Aging and sex differences in striatal dopaminergic function.

In this report the potassium- (30 mM) and amphetamine- (10 microM) stimulated responses of dopamine (DA) and 3,4-dihydroxy phenylacetic acid (DOPAC) from superfused striatal tissue of female and male mice as sampled at 2, 6, 18 and 24 months of age were compared. When assessed relative to responses obtained from 2-month-old female mice, potassium-stimulated DA output of female mice was significantly decreased at 18 months of age and significantly increased at 24 months of age. In male mice, the only statistically significant change was an increase in potassium-stimulated DA in the 24 versus 2-month-old mice. In response to amphetamine-stimulation, DA responses from striatal tissue of 18-month-old females were significantly decreased and that of 24-month-old mice significantly increased relative to that of the 2-month-old females. In the case of male mice, amphetamine-stimulated DA responses of 6- and 18-month-old mice were significantly decreased compared with responses observed in the 2-month-old males. In addition, amphetamine-stimulated DA responses of the 24-month-old females were significantly greater than the 24-month-old males. In general, the response profiles for DOPAC to potassium and amphetamine stimulation were similar to that of DA for male, but not female, mice. These results demonstrate that sex differences in striatal dopaminergic function are differentially affected by age. Overall, striatal DA responsiveness of female mice shows more extreme age-related changes, particularly between the 2- and 6-month versus the 18- and 24-month-old mice and a discord between DA and DOPAC responses. Such extreme changes may be related to the presence (at 2 and 6 months) versus absence (at 18 and 24 months) of estrous cycles/gonadal steroid hormonal functions in female mice.

Sex differences in K+-evoked striatal dopamine output from superfused striatal tissue fragments of reserpine-treated CD-1 mice.

Reserpine inhibits vesicular monoamine transporter-2 (VMAT-2) function and thereby impairs vesicular dopamine (DA) storage within nerve terminals. The present report compared the effects of reserpine treatment upon the striatal dopaminergic system in male and female mice as a means to assess potential sex differences in VMAT-2/DA storage function. After treatment with reserpine, male mice showed significantly greater striatal DA concentrations and K+ -evoked DA output from the striatum compared to females. By contrast, no statistically significant sex differences were observed in methamphetamine-evoked DA output in reserpine-treated mice. These results demonstrate a clear sex difference in the striatal dopaminergic responses to reserpine and suggest that females possess a more active VMAT-2/DA storage capacity, as indicated by the greater degree of deficits observed when VMAT-2/DA storage function is inhibited by reserpine. Such findings have important implications for understanding some of the bases for sex differences in neurotoxicity and neurodegeneration of the nigrostriatal dopaminergic system.

Age-related changes in nigrostriatal dopaminergic function are accentuated in +/- brain-derived neurotrophic factor mice.

The effects of a deletion for the brain derived neurotrophic factor (BDNF) allele (+/- BDNF) upon age-related changes in nigrostriatal dopaminergic (NSDA) function were assessed. Behavioral (beam crossing and spontaneous activity) and neurochemical (potassium-stimulated dopamine release from superfused striatum) measures were compared among Young (4-5 month), Middle (11-13 month) and Aged (19-21 month) +/- BDNF and their wild type littermate control (+/+ BDNF) mice. No statistically significant differences were obtained between +/+ and +/- BDNF mice at the Young age sampling period for any of the behavioral or neurochemical measures. Behavioral and neurochemical responses indices of NSDA function begin to diverge between +/+ and +/- Middle age BDNF mice and maximal differences were observed at the Aged period. For both movement and stereotypy times, scores obtained from +/+ mice were significantly decreased compared with +/- BDNF mice at the Aged period and center time scores of +/+ mice were decreased at both the Middle and Aged periods compared with +/- BDNF mice. Neurochemically, potassium-stimulated DA release of +/+ mice was significantly greater than +/- BDNF mice with maximal differences obtained at the Aged period. These results demonstrate marked differences in age-related changes of NSDA function between +/+ and +/- BDNF mice and suggest that the deletion of one allele for BDNF may make these mice more susceptible to age-related declines in NSDA function.

