Methodology and effects of repeated intranasal delivery of DNSP-11 in awake Rhesus macaques.

BACKGROUND: To determine if the intranasal delivery of neuroactive compounds is a viable, long-term treatment strategy for progressive, chronic neurodegenerative disorders, such as Parkinson's disease (PD), intranasal methodologies in preclinical models comparable to humans are needed. NEW METHOD: We developed a methodology to evaluate the repeated intranasal delivery of neuroactive compounds on the non-human primate (NHP) brain, without the need for sedation. We evaluated the effects of the neuroactive peptide, DNSP-11 following repeated intranasal delivery and dose-escalation over the course of 10-weeks in Rhesus macaques. This approach allowed us to examine striatal target engagement, safety and tolerability, and brain distribution following a single 125I-labeled DNSP-11 dose. RESULTS: Our initial data support that repeated intranasal delivery and dose-escalation of DNSP-11 resulted in bilateral, striatal target engagement based on neurochemical changes in dopamine (DA) metabolites-without observable, adverse behavioral effects or weight loss in NHPs. Furthermore, a 125I-labeled DNSP-11 study illustrates diffuse rostral to caudal distribution in the brain including the striatum-our target region of interest. COMPARISON WITH EXISTING METHODS: The results of this study are compared to our experiments in normal and 6-OHDA lesioned rats, where DNSP-11 was repeatedly delivered intranasally using a micropipette with animals under light sedation. CONCLUSIONS: The results from this proof-of-concept study support the utility of our repeated intranasal dosing methodology in awake Rhesus macaques, to evaluate the effects of neuroactive compounds on the NHP brain. Additionally, results indicate that DNSP-11 can be safely and effectively delivered intranasally in MPTP-treated NHPs, while engaging the DA system.

Age and lesion-induced increases of GDNF transgene expression in brain following intracerebral injections of DNA nanoparticles.

In previous studies that used compacted DNA nanoparticles (DNP) to transfect cells in the brain, we observed higher transgene expression in the denervated striatum when compared to transgene expression in the intact striatum. We also observed that long-term transgene expression occurred in astrocytes as well as neurons. Based on these findings, we hypothesized that the higher transgene expression observed in the denervated striatum may be a function of increased gliosis. Several aging studies have also reported an increase of gliosis as a function of normal aging. In this study we used DNPs that encoded for human glial cell line-derived neurotrophic factor (hGDNF) and either a non-specific human polyubiquitin C (UbC) or an astrocyte-specific human glial fibrillary acidic protein (GFAP) promoter. The DNPs were injected intracerebrally into the denervated or intact striatum of young, middle-aged or aged rats, and glial cell line-derived neurotrophic factor (GDNF) transgene expression was subsequently quantified in brain tissue samples. The results of our studies confirmed our earlier finding that transgene expression was higher in the denervated striatum when compared to intact striatum for DNPs incorporating either promoter. In addition, we observed significantly higher transgene expression in the denervated striatum of old rats when compared to young rats following injections of both types of DNPs. Stereological analysis of GFAP+ cells in the striatum confirmed an increase of GFAP+ cells in the denervated striatum when compared to the intact striatum and also an age-related increase; importantly, increases in GFAP+ cells closely matched the increases in GDNF transgene levels. Thus neurodegeneration and aging may lay a foundation that is actually beneficial for this particular type of gene therapy while other gene therapy techniques that target neurons are actually targeting cells that are decreasing as the disease progresses.

Oxidative stress and dopamine depletion in an intrastriatal 6-hydroxydopamine model of Parkinson's disease.

