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We previously showed that, in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD), vaccination with bacillus Calmette-Guerin (BCG) prior to MPTP exposure limited the loss of striatal dopamine (DA) and dopamine transporter (DAT) and prevented the activation of nigral microglia. Here, we conducted BCG dose studies and investigated the mechanisms underlying BCG vaccination's neuroprotective effects in this model. We found that a dose of 1 × 10(6) cfu BCG led to higher levels of striatal DA and DAT ligand binding (28% and 42%, respectively) in BCG-vaccinated vs. unvaccinated MPTP-treated mice, but without a significant increase in substantia nigra tyrosine hydroxylase-staining neurons. Previous studies showed that BCG can induce regulatory T cells (Tregs) and that Tregs are neuroprotective in models of neurodegenerative diseases. However, MPTP is lymphotoxic, so it was unclear whether Tregs were maintained after MPTP treatment and whether a relationship existed between Tregs and the preservation of striatal DA system integrity. We found that, 21 days post-MPTP treatment, Treg levels in mice that had received BCG prior to MPTP were threefold greater than those in MPTP-only-treated mice and elevated above those in saline-only-treated mice, suggesting that the persistent BCG infection continually promoted Treg responses. Notably, the magnitude of the Treg response correlated positively with both striatal DA levels and DAT ligand binding. Therefore, BCG vaccine-mediated neuroprotection is associated with Treg levels in this mouse model. Our results suggest that BCG-induced Tregs could provide a new adjunctive therapeutic approach to ameliorating pathology associated with PD and other neurodegenerative diseases., (Copyright © 2013 Wiley Periodicals, Inc.)

The continuing spread of methamphetamine (METH) abuse has stimulated research aimed at understanding consequences of its prolonged exposure. Alterations in nigrostriatal dopamine (DA) system parameters have been characterized in experimental studies after discontinuation of long-term METH but fewer studies have included similar assessments during METH exposure. Here, we report METH plasma pharmacokinetics and striatal DA system alterations in rat after noncontingent and contingent METH administration for 7.5 weeks. Escalating METH exposure was delivered by dynamic infusion (DI) that incorporated a "humanized" plasma METH half life or by intravenous self-administration (IVSA) that included binge intakes. Kinetic modeling of DI and IVSA for 24 h periods during the final week of METH exposure showed that plasma METH levels remained between 0.7 and 1.5 µM. Animals were sacrificed during their last METH administration for autoradiography assessment using [³H]ligands and D2 agonist-induced [³⁵S]GTPγS binding. DA transporter binding was decreased (DI, 34%; IVSA, 15%) while vesicular monoamine transporter binding and substantia nigra DA cell numbers were unchanged. Decreases were measured for D2 receptor (DI and IVSA, 15-20%) and [³⁵S]GTPγS binding (DI, 35%; IVSA, 18%). These similar patterns of DI and IVSA associated decreases in striatal DA markers reflect consequences of cumulative METH exposure and not the drug delivery method. For METH IVSA, individual differences were observed, yet each animal's total intake was similar within and across three 24-h binges. IVSA rodent models may be useful for identifying molecular mechanisms that are associated with METH binges in humans., (Copyright © 2013 Wiley Periodicals, Inc.)

Prior studies of appetite regulatory networks, primarily in rodents, have established that targeted electrical stimulation of ventromedial hypothalamus (VMH) can alter food intake patterns and metabolic homeostasis. Consideration of this method for weight modulation in humans with severe overeating disorders and morbid obesity can be further advanced by modeling procedures and assessing endpoints that can provide preclinical data on efficacy and safety. In this study we adapted human deep brain stimulation (DBS) stereotactic methods and instrumentation to demonstrate in a large animal model the modulation of weight gain with VMH-DBS. Female Göttingen minipigs were used because of their dietary habits, physiologic characteristics, and brain structures that resemble those of primates. Further, these animals become obese on extra-feeding regimens. DBS electrodes were first bilaterally implanted into the VMH of the animals (n = 8) which were then maintained on a restricted food regimen for 1 mo following the surgery. The daily amount of food was then doubled for the next 2 mo in all animals to produce obesity associated with extra calorie intake, with half of the animals (n = 4) concurrently receiving continuous low frequency (50 Hz) VMH-DBS. Adverse motoric or behavioral effects were not observed subsequent to the surgical procedure or during the DBS period. Throughout this 2 mo DBS period, all animals consumed the doubled amount of daily food. However, the animals that had received VMH-DBS showed a cumulative weight gain (6.1±0.4 kg; mean ± SEM) that was lower than the nonstimulated VMH-DBS animals (9.4±1.3 kg; p<0.05), suggestive of a DBS-associated increase in metabolic rate. These results in a porcine obesity model demonstrate the efficacy and behavioral safety of a low frequency VMH-DBS application as a potential clinical strategy for modulation of body weight.

