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.

Sex differences in dopamine- and vesicular monoamine-transporter functions.

Men and women differ with regard to their use of, and responses to, methamphetamine (MA). Analogous sex differences with regard to MA are observed in animal models. In this report, data from a series of experiments that focus upon dopamine transporter (DAT) and vesicular monoamine transporter2 (VMAT2) function are reviewed by way of providing some understanding for these sex differences to MA. The amount of dopamine (DA) recovered after infusion of DA into superfused striatal tissue was greater in females and an accentuated amount of extracellular DA was obtained from females after infusion of the DAT-blocking drug, nomifensine. These data suggest a higher level of DAT activity in females. To evaluate the implications of this sex difference in DAT function as related to MA, the amount of DA evoked by an infusion of MA into superfused striatal tissue was tested and found to be significantly greater in males. In contrast, potassium chloride-stimulated DA release was greater in females. The results of these DA-evoked experiments imply that the greater DAT activity of females, by itself, cannot explain the sex differences observed with MA, and our attention was then directed to the VMAT2. Administration of the VMAT2 blocker, reserpine, in vivo resulted in a significantly greater amount of striatal DA depletion within female mice and infusion of reserpine in vitro into striatal tissue produced significantly greater levels of extracellular DA in females. The data of these reserpine experiments suggest that females possess a more active/efficient VMAT2 function. Collectively, the data provide evidence for sex differences in both DAT and VMAT2 functioning, and we propose that the interaction of these two transporter systems contributes to the differences in response to MA between males and females.

Differences in reserpine-induced striatal dopamine output and content between female and male mice: implications for sex differences in vesicular monoamine transporter 2 function.

In this report a series of six in vitro experiments in which reserpine-evoked dopamine output and two in vivo experiments in which the effects of reserpine injections upon dopamine content from striatal tissue of female and male mice were performed as a means to assess possible sex differences in vesicular monoamine transporter 2 (VMAT2) function. Significantly greater amounts of dopamine were obtained from striatal tissue of female mice in response to either a brief (experiment 1) or continuous (experiment 2) infusion of reserpine. Similarly, reserpine-evoked dopamine output from striatal tissue of gonadectomized females was significantly greater that that of gonadectomized males (experiment 3). When reserpine-evoked dopamine responses were compared directly between intact versus gonadectomized females (experiment 4) or males (experiment 5) no statistically significant differences were obtained. Finally, comparisons of gonadectomized females treated or not with estrogen revealed no statistically significant differences in reserpine-evoked dopamine output (experiment 6). Injections of reserpine produced significantly greater depletions of striatal dopamine content within intact female versus male mice (experiment 7). Dopamine contents of gonadectomized females treated or not with estrogen did not differ following treatment with reserpine, but were significantly greater than that of gonadectomized males (experiment 8). Taken together, these results show that female striatal tissue is more responsive to reserpine-evoked dopamine output, and this sex difference appears to be estrogen independent. Similarly, the dopamine depleting effects of reserpine are greater in intact female mice, however, gonadectomy reverses this effect in an estrogen independent manner. The data suggest that female mice may have a greater amount/activity of VMAT2 function as revealed by the increased responsiveness to the VMAT2 blocking drug, reserpine. Such differences in VMAT2 function may be related to the gender differences observed in conditions like Parkinson's disease and drug addiction.

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.

Effects of neonatal and prepubertal hormonal manipulations upon estrogen neuroprotection of the nigrostriatal dopaminergic system within female and male mice.

Estrogen (E) can function as a neuroprotectant of the nigrostriatal dopaminergic (NSDA) system against methamphetamine (MA) neurotoxicity in female, but not male, mice. In the present report we examined whether the organizational effects of gonadal steroid hormones, as exerted in the early postnatal period, or developmental effects, as exerted during the pubertal period, would contribute to this sexually dimorphic neuroprotectant action of E. Neonatal gonadectomy and treatment with testosterone of female mice, retained the ability to show an E neuroprotectant response when tested as adults. However, females not treated with gonadal steroids failed to show an E-dependent neuroprotectant response. Neonatal gonadectomy of male mice, failed to result in the display of an E neuroprotectant response when tested as adults. Prepubertal gonadectomy of female mice, with or without testosterone treatment, abolished the capacity for E to produce neuroprotection against MA-induced NSDA neurotoxicity. Nor did prepubertal gonadectomy enable male mice to show an E neuroprotectant response. Taken together these results demonstrate that none of the manipulations performed within male mice enabled them to show an E-dependent neuroprotective response against MA-induced neurotoxicity of the NSDA system when tested as adults. For the female, it appears that the presences of gonadal steroids at these two developmental periods are needed for the display of an E-dependent neuroprotectant response within the adult.