Interactive effects of tamoxifen and oestrogen upon the nigrostriatal dopaminergic system: long-term treatments.

In the present report adult female rats were ovariectomized (OVX) and assigned to one of four treatment conditions. Treatments consisted of administering pellets containing 17beta-oestradiol (E), tamoxifen (TMX), a combination of TMX and E or no further treatment (OVX). Animals received these treatments immediately following OVX and were maintained in these conditions for a 40-day period. Subsequently, the corpus striatum (CS) was dissected from each animal and prepared for determinations of basal and amphetamine stimulated DA output using in-vitro superfusion. No statistically significant differences among the four treatment groups were obtained for basal dopamine output. The highest levels of amphetamine-stimulated dopamine responses were obtained from E treated rats. These values were significantly greater than that obtained from OVX rats and rats treated with a combination of TMX+E. The significance of these findings is that they indicate both a non-traditional central nervous system site and mechanism of action through which tamoxifen-oestrogen interactions can function. Such data may have important implications for administration of tamoxifen to premenopausal women as this anti-oestrogen may compromise nigrostriatal dopaminergic function under conditions where oestrogenic modulation is present.

Tamoxifen diminishes methamphetamine-induced striatal dopamine depletion in intact female and male mice.

It has been demonstrated that the nigrostriatal dopaminergic system of male mice is more sensitive to the neurotoxic effects of methamphetamine (MA). The basis for this difference can be related to oestrogen, which has the capacity to function as a neuroprotectant against neurotoxins that target the nigrostriatal dopaminergic system. We examined the effects of the anti-oestrogen, tamoxifen (TMX), upon MA-induced neurotoxicity of the nigrostriatal dopaminergic system in intact female and male CD-1 mice. Striatal dopamine concentrations of TMX-treated female and male mice receiving MA were significantly greater than mice receiving MA alone. In female, but not male, mice, oestrogen treatment also resulted in greater striatal dopamine concentrations compared to mice receiving MA alone. Interestingly, male mice treated with oestrogen were particularly sensitive to the acute toxic effects of MA and displayed no evidence of nigrostriatal neuroprotection. The dihydroxyphenylacetic acid/dopamine ratios following MA for female and male mice treated with TMX or females treated with oestrogen were significantly reduced compared to MA-treated mice and oestrogen + MA-treated male mice. No differences among the treatment groups were obtained for dopamine in the hypothalamus or olfactory bulb. These data demonstrate that TMX treatment of intact female and male mice diminishes striatal dopamine depletions to the nigrostriatal dopaminergic neurotoxin, MA. Oestrogen also displayed this capacity when administered to female, but accentuated acute toxicity in male mice. These effects are relatively specific for the nigrostriatal dopaminergic system. Such data suggest that TMX can function as a nigrostriatal dopaminergic neuroprotectant against MA-induced neurotoxicity in intact female and male mice.

Tamoxifen alters dopamine output through direct actions upon superfused corpus striatal tissue fragments.

Tamoxifen (10 pg/ml) was infused directly into superfused striatal tissue fragments of ovariectomized rats for a 50 min period. Immediately following the termination of tamoxifen there was a significant increase in dopamine output compared with non-infused controls. No such significant increase was observed with use of a 100 pg/ml tamoxifen dose. Although dopamine output was again increased upon termination of a 2 h infusion of tamoxifen, these levels failed to differ significantly from that of non-infused controls. Similarly, a shorter 10 min duration infusion of tamoxifen failed to alter dopamine output. Finally, we examined whether the tamoxifen-induced, post-infusion increase in dopamine output, as observed following a 50 min infusion of 10 pg/ml, involved a calcium dependent process. To achieve this goal, superfusions were performed with Calcium/Tamoxifen, No Calcium/Tamoxifen, No Calcium/No Tamoxifen and Calcium/No Tamoxifen. A significant increase in dopamine output post-tamoxifen infusion was obtained for the Calcium/Tamoxifen condition compared with the remaining three groups which failed to differ from one another. Taken together these results show that tamoxifen can alter dopamine output through direct, non-genomic effects upon striatal neurons. Responses to this anti-estrogen are intriguing since they are apparent following removal, but not during tamoxifen infusion and represent a calcium-dependent process. These data suggest that tamoxifen may represent an important modulator of nigrostriatal dopaminergic function.