A prototype Low Intensity Focused Ultrasound (LIFU) stimulator system was developed to evaluate non-invasive neuromodulation in a large animal model. We conducted a feasibility study on a Göttingen minipig, demonstrating reversible, targeted transcranial neuromodulation. The hypothalamus of the minipig was repeatedly stimulated with LIFU which evoked temporally correlated increases in both heart rate and blood pressure.

With the recent approval by the Food and Drug Administration (FDA) of Deep Brain Stimulation (DBS) for Parkinson's Disease, dystonia and obsessive compulsive disorder (OCD), vagus nerve stimulation (VNS) for epilepsy and depression, and repetitive transcranial magnetic stimulation (rTMS) for the treatment of depression, neuromodulation has become increasingly relevant to clinical research. However, these techniques have significant drawbacks (eg, lack of special specificity and depth for the rTMS, and invasiveness and cumbersome maintenance for DBS). This article reviews the background, rationale, and pilot studies to date, using a new brain stimulation method-low-intensity focused ultrasound pulsation (LIFUP). The ability of ultrasound to be focused noninvasively through the skull anywhere within the brain, together with concurrent imaging (ie, functional magnetic resonance imaging [fMRI]) techniques, may create a role for research and clinical use of LIFUP. This technique is still in preclinical testing and needs to be assessed thoroughly before being advanced to clinical trials. In this study, we review over 50 years of research data on the use of focused ultrasound (FUS) in neuronal tissue and live brain, and propose novel applications of this noninvasive neuromodulation method., (Copyright © 2011 Elsevier Inc. All rights reserved.)

There is a growing interest in using vaccination with CNS antigens to induce autoreactive T cell responses that home to damaged areas in the CNS and ameliorate neurodegenerative disease. Neuroprotective vaccine studies have focused on administering oligodendrocyte antigens or Copaxone® in complete Freund's adjuvant (CFA). Theoretical considerations, however, suggest that vaccination with a neuronal antigen may induce more robust neuroprotective immune responses. We assessed the neuroprotective potential of vaccines containing tyrosine hydroxylase (a neuronal protein involved in dopamine synthesis) or Copaxone® in CFA in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. Surprisingly, we observed that the main beneficial factor in these vaccines was the CFA. Since the major immunogenic component in CFA is Mycobacterium tuberculosis, which closely related to the bacille Calmette-Guérin (BCG) that is used in human vaccines, we tested BCG vaccination in the MPTP mouse model. We observed that BCG vaccination partially preserved markers of striatal dopamine system integrity and prevented an increase in activated microglia in the substantia nigra of MPTP-treated mice. These results support a new neuroprotective vaccine paradigm in which general (nonself-reactive) immune stimulation in the periphery can limit potentially deleterious microglial responses to a neuronal insult and exert a neurorestorative effect in the CNS. Accordingly, BCG vaccination may provide a new strategy to augment current treatments for a wide range of neuropathological conditions.

Parkinson's disease (PD) is characterized by dopaminergic neurodegeneration in the nigrostriatal region of the brain; however, the neurodegeneration extends well beyond dopaminergic neurons. To gain a better understanding of the molecular changes relevant to PD, we applied two-dimensional LC-MS/MS to comparatively analyze the proteome changes in four brain regions (striatum, cerebellum, cortex, and the rest of brain) using a MPTP-induced PD mouse model with the objective to identify potential nigrostriatal-specific and other region-specific protein abundance changes. The combined analyses resulted in the identification of 4,895 nonredundant proteins with at least two unique peptides per protein. The relative abundance changes in each analyzed brain region were estimated based on the spectral count information. A total of 518 proteins were observed with substantial MPTP-induced abundance changes across different brain regions. A total of 270 of these proteins were observed with specific changes occurring either only in the striatum and/or in the rest of the brain region that contains substantia nigra, suggesting that these proteins are associated with the underlying nigrostriatal pathways. Many of the proteins that exhibit changes were associated with dopamine signaling, mitochondrial dysfunction, the ubiquitin system, calcium signaling, the oxidative stress response, and apoptosis. A set of proteins with either consistent change across all brain regions or with changes specific to the cortex and cerebellum regions were also detected. Ubiquitin specific protease (USP9X), a deubiquination enzyme involved in the protection of proteins from degradation and promotion of the TGF-beta pathway, exhibited altered abundance in all brain regions. Western blot validation showed similar spatial changes, suggesting that USP9X is potentially associated with neurodegeneration. Together, this study for the first time presents an overall picture of proteome changes underlying both nigrostriatal pathways and other brain regions potentially involved in MPTP-induced neurodegeneration. The observed molecular changes provide a valuable reference resource for future hypothesis-driven functional studies of PD.

