TH activity seemed to be produced by differential effects on apparent K(m) and V(max). The stimulatory effect of the low dosages of OE(2) coincides with a decrease in the apparent K(m) values (increased affinity) for both substrate and cofactor and an increase in the V(max) of the enzyme. The inhibitory effect of the high dosages of OE(2) correlated with an increase in the apparent K(m) values (decreased affinity) for both substrate and cofactor, and a decrease in the V(max) compared with the lower dosage groups. The results strongly suggested that OE(2) can modulate brain catecholaminergic activity at the level of tyrosine hydroxylation which, in turn, may alter gonadotrophin secretion. OE(2) may elicit biphasic effects by differentially altering the enzyme affinity towards the substrate and cofactor.

Glucuronidation of odorant molecules in the rat olfactory system: activity, expression and age-linked modifications of UDP-glucuronosyltransferase isoforms, UGT1A6 and UGT2A1, and relation to mitral cell activity.

The aim of the present study was to examine the glucuronidation of a series of odorant molecules by homogenates prepared either with rat olfactory mucosa, olfactory bulb or brain. Most of the odorant molecules tested were efficiently conjugated by olfactory mucosa, whereas olfactory bulb and brain homogenates displayed lower activities and glucuronidated only a few molecules. Important age-related changes in glucuronidation efficiency were observed in olfactory mucosa and bulb. Therefore, we studied changes in expression of two UDP-glucuronosyltransferase isoforms, UGT1A6 and UGT2A1, in 1-day, 1- and 2-week-, 3-, 12- and 24-month-old rats. UGT1A6 was expressed at the same transcriptional level in the olfactory mucosa, bulb and brain, throughout the life period studied. UGT2A1 mRNA was expressed in both olfactory mucosa and olfactory bulb, in accordance with previous results [Mol. Brain Res. 90 (2001) 83], but UGT2A1 transcriptional level was 400-4000 times higher than that of UGT1A6. Moreover, age-dependent variations in UGT2A1 mRNA expression were observed. As it has been suggested that drug metabolizing enzymes could participate in olfactory function, mitral cell electrical activity was recorded during exposure to different odorant molecules in young, adult and old animals. Age-related changes in the amplitude of response after stimulation with several odorant molecules were observed, and the highest responses were obtained with molecules that were not efficiently glucuronidated by olfactory mucosa. In conclusion, the present work presents new evidence of the involvement of UGT activity in some steps of the olfactory process.

Impairment of adenylyl cyclase and of spatial memory function after microsphere embolism in rats.

The purpose of the present study was to characterize alterations in the adenylyl cyclase (AC), cyclic adenosine 3',5'-monophosphate (cAMP), and spatial memory function after sustained cerebral ischemia. Sustained cerebral ischemia was induced by injection of 900 microspheres (48 microm in diameter) into the right (ipsilateral) hemisphere of rats. Alterations in the AC and cAMP in the cerebral cortex and hippocampus were examined up to 7 days after the embolism. A decrease in the cAMP content was seen in the ipsilateral hemisphere throughout the experiment. Microsphere embolism (ME) decreased the activity of Ca(2+)/calmodulin (CaM)-sensitive AC in the ipsilateral hemisphere throughout the experiment, whereas the basal and 5'-guanylyl imidodiphosphate (Gpp(NH)p)-sensitive AC activities were not altered. Immunoblotting analysis of AC subtypes with specific antibodies showed a decrease in the immunoreactivity of AC-I in the ipsilateral hemisphere during these periods. No significant differences in the immunoreactivity of AC-V/VI and AC-VIII were observed after ME. The levels of GTP-binding proteins Galpha(s), Galpha(i), and Gbetawere unchanged. Furthermore, microsphere-embolized rats showed prolongation of the escape latency in the water maze task determined on the seventh to ninth day after the operation. These results suggest that sustained cerebral ischemia may induce the impairment of the AC, particularly a selective reduction in the AC-I level and activity, coupled with the decrease in cAMP content. This reduction may play an appreciable role in the disturbance in cAMP-mediated signal transduction system, possibly leading to learning and memory dysfunction.