Neuroprotective role of estrogen upon methamphetamine and related neurotoxins within the nigrostriatal dopaminergic system.

In this report we describe some of the data on the capacity for estrogen to function as a neuroprotectant of the nigrostriatal dopaminergic (NSDA) system. The data show that estrogen (E) can alter two different response characteristics to NSDA neurotoxins. The first being that striatal DA concentrations of ovariectomized rodents treated with E are consistently greater than non-E-treated animals in response to neurotoxins which produce degeneration of the NSDA system. The second being that E significantly reduces the amount of DA output upon initial exposure to the NSDA neurotoxin, 1-methyl-4-phenylpyridium ion (MPP+). At present, it is not known whether these two response characteristics are related. An intriguing possibility is that the E-dependent changes in initial DA output are related to the resultant neurotoxicity (attenuations in DA concentration reductions). So far our incipient findings do not seem to support this eventuality. However, additional testing on this topic is required. The present data suggest that one of the mechanisms by which E can exert these effects is through inhibition of DAT activity. This conclusion results from data which show that E produces: 1) an inhibition of [3H]DA uptake, 2) a reduction in DA clearance rates, and 3) an effect upon DA recovery that is similar to that observed to the putative DA uptake blocker, nomifensine. The capacity and significance for steroid hormones to modulate neurotransmitter transporters has been recently reviewed.

A novel and innovative quantitative kinetic software for virological colorimetric assays.

This study addresses the limited range of quantification with colorimetric assays (ELISA) starting from the analysis of color production in a reference external curve. An automatic ELISA management software, designated Quanti-Kin Detection System (QKDS) is described, which retains the sensitivity of the end-point reading and extends the dynamic range up to five logarithms with mathematical interpretation of color production. The QKDS software is a generic system suitable for different types of ELISA with substrate incubation at room temperature, does not require dedicated instruments, performs accurate quantification (including assay quality control) and has a user friendly interface. Specific applications were developed for three types of analytes: antibodies, viral antigens and nucleic acids. Data are presented on three representative QKDS applications to HIV antibodies, p24 antigen and proviral DNA kits. The precision of quantification is strictly correlated with the precision of the kit; however, for almost all samples with known analyte amount, the error percentage was below 10%, only for two cases in quantification of HIV proviral DNA the error percentage was around 25%. The necessity for a wide quantification range has been demonstrated by measuring clinical samples, which showed a distribution in all possible quantification ranges for all kits.

Effects of estrogen upon dopamine release from the corpus striatum of young and aged female rats.

In vitro superfusion was used to examine the effects of estrogen administration upon striatal dopamine release in ovariectomized young and aged female Fischer 344 rats in response to 10 microM amphetamine or 30 mM potassium stimulation. Estrogen treatment increased basal dopamine and decreased DOPAC release in young and aged females (10 micrograms estradiol benzoate given subcutaneously 24 and 48 h prior to superfusion). Amphetamine-stimulated dopamine release was significantly decreased in aged estrogen-treated females, but did not differ in young females as a function of estrogen treatment. Conversely, young females treated with estrogen showed significantly decreased striatal dopamine release in response to potassium stimulation, while aged females showed no differences as a function of hormone treatment. Striatal dopamine content was significantly decreased in all estrogen-treated young and aged females. It appears that estrogen is altering dopamine uptake mechanisms in both age groups, since basal DOPAC release is decreased and dopamine is increased. This estrogen effect depletes the readily releasable dopamine storage pool to a greater extent in the aged female as evidenced by reduced amphetamine-stimulated dopamine release. By contrast, estrogen does not alter vesicular dopamine storage pools in aged females, which are mobilized by potassium. These results may have important implications regarding sex differences in expression and treatment of age-related movement disorders.