Although the etiology of Parkinson's disease (PD) is unknown, a common element of most theories is the involvement of oxidative stress, either as a cause or effect of the disease. There have been relatively few studies that have characterized oxidative stress in animal models of PD. In the present study a 6-hydroxydopamine (6-OHDA) rodent model of PD was used to investigate the in vivo production of oxidative stress after administration of the neurotoxin. 6-OHDA was injected into the striatum of young adult rats and the production of protein carbonyls and 4-hydroxynonenal (HNE) was measured at 1, 3, 7, and 14 days after administration. A significant increase in both markers was found in the striatum 1 day after neurotoxin administration, and this increase declined to basal levels by day 7. There was no significant increase found in the substantia nigra at any of the time points investigated. This same lesion paradigm produced dopamine depletions of 90-95% in the striatum and 63-80% in the substantia nigra by 14-28 days post-6-OHDA. Protein carbonyl and HNE levels were also measured in middle-aged and aged animals 1 day after striatal 6-OHDA. Both protein carbonyl and HNE levels were increased in the striatum of middle-aged and aged animals treated with 6-OHDA, but the increases were not as great as those observed in the young adult animals. Similar to the young animals, there were no increases in either marker in the substantia nigra of the middle-aged and aged animals. There was a trend for an age-dependent increase in basal amounts of oxidative stress markers when comparing the non-lesioned side of the brains of the three age groups. These results support that an early event in the course of dopamine depletion following intrastriatal 6-OHDA administration is the generation of oxidative stress.

Methamphetamine and human immunodeficiency virus protein Tat synergize to destroy dopaminergic terminals in the rat striatum.

Dysfunction of the dopaminergic system accompanied by loss of dopamine in the striatum is a major feature of human immunodeficiency virus-1-associated dementia. Previous studies have shown that human immunodeficiency virus-1-associated dementia patients with a history of drug abuse have rapid neurological progression, prominent psychomotor slowing, more severe encephalitis and more severe dendritic and neuronal damage in the frontal cortex compared with human immunodeficiency virus-1-associated dementia patients without a history of drug abuse. In a previous study, we showed that methamphetamine and human immunodeficiency virus-1 protein Tat interact to produce a synergistic decline in dopamine levels in the rat striatum. The present study was carried out to understand the underlying cause for the loss of dopamine. Male Sprague-Dawley rats were administered saline, methamphetamine, Tat or Tat followed by methamphetamine 24 h later. Two and seven days later the animals were killed and tissue sections from striatum were processed for silver staining to examine terminal degeneration while sections from striatum and substantia nigra were processed for tyrosine hydroxylase immunoreactivity. Striatal tissue was also analyzed by Western blotting for tyrosine hydroxylase protein levels. Compared with controls, methamphetamine+Tat-treated animals showed extensive silver staining and loss of tyrosine hydroxylase immunoreactivity and protein levels in the ipsilateral striatum. There was no apparent loss of tyrosine hydroxylase in the substantia nigra. Markers for oxidative stress were significantly increased in striatal synaptosomes from Tat+methamphetamine group compared with controls. The results indicate that methamphetamine and Tat interact to produce an enhanced injury to dopaminergic nerve terminals in the striatum with sparing of the substantia nigra by a mechanism involving oxidative stress. These findings suggest a possible mode of interaction between methamphetamine and human immunodeficiency virus-1 infection to produce enhanced dopaminergic neurotoxicity in human immunodeficiency virus-1 infected/methamphetamine-abusing patients.

The effect of neurotoxic doses of methamphetamine on methamphetamine-conditioned place preference in rats.

RATIONALE: Methamphetamine has been shown to produce neurotoxicity demonstrated by depletions of dopamine and serotonin in the striatum and nucleus accumbens. OBJECTIVE: The current study examined the effects of neurotoxic doses of methamphetamine on the rewarding effect of subsequent administration of methamphetamine assessed by the conditioned place preference (CPP) procedure. METHODS: Male and female rats were treated with a neurotoxic regimen of methamphetamine (4 x 10 mg/kg, s.c., once every 2 h) or saline, and concentrations of dopamine, 3,4-dihydroxyphenylacetic acid, serotonin, and 5-hydroxyindoleacetic acid were measured 15 days later in the striatum, nucleus accumbens, and prefrontal cortex (PFC). In another experiment, male rats were given methamphetamine neurotoxic treatment or saline and were then conditioned 7 days later with methamphetamine (0.1, 0.3, or 1.0 mg/kg, s.c.) or saline using a four-trial CPP procedure. Locomotor activity was also measured during the conditioning sessions to investigate whether or not the neurotoxic methamphetamine treatment altered locomotor activity following a subsequent methamphetamine challenge. RESULTS: Males and females did not differ significantly in the amount of neurochemical depletion produced by methamphetamine in any brain region. Collapsed across sex, dopamine was significantly depleted in nucleus accumbens (25%) and striatum (51%); serotonin was significantly depleted in nucleus accumbens (35%), striatum (34%) and PFC (33%). The methamphetamine challenge dose dependently increased locomotor activity, but the increase was not affected by treatment with neurotoxic doses of methamphetamine. In contrast, treatment with neurotoxic doses of methamphetamine enhanced CPP at the intermediate conditioning dose (0.3 mg/kg). CONCLUSIONS: These results indicate that the rewarding effect of methamphetamine is enhanced by prior treatment with neurotoxic doses of methamphetamine, suggesting either a compensatory hyperfunctioning of spared dopamine neurons or a loss of inhibitory control from serotonergic input.