Bingeing is one pattern of high-dose methamphetamine (METH) abuse, which involves continuous drug taking over several days and can result in psychotic behaviors for which the brain pathology remains poorly defined. A corresponding animal model of this type of METH exposure may provide novel insights into the neurochemical and behavioral sequelae associated with this condition. Accordingly, to simulate the pharmacokinetic profile of a human METH binge exposure in rats, we used a computer-controlled, intravenous METH procedure (dynamic infusion, DI) to overcome species differences in METH pharmacokinetics and to replicate the human 12-h plasma METH half-life. Animals were treated over 13 weeks with escalating METH doses, using DI, and then exposed to a binge in which drug was administered every 3 h for 72 h. Throughout the binge, behavioral effects included unabated intense oral stereotypies in the absence of locomotion and in the absence of sleep. Decrements in regional brain dopamine, norepinephrine, and serotonin levels, measured at 1 and 10 h after the last injection of the binge, had, with the exception of caudate-putamen dopamine and frontal cortex serotonin, recovered by 48 h. At 10 h after the last injection of the binge, [(3)H]ligand binding to dopamine and vesicular monoamine transporters in caudate-putamen were reduced by 35 and 13%, respectively. In a separate METH binge-treated cohort, post-binge behavioral alterations were apparent in an attenuated locomotor response to a METH challenge infusion at 24 h after the last injection of the binge. Collectively, the changes we characterized during and after a METH binge suggest that for human beings under similar exposure conditions, multiple time-dependent neurochemical deficits contribute to their behavioral profiles.

The area and volume of brain structural features, as assessed by high-resolution three-dimensional magnetic resonance imaging (MRI), are among the most heritable measures relating to the human CNS. We have conducted MRI scanning of all available monkeys >2 years of age (n = 357) from the extended multigenerational pedigree of the Vervet Research Colony (VRC). Using a combination of automated and manual segmentation we have quantified several correlated but distinct brain structural phenotypes. The estimated heritabilities (h(2)) for these measures in the VRC are higher than those reported previously for such features in humans or in other nonhuman primates: total brain volume (h(2) = 0.99, SE = 0.06), cerebral volume (h(2) = 0.98, SE = 0.06), cerebellar volume (h(2) = 0.86, SE = 0.09), hippocampal volume (h(2) = 0.95, SE = 0.07) and corpus callosum cross-sectional areas (h(2) = 0.87, SE = 0.07). These findings indicate that, in the controlled environment and with the inbreeding structure of the VRC, additive genetic factors account for almost all of the observed variance in brain structure, and suggest the potential of the VRC for genetic mapping of quantitative trait loci underlying such variance.