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.

Acute effects of estrogen upon methamphetamine induced neurotoxicity of the nigrostriatal dopaminergic system.

Estrogen acts as a neuroprotectant of the nigrostriatal dopaminergic system when given chronically to female mice prior to Methamphetamine (MA) insult. In this report, we tested the acute effects of Estradiol Benzoate (EB-10 microg in Oil) in ovariectomized CD-1 mice to function as a neuroprotectant when administered prior to (Experiment 1) or after (Experiment 2) MA treatment. Striatal dopamine (DA) concentrations and DOPAC/DA ratios were measured to assess the neuroprotective effects of EB. In Experiment 1, we observed that EB exerted a neuroprotective effect upon striatal dopamine concentrations when administered at 24 and 12, but not at 0.5, hours prior to MA injection and upon DOPAC/DA ratios when administered at 24, 12 and 0.5 hours prior to MA. In Experiment 2, no evidence for estrogen to protect the striatum from MA insult was obtained when EB was administered at 15, 30, 60 or 120 minutes after MA. These results show that EB can act as a modulator of MA-induced nigrostriatal dopaminergic neurotoxicity suggestive of a neuroprotectant, when administered within 0.5 hour of MA insult as assessed by measures of DOPAC/DA, but fails to prevent depletion of DA when given after MA insult. The data suggest that estrogen may exert this rapid neuroprotective effect through a non-genomic mechanism.

Estrogen, but not testosterone, attenuates methamphetamine-evoked dopamine output from superfused striatal tissue of female and male mice.

The gonadal steroid hormone, estrogen, has the capacity to function as a neuroprotectant against methamphetamine (MA)-induced neurotoxicity of the nigrostriatal dopaminergic system within female, but not male, mice. In an attempt to understand some of the bases for this effect of estrogen, the incipient effects of MA upon evoked dopamine output from superfused striatal tissue fragments of gonadectomized female and gonadectomized as well as intact male mice were evaluated under conditions where estrogen (or testosterone) was present in the medium. The amount of dopamine evoked by MA was significantly reduced when estrogen was co-infused with MA. This attenuation was obtained with striatal tissue fragments of gonadectomized female and gonadectomized and intact male mice. In contrast to estrogen, co-infusion of testosterone failed to produce an overall statistically significant change in MA-evoked dopamine output within superfused striatal tissue fragments of gonadectomized female and male mice. In this way, the gonadal steroid hormones, estrogen and testosterone, exert differential modulatory effects upon MA-evoked dopamine output from superfused striatal tissue fragments. However, similar effects to these gonadal steroid hormones were observed between gonadectomized female and gonadectomized or intact male mice. These data reveal an absence of a sexual dimorphism in striatal responsiveness with regard to estrogen's ability to alter MA-evoked DA output. Accordingly, the sexually dimorphic capacity for estrogen to function as a neuroprotectant may involve a composite of actions upon the nigrostriatal dopaminergic system involving events/sites other than the initial stimulation of dopamine output.

Methamphetamine produces subsequent reductions in running time to exhaustion in mice.

Treatment with methamphetamine (MA - 20 mg/kg x 4 i.p. at 2-h intervals) or its vehicle at 7 days prior to tests of running time to exhaustion were evaluated in 60-day-old CD-1 male mice. Pre-treatment running times were not significantly different between the two groups. MA resulted in significantly decreased running times to exhaustion compared to vehicle-treated controls and a significant reduction in corpus striatal dopamine and DOPAC but not norepinephrine. No statistically significant differences in catecholamines were obtained within the hypothalamus, or left ventricle of the heart, nor were body weights significantly different between these groups. The data show that a regimen of MA, which results in an approximate 90% depletion of striatal DA, produces a reduction in running time to exhaustion.