Neuroprotective effects of GDNF against 6-OHDA in young and aged rats.

In young adult rats, glial cell line-derived neurotrophic factor (GDNF) can completely protect against 6-hydroxydopamine-induced loss of nigral dopamine neurons when administered 6 h prior to the 6-hydroxydopamine. The present study was undertaken to determine if GDNF would provide similar protective effects in aged rats. Male, Fischer 344 x Brown Norway hybrid rats of 3, 18 and 24 months of age were given an intranigral injection of GDNF or vehicle followed 6 h later with an intranigral injection of 6-hydroxydopamine. Nigral dopamine neuron cell survival, and striatal and nigral dopamine and DOPAC levels, were evaluated 2 weeks after the lesions. In vehicle treated animals cell survival on the lesioned side ranged from 15 to 27%. GDNF promoted significant cell survival in the nigra of all three age groups; however, the percent survival was lowest in the 24-month-old animals (85% at 3 months, 75% at 18 months, 56% at 24 months). Similarly, dopamine levels in the striatum and substantia nigra on the lesioned side remained significantly greater in the GDNF treated animals compared to the vehicle treated animals. As with the cell survival experiment, the protective effects of GDNF on dopamine levels were less in the 24-month-old animals. GDNF pretreatment also protected against 6-hydroxydopamine-induced reductions in striatal DOPAC levels in all age groups. Overall, these results indicate that GDNF can protect nigrostriatal dopamine neurons against the effects of 6-hydroxydopamine in aged as well as young adult rats. However, the extent of protection is less in the aged (24-month-old) animals.

Restorative effects of GDNF on striatal dopamine release in rats treated with neurotoxic doses of methamphetamine.

Repeated methamphetamine (METH) administration to animals can result in long-lasting decreases in striatal dopamine (DA) release and content. Glial cell line-derived neurotrophic factor (GDNF) has pronounced effects on dopaminergic systems in vivo, including neuroprotective effects against METH. The present experiments were designed to examine the ability of GDNF to reverse, or accelerate recovery from, METH-induced alterations in striatal DA release. Male Fischer-344 rats were administered METH (5 mg/kg, s.c.) or saline 4 times in one day at 2-hour intervals. Seven days later the animals were anesthetized and given a single injection of 10 microg GDNF, or vehicle, into the right striatum. Three weeks later microdialysis experiments were carried out in both the right and left striata to examine basal and evoked levels of DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). In animals treated with METH followed by vehicle 7 days later, there were significant reductions in potassium- and amphetamine-evoked overflow of DA, and in basal levels of DOPAC and HVA, compared to control animals. In rats treated with METH followed 7 days later with GDNF, there were significant increases in potassium- and amphetamine-evoked overflow of DA on the right, GDNF-treated, side of the brain compared to the left side. Basal levels of DOPAC and HVA were also elevated on the GDNF-treated side of the brain. These results suggest that GDNF can accelerate recovery of dopaminergic release processes in the striatum of rats treated with neurotoxic doses of METH.

Methamphetamine toxicity is attenuated in mice that overexpress human manganese superoxide dismutase.

We have investigated methamphetamine (MA) toxicity in transgenic mice that overexpress the human form of mitochondrial manganese superoxide dismutase (MnSOD). Our results reveal a significant reduction in the long-term depletion of striatal dopamine and protein oxidation following repeated administration of MA in transgenic vs. non-transgenic littermates. These findings support the notion that ROS contribute to MA-induced brain damage and suggest that mitochondria may play an important role in this form of neurodegeneration.

Attenuation and recovery of evoked overflow of striatal serotonin in rats treated with neurotoxic doses of methamphetamine.