Tamoxifen treatment of ovariectomized mice alters dopamine release from striatal tissue fragments superfused in vitro.

In this report we examined the effect of tamoxifen upon the nigrostriatal dopaminergic system. Ovariectomized mice were subjected to one of the following treatments: two subcutaneous injections administered on successive days of the sesame oil vehicle (control), estradiol benzoate (EB-10 micrograms), tamoxifen citrate (TMX 125 micrograms) or a combination of EB+TMX. At 24 h after the second injection, the caudate nucleus was superfused in vitro to evaluate the effects of these treatments upon basal as well as potassium stimulated (30 mM) dopamine release rates. In addition, uteri were weighed from each animal. Basal and total fractional dopamine release rates from the caudate nucleus of control mice were significantly lower than those of the other three treatments, which failed to differ among each other. Potassium minus (-) basal stimulated dopamine release rates failed to differ significantly among the four treatment conditions. Uterine weights of the TMX treated mice were significantly greater than controls, but significantly lower than EB and EB+TMX animals. These data show that TMX can significantly increase caudate nucleus dopamine release to levels observed in EB treated mice. These agonistic effects of TMX upon nigrostriatal dopaminergic function can be contrasted with its relatively weak estrogenic effects upon uterine weights and indicate the discriminatory, system specific effects that can be exerted by this anti-estrogen. This demonstration of TMX's ability to modulate central nervous system function is of particular relevance in light of pending clinical trials for the prophylactic use of TMX in the treatment of women for breast cancer.

Tamoxifen eliminates estrogen's neuroprotective effect upon MPTP-induced neurotoxicity of the nigrostriatal dopaminergic system.

The capacity for 17-alpha and 17-Beta estradiol to modulate MPTP-induced neurotoxicity of the nigrostriatal dopaminergic system and potential antagonism of this modulation by the anti-estrogen, tamoxifen, were evaluated. Treatment of retired breeder ovariectomized C57/Bl mice with 17-Beta estradiol diminished the amount of striatal dopamine reduction resulting from MPTP treatment with striatal dopamine concentrations of these 17-Beta estradiol treated mice failing to differ significantly from vehicle treated controls. A combined administration of 17-Beta estradiol with tamoxifen abolished this neuroprotective effect of estrogen as striatal dopamine concentrations of this group were significantly lower than vehicle treated controls. Results to 17-alpha estradiol were less effective since striatal dopamine concentrations of these mice following MPTP treatment were significantly decreased as compared with vehicle controls. In contrast to the nigrostriatal dopaminergic system, no statistically significant effects of these treatments were observed upon olfactory bulb dopamine concentrations. Taken together, these results show that 17-Beta, but not an equivalent concentration of 17-alpha estradiol was effective in decreasing striatal dopamine neurotoxicity to MPTP. This effect of 17-Beta estradiol was abolished by tamoxifen. These data have important implications regarding the mechanisms of estrogen-tamoxifen interactions within the nigrostriatal dopaminergic system as well as for clinical applications of tamoxifen within pre-menopausal women.

Growth and penetration of Salmonella enteritidis, Salmonella heidelberg and Salmonella typhimurium in eggs.