Purpose: Pretreatment with cyclosporine, a P-glycoprotein (P-gp) modulator increases brain uptake of 4-(2'-methoxyphenyl)-1-[2'-(N-2"-pyridinyl)-p-[(18)F]fluorobenzamido]ethylpiperazine ([(18)F]MPPF) for binding to hydroxytryptamine(1A) (5-HT(1A)) receptors. Those increases were quantified in rat brain with in vivo microPET and ex vivo tissue studies., Materials and Methods: Each Sprague-Dawley rat (n = 4) received a baseline [(18)F]MPPF microPET scan followed by second scan 2-3 weeks later that included cyclosporine pretreatment (50 mg/kg, i.p.). Maximum a posteriori reconstructed images and volumetric ROIs were used to generate dynamic radioactivity concentration measurements for hippocampus, striatum, and cerebellum, with simplified reference tissue method (SRTM) analysis. Western blots were used to semiquantify P-gp regional distribution in brain., Results: MicroPET studies showed that hippocampus uptake of [(18)F]MPPF was increased after cyclosporine; ex vivo studies showed similar increases in hippocampus and frontal cortex at 30 min, and for heart and kidney at 2.5 and 5 min, without concomitant increases in [(18)F]MPPF plasma concentration. P-gp content in cerebellum was twofold higher than in hippocampus or frontal cortex., Conclusions: These studies confirm and extend prior ex vivo results (J. Passchier, et al., Eur J Pharmacol, 2000) that showed [(18)F]MPPF as a substrate for P-gp. Our microPET results showed that P-gp modulation of [(18)F]MPPF binding to 5-HT(1A) receptors can be imaged in rat hippocampus. The heterogeneous brain distribution of P-gp appeared to invalidate the use of cerebellum as a nonspecific reference region for SRTM modeling. Regional quantitation of P-gp may be necessary for accurate PET assessment of 5-HT(1A) receptor density when based on tracer uptake sensitive to P-gp modulation.

Background: Kaposi's sarcoma-associated herpesvirus (KSHV) possesses two distinct life cycles, lytic replication and latency. An immediate early viral protein, Replication and transcription activator (RTA), is responsible for the virus switch from latency to active replication., Methods: To identify cellular pathways that reactivate KSHV replication, an RTA-responsive viral early promoter, PAN, coupled with an enhanced green fluorescent protein (EGFP) reporter was delivered into a KSHV latently infected B cell line. Five different chemical libraries with defined cellular targets were screened for their ability to induce the PAN promoter as an indication of lytic replication., Results: We identified seven chemicals that disrupted latency in KSHV latently infected B cells, five being N-acyl-dopamine derivatives. We showed that these chemicals reactivate KSHV through interacting with dopamine receptors, and that KSHV utilizes dopamine receptors and the associated PKA and MAP kinase pathways to detect and transmit stress signals for reactivation., Conclusion: Our study identified two cellular signaling pathways that mediate KSHV reactivation and provided a chemical genetics approach to identify new endogenous activators with therapeutic potential against herpesvirus associated malignancies.

Methamphetamine (METH)-associated alterations in the human striatal dopamine (DA) system have been identified with positron emission tomography (PET) imaging and post-mortem studies but have not been well correlated with behavioral changes or cumulative METH intake. Animal studies that model some aspects of human long-term METH abuse can establish dose-dependency profiles of both behavioral changes and potential brain neurotoxicities for identifying consequences of particular cumulative exposures. Based on parameters from human and our monkey pharmacokinetic studies, we modeled a prevalent human METH exposure of daily multiple doses in socially housed vervet monkeys. METH doses were escalated over 33 weeks, with final dosages resulting in estimated peak plasma METH concentrations of 1-3 microM, a range measured in human abusers. With larger METH doses, progressive increases in abnormal behavior and decreases in social behavior were observed on 'injection' days. Anxiety increased on 'no injection' days while aggression decreased throughout the study. Thereafter, during 3 weeks abstinence, differences in baseline vs post-METH behaviors were not observed. Post-mortem analysis of METH brains showed 20% lower striatal DA content while autoradiography studies of precommissural striatum showed 35% lower [3H]WIN35428 binding to the DA transporter. No statistically significant changes were detected for [3H]dihydrotetrabenazine binding to the vesicular monoamine transporter (METH-lower by 10%) or for [3H]SCH 23390 and [3H]raclopride binding to DA D1 and D2 receptors, respectively. Collectively, this long-term, escalating dose METH exposure modeling a human abuse pattern, not associated with high-dose binges, resulted in dose-dependent behavioral effects and caused persistent changes in presynaptic striatal DA system integrity.

The molecular mechanisms underlying the changes in the nigrostriatal pathway in Parkinson's disease (PD) are not completely understood. Here, we use mass spectrometry and microarrays to study the proteomic and transcriptomic changes in the striatum of two mouse models of PD, induced by the distinct neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine (METH). Proteomic analyses resulted in the identification and relative quantification of 912 proteins with two or more unique peptides and 86 proteins with significant abundance changes following neurotoxin treatment. Similarly, microarray analyses revealed 181 genes with significant changes in mRNA, following neurotoxin treatment. The combined protein and gene list provides a clearer picture of the potential mechanisms underlying neurodegeneration observed in PD. Functional analysis of this combined list revealed a number of significant categories, including mitochondrial dysfunction, oxidative stress response, and apoptosis. These results constitute one of the largest descriptive data sets integrating protein and transcript changes for these neurotoxin models with many similar end point phenotypes but distinct mechanisms.