Repeated administration of methamphetamine to animals can lead to long-lasting decreases in striatal monoamine content. In the present study, the effects of neurotoxic doses of methamphetamine on basal and evoked overflow of striatal serotonin and of its primary metabolite 5-hydroxyindoleacetic acid were examined in awake rats using in vivo microdialysis. Male Fischer-344 rats were administered methamphetamine (5 mg/kg, s.c.) or saline four times in 1 day at 2-h intervals. Microdialysis studies were carried out 1 week, 1 month, and 6 months later. At 1 week posttreatment there were significant decreases in potassium- and amphetamine-evoked overflow of serotonin in the striatum of the methamphetamine-treated animals. Basal extracellular levels of 5-hydroxyindoleacetic acid but not of serotonin were also decreased. Evoked overflow of serotonin recovered by 1 month, and extracellular levels of 5-hydroxyindoleacetic acid had recovered by 6 months. Tissue levels of serotonin and 5-hydroxyindoleacetic acid were decreased at 1 week posttreatment but back to control levels by 1 month after treatment. These results indicate that presynaptic serotonergic functioning is attenuated in the striatum of rats treated 1 week earlier with neurotoxic doses of methamphetamine. However, in the model used, the changes are transient, and recovery can occur within 1-6 months posttreatment.

Recovery of presynaptic dopaminergic functioning in rats treated with neurotoxic doses of methamphetamine.

Repeated administration of methamphetamine (METH) to animals can result in long-lasting decreases in striatal dopamine (DA) content. In addition, the evoked overflow of striatal DA is reduced in rats 1 week after neurotoxic doses of METH. However, whether these functional changes in DA release are permanent or tend to recover over time has not been established. In the present study we used in vivo electrochemistry and microdialysis to examine evoked overflow of DA in the striatum of METH-treated rats at several time points after treatment to determine if DA overflow would spontaneously recover. Male Fischer-344 rats were administered METH (5 mg/kg, s.c. ) or saline four times in one day at 2 hr intervals. In vivo electrochemistry experiments in anesthetized rats, and in vivo microdialysis studies in awake rats, were carried out 1 week, 1 month, 6 months, and 12 months after treatment. At 1 week after treatment there were significant decreases in potassium- and amphetamine-evoked overflow of DA, and in clearance of DA, in the striatum of the METH-treated animals. Basal extracellular levels of DA and its metabolites were also decreased. Evoked overflow had partially recovered by 1 month. By 6 months evoked overflow of DA appeared to be normal in the METH-treated rats. However, whole tissue levels of striatal DA were still significantly decreased. All parameters were back to control values by 12 months. These results suggest that presynaptic dopaminergic functioning can recover to normal levels in the striatum of METH-treated rats by 12 months after treatment.

Functional MRI of basal ganglia responsiveness to levodopa in parkinsonian rhesus monkeys.

Functional MRI (fMRI) was used to study striatal sensitivity to levodopa in hemiparkinsonian rhesus monkeys. Responses consistent with increased neuronal activity were seen in areas whose normal dopaminergic input from the substantia nigra pars compacta had been ablated by MPTP. Sites of increased activity following levodopa included the lateral putamen, the ventral region of the caudate head, septal areas, and midlateral amygdala in the MPTP-lesioned hemisphere. Increased activity was also observed in the same areas in the nonlesioned hemisphere, but was less pronounced in spatial extent and magnitude, suggesting either subclinical contralateral damage and/or functional adaptations in the contralateral dopamine systems. The increases in neuronal activity following levodopa treatment were temporally correlated with increases in striatal dopamine levels. Chronic levodopa treatment reduced behavioral responsiveness to levodopa and abolished the fMRI response. These results suggest that fMRI can detect changes in dopamine receptor-mediated neuronal sensitivity to dopaminergic agents.

Excitatory influence of the locus coeruleus in hypothalamic-pituitary-adrenocortical axis responses to stress.