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.

Nisoxetine infusion into the olfactory bulb enhances the capacity for male rats to identify conspecifics.

In the present report, the norepinephrine uptake inhibitor nisoxetine as well as a cocktail of nisoxetine and the alpha-adrenergic receptor antagonist phentolamine were infused unilaterally into the olfactory bulb during microdialysis to assess their effects upon the capacity of male rats to identify conspecifics. A social discrimination test was conducted while simultaneously measuring olfactory bulb norepinephrine output in the dialysate before, during, and after behavioral testing. Nisoxetine significantly increased norepinephrine levels in the olfactory bulb compared with the Ringer's solution control group. Following such increases in olfactory bulb norepinephrine, identification responses were enhanced compared with that observed in the Ringer's control. In the presence of phentolamine, nisoxetine elevated olfactory bulb norepinephrine to levels similar to that obtained in the nisoxetine alone group, however, investigatory responses directed to the conspecifics indicated an absence of identification capacity similar to that observed in the Ringer's control group. These results reveal a direct link between norepinephrine transmission in the olfactory bulb and enhanced identification via its activation of postsynaptic alpha-adrenergic receptors. These results also show that inhibition of norepinephrine uptake may represent an important mechanism involved with the enhancement of social identification and suggest a possible novel effect for the antidepressant nisoxetine.

Evaluation of nigrostriatal dopaminergic function in adult +/+ and +/- BDNF mutant mice.

Deletion of a single copy of the BDNF gene has been shown to affect the nigrostriatal dopaminergic system of young adult BDNF mice. In the present report we evaluated various indices of nigrostriatal dopaminergic function between 9-month-old wild-type (+/+) and heterozygous (+/-) BDNF mutant mice. Performance in a sensorimotor beam walking task was significantly decreased in +/- mice as indicated by increased times required to traverse both a wide (21 mm) and narrow (6 mm) beam. No differences in spontaneous locomotor behavior were observed between the +/+ and +/- mice. Amphetamine-stimulated (5 mg/kg) locomotor behavior was increased to a greater degree in the +/- mice, with the number of movements performed by these mice being significantly greater than their +/+ controls. Corpus striatal dopamine concentrations were significantly greater in the +/- BDNF mice. The absence of any significant differences for dopamine concentrations within the hypothalamus and olfactory bulb of these mice, as well as an absence of any difference in striatal norepinephrine concentrations, suggested a relative specificity of these effects to the corpus striatum. Both the +/- and +/+ mice showed similar reductions in striatal dopamine concentrations in response to a neurotoxic regimen of methamphetamine (20 mg/kg). Collectively these data show increased levels of striatal dopamine concentrations associated with altered behavioral responses involving the nigrostriatal dopaminergic system within the heterozygous BDNF mutant mice.

Tamoxifen abolishes estrogen's neuroprotective effect upon methamphetamine neurotoxicity of the nigrostriatal dopaminergic system.

The effects of 17beta-estradiol and the anti-estrogen, tamoxifen, on methamphetamine-induced neurotoxicity of the nigrostriatal dopaminergic system were examined in ovariectomized CD-1 mice. In Experiment 1, striatal dopamine concentrations from estrogen treated mice were significantly greater than that from non-estrogen treated mice following methamphetamine. Dopamine concentrations from estrogen+tamoxifen+methamphetamine treated mice were decreased compared to estrogen+methamphetamine treated mice and not significantly different from those of tamoxifen+methamphetamine treated mice or mice receiving methamphetamine alone. These results suggest that estrogen is functioning as a neuroprotectant of methamphetamine-induced nigrostriatal dopaminergic neurotoxicity and that this neuroprotective effect of estrogen is abolished in the presence of tamoxifen. In Experiment 2, estrogen administration after methamphetamine treatment did not produce any significant changes in dopamine concentrations compared with methamphetamine treatment alone. The data from Experiment 2 show that estrogen cannot reverse the methamphetamine-induced neurotoxicity upon the nigrostriatal dopaminergic system. Similar results were observed for the potassium-stimulated dopamine outputs from these treatment conditions as evaluated with in vitro superfusion, although a difference between the two measures for the estrogen+methamphetamine treated group was obtained in Experiment 1. These results have important implications for estrogen-tamoxifen interactions upon the nigrostriatal dopaminergic system and the gender differences which are observed in Parkinson's disease and animal models of nigrostriatal dopaminergic neurotoxicity as well as for the proposed use of tamoxifen in pre-menopausal women at risk for breast cancer.