Object: Deep brain stimulation (DBS) has become an effective therapy for an increasing number of brain disorders. Recently demonstrated DBS of the posterior hypothalamus as a safe treatment for chronic intractable cluster headaches has drawn attention to this target, which is involved in the regulation of diverse autonomic functions and feeding behavior through complex integrative mechanisms. In this study, the authors assessed the feasibility of ventromedial hypothalamus (VMH) DBS in freely moving vervet monkeys to modulate food intake as a model for the potential treatment of eating disorders., Methods: Deep brain stimulation electrodes were bilaterally implanted into the VMH of 2 adult male vervet monkeys by using the stereotactic techniques utilized in DBS in humans. Stimulators were implanted subcutaneously on the upper back, allowing ready access to program stimulation parameters while the animal remained conscious and freely moving. In anesthetized animals, intraoperatively and 6-10 weeks postsurgery, VMH DBS parameters were selected according to minimal cardiovascular and autonomic nervous system responses. Thereafter, conscious animals were subjected to 2 cycles of VMH DBS for periods of 8 and 3 days, and food intake and behavior were monitored. Animals were then killed for histological verification of probe placement., Results: During VMH DBS, total food consumption increased. The 3-month bilateral implant of electrodes and subsequent periods of high-frequency VMH stimulation did not result in significant adverse behavioral effects., Conclusions: This is the first study in which techniques of hypothalamic DBS in humans have been applied in freely moving nonhuman primates. Future studies can now be conducted to determine whether VMH DBS can change hypothalamic responsivity to endocrine signals associated with adiposity for long-term modulation of food intake.

Increases in basal ganglia iron are well documented for neurodegenerative diseases but have not been associated with methamphetamine (METH). In this study, vervet monkeys that received two doses of METH (2 mg/kg, intramuscularly, 6 h apart) showed at 1 month, iron increases in substantia nigra pars reticulata and globus pallidus, with concurrent increases of ferritin-immunoreactivity and decreases of tyrosine hydroxylase-immunoreactivity in substantia nigra. At 1.5 years, substantia nigra tyrosine hydroxylase-immunoreactivity had recovered while iron and ferritin-immunoreactivity increases persisted. Globus pallidus and substantia nigra iron levels of the adult METH-exposed animals (age 5-9 years) were now comparable with those of drug-naive, aged animals (19-22 years), suggesting an aging-related condition that might render those regions more vulnerable to oxidative stress.

To elucidate the organization of the serotoninergic innervation within the orbitofrontal cortex (OFC), serotonin transporter (SERT) density was quantified by autoradiography using [(3)H]cyanoimipramine binding. In six adult vervet monkeys, 15 architectonic areas were delineated according to cytoarchitectonic (Nissl), myeloarchitectonic (Gallyas) and chemoarchitectonic (acetylcholinesterase) criteria to assess SERT distribution at two levels of organization: cortical area and cortical type. For cortical type, the 15 areas were evenly divided into three different categories primarily based upon the degree of granularization of layer IV: agranular, dysgranular, and granular. Within agranular and dysgranular, but not granular cortical types, SERT density was area-specific and progressively decreased in a medial to lateral gradient. Across cortical types, SERT density decreased in a caudal to rostral gradient: agranular>dysgranular>granular. A similar caudal to rostral gradient was seen when serotonin content was measured (using high performance liquid chromatography) in areas representative of each cortical type. Collectively, these results suggest that the serotoninergic innervation is organized according to both cortical type and area, and is thus structured to differentially modulate information processing within the OFC.