The locus coeruleus (LC) is a key brainstem region involved in arousal and is highly responsive to alerting/stressful stimuli, including those that activate the hypothalamic-pituitary-adrenocortical (HPA) axis. It is currently unclear whether the LC exerts any regulatory influence on the HPA axis and, consequently, on neuroendocrine responses to stress. The present studies were designed to test the hypothesis that the LC promotes HPA axis responses to acute and chronic stress. Adult male rats received bilateral (6-hydroxydopamine) lesions of the LC that produced severe cell loss in the LC and 80% depletion of noradrenaline in medial prefrontal cortex. Notably, lesions did not affect dopamine-beta-hydroxylase protein content in the parvocellular paraventricular nucleus (PVN), indicating a lack of collateral damage to other ascending noradrenergic pathways. LC lesions significantly reduced peak adrenocorticotropic hormone (ACTH) and corticosterone responses to 30 min acute restraint stress. However, LC lesions did not significantly attenuate neuroendocrine or other physiological responses to a 4-week chronic variable stress regimen. LC lesions did not substantially affect basal concentrations of plasma corticosterone or corticotropin-releasing hormone mRNA expression in the hypothalamic paraventricular nucleus following chronic stress. We conclude that the LC is a HPA-excitatory brain region, promoting neuroendocrine and physiological responses primarily to acute stress. However, a potential role for the LC in the induction of HPA axis hyperactivity following chronic stress can not be ruled out.

Augmented methamphetamine-induced overflow of striatal dopamine 1 day after GDNF administration.

Glial cell line-derived neurotrophic factor (GDNF) can attenuate the dopamine (DA)-depleting effects of neurotoxic doses of methamphetamine (METH) when given 1 day prior to the METH. The neurotoxic effects of METH may be due, in part, to sustained increases in extracellular levels of DA. It is therefore possible that GDNF may be altering the effects of METH by influencing extracellular levels of DA during the METH treatment. The purpose of the present study was to determine if GDNF has effects on extracellular levels of DA in the striatum by 24-h post-administration. GDNF (10 microgram in 2 microliter vehicle) or vehicle was injected into the right striatum or substantia nigra of anesthetized male rats. The next day the animals were anesthetized again and dialysis probes were positioned in both the right and left striata and perfused with artificial cerebrospinal fluid. Following the collection of baseline samples the rats were administered METH (5 mg/kg, s.c.). The METH injections dramatically increased extracellular DA levels on both sides of the brain. However, levels on the GDNF injected side were significantly greater than levels on the contralateral side. Basal levels of DA were not significantly different between the two sides, but levels of DA metabolites were elevated on the GDNF side. Post-mortem tissue levels of DA metabolites, but not DA, were also elevated in the striatum and substantia nigra. These results indicate that GDNF has significant effects on DA neuron functioning within 24 h of administration and that GDNF can augment DA overflow while inhibiting the neurotoxic effects of METH.

GDNF protection against 6-OHDA-induced reductions in potassium-evoked overflow of striatal dopamine.

Glial cell line-derived neurotrophic factor (GDNF), when administered before 6-hydroxydopamine (6-OHDA), has been shown to prevent the reduction in nigral dopamine (DA) levels and tyrosine hydroxylase-positive neurons normally observed after 6-OHDA lesions. The present study examined the ability of GDNF to prevent 6-OHDA-induced reductions in striatal DA release and reductions in striatal and nigral DA levels. GDNF (10 micrograms), or vehicle, was injected into the right nigra of anesthetized male Fischer-344 rats and was followed 6 hr later by intranigral 6-OHDA or saline. Three to four weeks later the animals were anesthetized with urethane and prepared for in vivo electrochemistry. Potassium-evoked overflow of DA was dramatically decreased in the right striatum of the vehicle + 6-OHDA-treated animals. GDNF appeared to prevent the reduction in evoked overflow of DA in the right striatum of the 6-OHDA-treated animals. However, in comparison with that in animals that received GDNF + saline, the overflow of DA was significantly reduced in the GDNF + 6-OHDA animals. Similarly, although nigral levels of DA were above normal in the GDNF + 6-OHDA-treated animals, they were below DA levels found in GDNF + saline-treated rats. Striatal DA levels were partially protected by GDNF. In animals examined 10-12 weeks after the GDNF and 6-OHDA treatments, the apparent protective ability of GDNF on the evoked overflow of DA in the striatum was diminished. Thus, although intranigral GDNF can prevent 6-OHDA-induced reductions in nigral DA levels, long-term protection of the evoked overflow of DA in the striatum is minimal.