Eggs and egg dishes are important vehicles for Salmonella infections. Salmonella enteritidis, Salmonella typhimurium and Salmonella heidelberg, which can be isolated from chicken ovaries and feces, have been implicated in approximately 50% of the foodborne salmonellosis outbreaks in the United States. In this study, the growth of these three organisms, inoculated into yolks and albumen, was compared at 4, 10 and 25 degrees C. Regardless of whether 10(2) cfu/g or 10(4) cfu/g was inoculated into the yolk or albumen, populations of all strains increased 3 logs or more in number in one day when incubated at 25 degrees C. Maximum numbers of Salmonella ranged from 10(8) to 10(10) cfu/g. All strains grew at 10 degrees C, but peak numbers were lower and occurred later than those at 25 degrees C. Populations of the three Salmonella strains inoculated into eggs stored at 4 degrees C grew sporadically; in some test groups populations declined. The potential for Salmonella in contaminated feces to establish in the interior of eggs was examined by monitoring shell penetration. At 25 degrees C, all three Salmonella strains penetrated the shell in 3 days, but at 4 degrees C, only S. typhimurium was found in one membrane sample. When hatchery conditions were simulated by incubating eggs at 35 degrees C for 30 min followed by storage at 4 degrees C, penetration was enhanced. Penetration was observed by day 1-3 when eggs were exposed to 10(4) cfu Salmonella/g feces. Increasing the inoculum to 10(6) cfu/g feces resulted in 50-75% of the contents of eggs to be contaminated by day 1. All Salmonella-positive samples were detected by enrichment. Results of this study indicate that S. enteritidis, S. typhimurium, or S. heidelberg present in feces can penetrate to the interior of eggs and grow during storage.

Estrogen as a neuroprotectant against MPTP-induced neurotoxicity in C57/B1 mice.

Castrated retired breeder male and female mice were treated or not with a 17 beta-estradiol pellet. At 10 days postcastration +/- estrogen treatment all animals were treated with MPTP. Five days later, concentrations of dopamine were determined from the corpus striatum and olfactory tubercle. Both castrated male and female mice treated with estrogen had significantly greater concentrations of dopamine within the corpus striatum compared with their respective gender controls, which did not receive estrogen. By contrast, no statistically significant differences in olfactory tubercle dopamine concentrations were obtained. Overall concentrations of dopamine within the corpus striatum, but not olfactory tubercle, were substantially greater in female vs. male mice. These data demonstrate that treatment with estrogen prevents reductions in corpus striatal dopamine concentrations in castrated mice treated with MPTP. Interestingly, this effect of estrogen was observed in both male and female mice. These results suggest that estrogen may serve as a neuroprotectant against an agent that is toxic to the nigrostriatal dopaminergic system in both male and female animal models of Parkinsonism.

Suppression of reproductive maturation in male-stimulated virgin female microtus by a female urinary chemosignal.

Urine from female Microtus ochrogaster possesses a chemosignal that suppresses reproductive maturation in other females. Uterine enlargement in virgin females stimulated by a male was suppressed by subsequent association with another female or by application of female urine on the nose. Females so suppressed are not able to achieve estrus. Urine from virgin sibling and non-sibling females and from pregnant females possesses the suppressing effect.

ArchAIDS connects a clinical department and a virological laboratory to automatize the follow up of AIDS patients.

A communication system for the automation of the follow up of AIDS patients set up by DIST at the Molecular Virology Unit in the Advanced Biotechnology Centre of Genova and at the Department of Internal Medicine of the Medical School of Genova is presented. This system includes a distributed database to store both clinical and virological data and a set of procedures to transfer patient data with a complete respect of requirements about completeness and privacy.

Communication and education. Developing the role of the diabetes specialist nurse.

1. Specialist nurses should not de-skill their generalist colleagues but should provide education and support to enhance their skills. 2. General nurses are not equipped to educate patients with diabetes, and require education and support. 3. Ward-based key workers and a referral system are transferable to other hospitals and to the management of other chronic diseases.

Urinary JCV-DNA testing during natalizumab treatment may increase accuracy of PML risk stratification.