Characterization of methamphetamine's (METH) dose-dependent effects on brain neurochemistry may represent a critical component for better understanding the range of resultant behavioral pathologies. Most human studies, however, have assessed only the effects of long term, high dose METH abuse (e.g., greater than 1000 mg/day) in individuals meeting DSM-IV criteria for METH dependence. Yet, for the majority of METH abusers, their patterns of METH exposure that consist of lower doses remain less well-characterized. In this study, blood samples were obtained from 105 individuals detained by police for possible criminal activity and testing positive for stimulants by EMIT assay. METH blood concentrations were subsequently quantified by GC-MS and were predominantly in the low micromolar range (0.1-11.1 microM), with median and mean values of 1.3 microM (0.19 mg/l) and 2 microM (0.3 mg/l), respectively. Pharmacokinetic calculations based on these measured values were used to estimate initial METH body burdens, the median value being 52 mg. Modeling a 52 mg dose for a 4 day-METH maintenance exposure pattern of 4 doses/day at 4 h intervals showed that blood concentrations remained between 1 and 4 microM during this period. Collectively, these data present evidence for a METH exposure pattern distinct from high dose-METH abuse and provide the rationale for assessing potential brain pathology associated with such lower dose-METH exposure.

A major feature of human methamphetamine (METH) abuse is the gradual dose escalation that precedes high-dose exposure. The period of escalating doses (EDs) is likely associated with development of tolerance to aspects of METH's pharmacologic and toxic effects but the relative contributions of pharmacokinetic and pharmacodynamic factors have not been well defined. In our prior studies in rats, we showed that pretreatment with an ED-METH regimen (0.1-4.0 mg/kg over 14 days) attenuated the toxicity of a subsequently administered high-dose METH binge (4 x 6 mg/kg at 2 h interval) that itself produced behavioral stereotypy, increases in core temperature, and decreases in DA system phenotypic markers in caudate-putamen (CP). Using those ED-METH and binge protocols in the present studies, pharmacokinetic and pharmacodynamic parameters that may have contributed to the apparent neuroprotection afforded by ED-METH were assessed. The ED-METH regimen itself reduced [(3)H]WIN35,428 (WIN) binding to the dopamine transporter (DAT) by 15% in CP, but did not affect DA content. During the METH binge, ED-METH pretreated animals showed attenuated increases in core temperature while concurrent microdialysis studies in CP showed a reduced DA response despite unaltered extracellular levels of METH. At 1 h after the binge, concentrations of METH and its metabolite amphetamine in brain and plasma were unaffected by the ED-METH. The results show that ED-METH pretreatment produces reductions in DAT binding and the DA response during a subsequent METH binge by altering pharmacodynamic and not pharmacokinetic parameters.

Increased abundance of nitrotyrosine modifications of proteins have been documented in multiple pathologies in a variety of tissue types and play a role in the redox regulation of normal metabolism. To identify proteins sensitive to nitrating conditions in vivo, a comprehensive proteomic data set identifying 7792 proteins from a whole mouse brain, generated by LC/LC-MS/MS analyses, was used to identify nitrated proteins. This analysis resulted in the identification of 31 unique nitrotyrosine sites within 29 different proteins. More than half of the nitrated proteins that have been identified are involved in Parkinson's disease, Alzheimer's disease, or other neurodegenerative disorders. Similarly, nitrotyrosine immunoblots of whole brain homogenates show that treatment of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an experimental model of Parkinson's disease, induces an increased level of nitration of the same protein bands observed to be nitrated in brains of untreated animals. Comparing sequences and available high-resolution structures around nitrated tyrosines with those of unmodified sites indicates a preference of nitration in vivo for surface accessible tyrosines in loops, a characteristic consistent with peroxynitrite-induced tyrosine modification. In addition, most sequences contain cysteines or methionines proximal to nitrotyrosines, contrary to suggestions that these amino acid side chains prevent tyrosine nitration. More striking is the presence of a positively charged moiety near the sites of nitration, which is not observed for non-nitrated tyrosines. Together, these observations suggest a predictive tool of functionally important sites of nitration and that cellular nitrating conditions play a role in neurodegenerative changes in the brain.

The translational value of preclinical models of methamphetamine abuse depends in large part on the degree to which the drug regimens used in animals produce methamphetamine exposure patterns similar to those experienced by human methamphetamine abusers. To approximate one common form of methamphetamine abuse, we studied the effects of a schedule of intravenous methamphetamine administration in rats which included 2 weeks of progressively more frequent drug injections (0.125 mg/kg/injection) followed by 40 maintenance days during which animals received 40 daily injections (at 15-min intervals), with the dose gradually increasing (0.125-0.25 mg/kg per injection) every 5-10 days. This treatment produced an emerging behavioral profile characterized by gradually more continuous periods of activation consisting of progressively more intense, focused stereotypy interrupted by episodic bursts of locomotion. We also assessed markers of dopamine neurotransmission (dopamine transporter, vesicular monoamine transporter, and dopamine D1 and D2 receptors) at 15 min and (including dopamine levels) at 6 and 30 days following cessation of methamphetamine treatment. All dopamine components measured in caudate-putamen were significantly reduced at 15 min and 6 days after the final methamphetamine injection. Dopamine D1 and D2 receptors fully recovered after 30 days of drug abstinence, whereas dopamine and the dopamine transporter exhibited significant but incomplete recovery by this time point. In contrast, only the vesicular monoamine transporter exhibited no evidence of recovery over the 30-day withdrawal period. These data are discussed in terms of damage to dopamine terminals and compensatory adjustments in mechanisms maintaining functional dopaminergic transmission.