GDNF improves dopamine function in the substantia nigra but not the putamen of unilateral MPTP-lesioned rhesus monkeys.

Microdialysis measurements of dopamine (DA) and DA metabolites were carried out in the putamen and substantia nigra of unilateral 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned rhesus monkeys that received intraventricular injections of vehicle or glial-derived neurotrophic factor (GDNF, 300 microg) 3 weeks prior to the microdialysis studies. Following behavioral measures in the MPTP-lesioned monkeys, they were anesthetized with isoflurane and placed in a stereotaxic apparatus. Magnetic resonance imaging (MRI)-guided sterile stereotaxic procedures were used for implantations of the microdialysis probes. Basal extracellular levels of DA and the DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were found to be decreased by >95% in the right putamen of the MPTP-lesioned monkeys as compared to normal animals. In contrast, basal DA levels were not significantly decreased, and DOPAC and HVA levels were decreased by only 65% and 30%, respectively, in the MPTP-lesioned substantia nigra. Significant reductions in d-amphetamine-evoked DA release were also observed in the MPTP-lesioned substantia nigra and putamen of the monkeys as compared to normal animals. A single intraventricular administration of GDNF into one group of MPTP-lesioned monkeys elicited improvements in the parkinsonian symptoms in these animals at 2-3 weeks post-administration. In addition, d-amphetamine-evoked overflow of DA was significantly increased in the substantia nigra but not the putamen of MPTP-lesioned monkeys that had received GDNF. Moreover, post-mortem brain tissue studies showed increases in whole tissue levels of DA and DA metabolite levels primarily within the substantia nigra in MPTP-lesioned monkeys that had received GDNF. Taken together, these data support that single ventricular infusions of GDNF produce improvements in motoric behavior in MPTP-lesioned monkeys that correlate with increases in DA neuronal function that are localized to the substantia nigra and not the putamen.

Effects of neurotoxic doses of methamphetamine on potassium and amphetamine evoked overflow of dopamine in the striatum of awake rats.

The effects of neurotoxic doses of methamphetamine on basal and evoked overflow of dopamine in the striatum were examined in awake rats using microdialysis. Male Fischer-344 rats were administered methamphetamine (5 mg/kg s.c.) or saline four times in 1 day at 2-h intervals. Microdialysis experiments were carried out 1 week later. Basal levels of dopamine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid were reduced in the striatum of the methamphetamine-treated animals. Local application of excess potassium (100 mM) and amphetamine (100 microM), and intraperitoneal injection of amphetamine (1.5 mg/ kg), led to increased levels of extracellular dopamine in the striatum of both methamphetamine- and saline-treated rats. However, the increase was significantly less in the methamphetamine-treated animals. Tissue levels of dopamine and metabolites were reduced in the striata of rats treated with methamphetamine. These results indicate that treatment with neurotoxic doses of methamphetamine can lead to functional changes in dopamine release in the striatum of Fischer-344 rats.

Development of immune hyperinnervation in NGF-transgenic mice.

Sympathetic innervation of lymphoid tissues is localized to specific tissue compartments, but little is known of the "factors" that are important in establishing this pattern during development. Numerous studies have shown interactions of nerve growth factor (NGF) with the immune system, which may include modulation of immune innervation. We previously have shown that NGF transgenic mice, which overexpress NGF in skin and not immune tissues, have a dramatic hyperinnervation of splenic marginal zone and peripheral lymph node medulla and capsule. The purpose of the current studies was to determine if the presence of elevated NGF would alter immune system development and the process of sympathetic ingrowth. The results show that the splenic innervation in NGF transgenics gradually diverged from controls during the first two postnatal weeks, with the greatest change occurring between postnatal days 13 and 16 when the splenic organization was reaching the adult pattern. In contrast, the peripheral lymph nodes were hyperinnervated at an earlier age. mesenteric lymph nodes never diverged from the normal pattern. NGF levels in transgenic spleen were much higher than controls at postnatal days 1 and 2, when little innervation was present, and declined as the tissue matured, possibly because of NGF uptake by the ingrowing sympathetic fibers. This suggests that immune tissues are capable of concentrating NGF, which in turn may modulate the level of innervation by the sympathetic nervous system.