The effect of testosterone upon methamphetamine neurotoxicity of the nigrostriatal dopaminergic system.

The gonadal steroid hormone estrogen (E) can function as a neuroprotectant of nigrostriatal dopaminergic (NSDA) neurotoxicity, however, there exists very limited information on the role of testosterone (T) in this capacity. In the present report, the effects of T on methamphetamine (MA) induced neurotoxicity of the NSDA system were examined in gonadectomized female and male CD-1 mice. In Experiment 1, striatal dopamine (DA) concentrations and output from T-treated ovariectomized mice were not significantly different from that of non-T-treated mice following MA. These results suggest that T is not functioning as a modulator of MA-induced NSDA neurotoxicity in ovariectomized CD-1 mice. In Experiment 2, there were no significant differences in DA concentrations or output among T-treated, non-T-treated as well as E-treated orchidectomized mice following MA. The results of Experiment 2 indicate that the neuroprotective effect of E reported within ovariectomized mice is not seen in male mice. Nor does T appear to function as a modulator of MA neurotoxicity in male mice. These effects of T and E upon the MA induced neurotoxicity of the NSDA system have important implications for the gender differences which are observed in animal models of NSDA neurotoxicity and in Parkinson's disease.

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.

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.

The effect of GDNF on nigrostriatal dopaminergic function in response to a two-pulse K(+) stimulation.

We examined the effect of glial cell line-derived neurotrophic factor (GDNF) upon nigrostriatal dopaminergic function in response to two-pulse potassium (K(+)) stimulation in rats under in vivo microdialysis conditions. The two-pulse infusion protocol permits us to focus upon the role of this neurotrophin as related to vesicular storage and release of dopamine (DA). The effects of two 20-min infusions of K(+) (70 mM) upon DA and dihydroxyphenylacetic acid (DOPAC) output from the striatum of rats which received a single ipsilateral intrastriatal injection of vehicle, 1 microgram GDNF, or 10 microgram GDNF 1 week before microdialysis were examined. In the 10 microgram GDNF-treated rats, there was a significant increase in the peak DA output in response to the second versus first K(+) infusion, a significant increase in both basal and overall K(+)-evoked DOPAC responses, and significantly increased striatal DA and DOPAC contents compared with vehicle- or 1 microgram GDNF-treated rats. These data demonstrate that two-pulse K(+) stimulation is a useful means to detect significant mechanistic changes in DA neurons resulting from GDNF treatment. These results suggest the possibility for GDNF to modulate vesicular pools of DA for release.

Inactivation of one copy of the mouse neurotrophin-3 gene induces cardiac sympathetic deficits.

Whether two copies of the neurotrophin-3 (NT3) gene are necessary for proper development of cardiac sympathetic innervation was investigated in mice carrying a targeted inactivation of the NT3 gene. Heterozygous (+/-) and null (-/-) mutant mice had fewer stellate ganglion neurons than did wild-type (+/+) mice at postnatal day 0 (P0 or birth), and this deficit was maintained between adult (P60) +/- and +/+ mice. The sympathetic innervation of the heart matured postnatally in +/+ and +/- mice. Tyrosine hydroxylase (TH)-positive axons were restricted largely to the epicardium at P0, were concentrated around large blood vessels in the myocardium at P21, and were present among cardiac myocytes at P60. Cardiac norepinephrine (NE) concentrations paralleled the growth of the sympathetic axons into the heart. NE concentrations were equivalent among +/+, +/-, and -/- mice at birth, but differences between +/- and +/+ mice increased with age. Adult +/- mice also exhibited lower resting heart rates and sympathetic tonus than +/+ mice. Thus deletion of one copy of the NT3 gene translates into anatomical, biochemical, and functional deficits in cardiac sympathetic innervation of postnatal mice, thereby indicating a gene-dosage effect for the NT3 gene.