An automated method for placement of 3D rat brain atlas-derived volumes of interest (VOIs) onto PET studies has been designed and evaluated. VOIs representing major structures of the rat brain were defined on a set of digitized cryosectioned images of the rat brain. For VOI placement, each PET study was registered with a synthetic PET target constructed from the VOI template. Registration was accomplished with an automated algorithm that maximized the mutual information content of the image volumes. The accuracy and precision of this method for VOI placement was determined using datasets from PET studies of the striatal dopamine and hippocampal serotonin systems. Each evaluated PET study could be registered to at least one synthetic PET target without obvious failure. Registration was critically dependent upon the initial position of the PET study relative to the synthetic PET target, but not dependent on the amount of synthetic PET target smoothing. An evaluation algorithm showed that resultant radioactivity concentration measurements of selected brain structures had errors=2% due to misalignment with the corresponding VOI. Further, radioligand binding values calculated from these measurements were found to be more precise than those calculated from measurements obtained with manually drawn regions of interest (ROIs). Overall, evaluation results demonstrated that this atlas-derived VOI method can be used to obtain unbiased measurements of radioactivity concentration from PET studies. Its automated features, and applicability to different radioligands and brain regions, will facilitate quantitative rat brain PET assessment procedures.

The neurotoxic effects of methamphetamine (METH) have been characterized primarily from the study of high-dose binge regimens in rodents. However, this drug administration paradigm does not include a potentially important feature of stimulant abuse in humans, that is, the gradual escalation of stimulant doses that frequently occurs prior to high-dose exposure. We have argued that pretreatment with escalating doses (EDs) might significantly alter the neurotoxic profile produced by a single high-dose binge. In the present study, we tested this hypothesis by pretreating rats with saline or gradually increasing doses of METH (0.1-4.0 mg/kg over 14 days), prior to an acute METH binge (4 x 6 mg/kg at 2 h intervals). These animals, whose behavior was continuously monitored throughout drug treatment, were then killed 3 days later for determination of caudate-putamen dopamine (DA) content, levels of [(3)H]WIN 35,428 binding to the DA transporter, and levels of [(3)H]dihydrotetrabenazine ([(3)H]DTBZ) binding to the vesicular monoamine transporter. ED pretreatment markedly attenuated the stereotypy response, as well as the hyperthermia and indices of sympathetic activation associated with the acute binge. In addition, ED pretreatment prevented the decline in [(3)H]WIN 35,428 binding, and significantly diminished the decrease in DA levels, but did not affect the decrease in [(3)H]DTBZ binding associated with the acute binge. We suggest that further study of the effects produced by a regimen which includes a gradual escalation of doses prior to high-dose METH binge exposure could more accurately identify the neurochemical and behavioral changes relevant to those that occur as a consequence of high-dose METH abuse in humans.

The protein neuronatin is expressed in the nervous system of the fetus and neonate at a much higher level than in the adult. Its function is unknown. As a result of variable splicing, neuronatin mRNA exists in two forms, alpha and beta. Wild type PC12 cells express neuronatin-alpha. We have isolated a PC12 variant, called 1.9, that retains many of the neuron-like properties of wild type PC12 cells, but it does not express neuronatin and it exhibits markedly increased sensitivity to the toxic effects of nigericin, rotenone and valinomycin. Pretreatment of the 1.9 cells with alpha-methyltyrosine, which inhibits dopamine synthesis, had little effect on the cells' sensitivity to nigericin, rotenone or valinomycin indicating that dopamine-induced oxidative stress was not involved in the toxicity of these compounds. However, flattened cell subvariants of the 1.9 cells, which do not have any neuron-specific characteristics, did not exhibit increased sensitivity to nigericin indicating that some neuronal characteristic of the 1.9 cells contributed to the toxicity of nigericin. After the neuronatin-beta gene was transfected into and expressed in the 1.9 cells, they regained wild type PC12 levels of resistance to nigericin, rotenone and valinomycin. These studies suggest that the function of neuronatin during development could be to protect developing cells from toxic insult occurring during that period.