The risk of progressive multifocal leukoencephalopathy (PML) in patients treated with natalizumab for multiple sclerosis (MS) is a serious concern. The presence of anti-JC virus antibodies is a risk factor for PML development, but 2.5 % of the patients result falsely-negative, while the prognostic relevance of testing JCV-DNA in biological fluids of treated patients is debated. Aim of this work was to evaluate the utility of testing JCV-DNA, together with anti-JCV antibodies, in biological samples of treated patients as a tool for PML risk stratification. 126 subjects from 5 MS Centers in Italy were included in the study. We performed a cross-sectional study in 63 patients testing JCV-DNA in blood, peripheral blood cells and urine. We longitudinally assessed the presence of JCV-DNA in a cohort of 33 subjects, one of which developed PML. We could test retrospectively serum samples from another PML case occurred during natalizumab therapy. Anti-JCV antibodies and urinary JCV-DNA were both tested in 73 patients. No changes in JCV-DNA status occurred during natalizumab treatment. The subject who developed PML in the longitudinal cohort had detectable JCV-DNA in urine at all time-points while serum or blood from both PML patients were always negative before the onset of disease and, in one case, after. Four subjects with JCV-DNA in urine and undetectable anti-JCV antibodies were retested for anti-JCV antibodies and three out of four resulted positive. In conclusion, testing JCV-DNA in urine is complementary to testing anti-JCV antibodies in identifying patients at risk of PML.

Markers associated with sex differences in methamphetamine-induced striatal dopamine neurotoxicity.

Three different approaches were employed to assess various markers associated with sex differences in responses to methamphetamine (MA). Bioassay measures reveal that MA treatment results in significantly greater reductions in body weight and increases in body temperature in male mice. Protein and mRNA determinations show significant increases in Bcl-2 and PAI-1 in male mice, while females show significant increases in GFAP and decreases in IGF-1R following treatment with MA. In mice with a heterozygous mutation of their dopamine transporter (+/- DAT), only female mice show significant differences in dopamine transporter binding and mRNA and associated reductions in striatal dopamine content along with increases in MA-evoked striatal dopamine output. The identification of these sex-dependent differences in markers provides a foundation for more exhaustive evaluation of their impact upon, and treatment of, disorders/neurotoxicity of the nigrostriatal dopaminergic system and the bases for the differences that exist between females and males.

Relationships among gender, age, time, and temperature in methamphetamine-induced striatal dopaminergic neurotoxicity.

A neurotoxic regimen of methamphetamine (MA-40 mg/kg ip) administered at 0 (control-MA vehicle), 0.5 and 72 h prior to determinations of striatal dopamine (DA) and DOPAC (3,4-dihydroxyphenylacetic acid)/DA ratios were compared among juvenile and adult female and male mice. Adult females and males showed similar depletions in striatal DA at 0.5 h post-MA, but males showed greater DA depletions and DOPAC/DA ratios at 72 h post-MA. Juvenile mice showed neither sex differences, nor any MA neurotoxicity upon striatal DA or DOPAC/DA ratios. Following MA, body temperatures increased in all mice, but increases in adult males were greater than adult females; juveniles showed no sex differences and body temperature increases were similar to that of adult males. MA-evoked DA output was greater in adult compared to juvenile males and a biologically effective regimen of testosterone to juvenile males neither increased MA-evoked DA output nor decreased MA-induced striatal DA like that observed in adult males. These results demonstrate: (1) Unlike adults, juvenile mice show neither a sex difference for MA-induced neurotoxicity or body temperature increases, nor MA neurotoxicity, (2) Initial effects of MA (0.5 h) in adult females and males are similar, but at 72 h post-MA females show no further striatal DA depletion, (3) Increased striatal DA depletion within adult versus juvenile males may be related to initially higher MA-evoked DA responses, and (4) Testosterone fails to convert juvenile males into adults with regard to MA effects.