GDNF protection against 6-OHDA: time dependence and requirement for protein synthesis.

Glial cell line-derived neurotrophic factor (GDNF) injected intranigrally protects midbrain dopamine neurons against 6-hydroxydopamine (6-OHDA) toxicity. The timing between GDNF administration and exposure to 6-OHDA is critical in achieving optimal protection. When injected 6 hr before an intranigral injection of 6-OHDA, GDNF provides complete protection as measured by the number of surviving neurons in the substantia nigra of adult rats. The surviving neuronal population decreases by approximately 50% with 12 and 24 hr separating GDNF and 6-OHDA administrations. In controls with 6-OHDA lesions, there is <10% survival of nigral dopamine neurons. No significant increase in survival is seen with either concurrent injections of GDNF and 6-OHDA or 1 hr GDNF pretreatment. Based on HPLC measurements, striatal and midbrain dopamine levels are at least twofold higher on the lesioned side in animals receiving GDNF 6 hr before a 6-OHDA lesion compared with vehicle recipients. Protein synthesis is necessary for GDNF-induced neuroprotective effects because cycloheximide pretreatment that inhibits protein synthesis also blocks neuroprotection.

Decreases in evoked overflow of dopamine in rat striatum after neurotoxic doses of methamphetamine.

The repeated administration of methamphetamine (METH) can result in long-lasting decreases in dopamine (DA) levels, tyrosine hydroxylase activity and DA uptake sites in the striatum. However, whether these changes lead to functional alterations in the dynamics of DA release and uptake has not been extensively examined. The present study used in vivo electrochemistry and microdialysis to examine potassium- and amphetamine-evoked release of DA in the striatum and nucleus accumbens (NAc) of METH-treated rats. Male Fischer-344 rats were administered METH (5 mg/kg s.c.) or saline four times in 1 day, at 2-hr intervals. One week later the animals were anesthetized with urethane and prepared for in vivo electrochemical recordings. The METH treatment resulted in dramatic decreases in potassium-evoked release of DA and in the rate of DA clearance in the striatum, whereas the NAc was not significantly affected. In vivo microdialysis studies demonstrated significant decreases in basal DA levels and in potassium- and amphetamine-evoked overflow of DA in the striatum of METH-treated animals. Basal and evoked DA levels in the NAc were not altered. Post-mortem levels of tissue DA were decreased by 41 to 67% in the striatum and 25 to 31% in the NAc. These results indicate that the striatum is more sensitive than the NAc to the neurotoxic effects of METH, both in measures of functional dynamics of DA signaling and in tissue levels of DA. It remains to be determined whether these functional changes in DA release and uptake are permanent or tend to recover over time.

GDNF selectively protects dopamine neurons over serotonin neurons against the neurotoxic effects of methamphetamine.

Repeated methamphetamine (METH) administration to animals can result in long-lasting decreases in striatal dopamine (DA) and serotonin (5-HT) levels. Glial cell line-derived neurotrophic factor (GDNF) has pronounced effects on dopaminergic systems in vivo, including partial neuroprotective effects against 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine -induced lesions. The present study examined the ability of GDNF to prevent METH-induced reductions in potassium-evoked overflow of DA, and DA and 5-HT content, in striatum. GDNF (10 microg) or vehicle was injected into the right striatum of anesthetized rats. Twenty-four hours later, the rats were injected four times at 2 hr intervals with METH (5 mg/kg, s.c.) or saline. One week later, in vivo electrochemistry was used to monitor the overflow of DA evoked by local potassium application. Evoked overflow of DA was dramatically decreased in the striatum of METH-treated animals. GDNF prevented the reduction in evoked overflow of DA in the right striatum of the METH-treated animals. After each experiment, the animals were killed, and striatal DA and 5-HT levels determined by HPLC. The METH treatment produced significant decreases in both neurotransmitters. GDNF administration prevented the reduction in striatal DA levels on the treated side of the brain, whereas levels on the contralateral side were still decreased. In dose-response studies, 1 microg of GDNF was as protective as 10 microg, whereas 0.1 microg was only partially protective. In contrast, 5-HT levels were only minimally protected by previous administration of GDNF. These results suggest that GDNF can selectively protect DA neurons, compared with 5-HT neurons, against the neurotoxic effects of METH.