To facilitate high-throughput 3D imaging of brain gene expression, a new method called voxelation has been developed. Spatially registered voxels (cubes) are analyzed, resulting in multiple volumetric maps of gene expression analogous to the images reconstructed in biomedical imaging systems. Using microarrays, 40 voxel images for 9000 genes were acquired from brains of both normal mice and mice in which a pharmacological model of Parkinson's disease (PD) had been induced by methamphetamine. Quality-control analyses established the reproducibility of the voxelation procedure. The investigation revealed a common network of coregulated genes shared between the normal and PD brain, and allowed identification of putative control regions responsible for these networks. In addition, genes involved in cell/cell interactions were found to be prominently regulated in the PD brains. Finally, singular value decomposition (SVD), a mathematical method used to provide parsimonious explanations of complex data sets, identified gene vectors and their corresponding images that distinguished between normal and PD brain structures, most pertinently the striatum.

The abuse of methamphetamine (METH) continues to increase throughout all age groups in different regions of the United States. "Ice," the popularized jargon for (+) methamphetamine hydrochloride, is the predominant drug form that is now consumed. "Ice" is effectively absorbed after either smoking or snorting and it is this rapid influx of drug that produces effects similar to those after intravenous administration. The intensity of METH actions in the central and peripheral nervous system shows tolerance after chronic administration, indicating that neuroadaptations have occurred. Thus, the physiological processes and corresponding biochemical mechanisms that regulate neuronal function have been changed by METH exposure. These biological alterations contribute to the craving and dependence associated with METH abuse and the withdrawal syndrome upon abstinence. However, these changes in behavior may also result from METH-induced neurotoxicity. This article reviews aspects of METH pharmacokinetics and related molecular pharmacodynamics that represent METH pharmacology and then relates those actions to their potential to produce neurotoxicity in humans.

Object: Radiosurgery for functional neurosurgery performed using a linear accelerator (LINAC) has not been extensively characterized in preclinical studies. In the present study, the properties of a newly designed 3-mm-diameter collimator were evaluated in a dedicated LINAC, which produced lesions in the basal ganglia of vervet monkeys. Lesion formation was determined in vivo in three animals by examining magnetic resonance (MR) images to show the dose-delivery precision of targeting and the geometry and extent of the lesions. Postmortem immunohistochemical studies were conducted to determine the extent of lesion-induced radiobiological effects., Methods: In three male vervet monkeys, the subthalamic nucleus (STN; one animal) and the pars compacta of the lateral substantia nigra (SN; two animals) were targeted by a Novalis Shaped Beam Surgery System that included a 3-mm collimator and delivered a maximum dose of 150 Gy. Magnetic resonance images obtained 4, 5, and 9 months posttreatment were reviewed, and the animals were killed so that immunohistological characterizations could be made., Conclusions: The generation of precise radiosurgical lesions by a 3-mm collimator was validated in studies that targeted the basal ganglia of the vervet monkey. The extent of the lesions created in all animals remained restricted in diameter (< 3 mm) throughout the duration of the studies, as assessed by reviewing MR images. Histological studies showed that the lesions were contained within the STN and SN target areas and that there were persistent increases in glial fibrillary acidic protein immunoreactivity. Increases in immunoreactivity for tyrosine hydroxylase, the serotonin transporter, and the GluR1 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate glutamate receptor in penumbral regions of the lesion were suggestive of compensatory neuronal adaptations. This radiosurgical approach may be of particular interest for the induction of lesions of the STN and SN in studies of experimental parkinsonism, as well as for the development of potential radiosurgical treatments for Parkinson disease.

For serial imaging studies of the rat brain with positron emission tomography (PET), reproducible positioning of the head can facilitate spatial alignment of images and quantitative analysis. To achieve this aim, we constructed a plastic head frame and tested the positioning reproducibility on a high-resolution small-animal PET scanner, microPET. Two sets of ear bars, with tapers of either 18 degrees (sharp) or 45 degrees (blunt), were evaluated for their relative precision in securing the animal to the frame. For sequential positioning of an animal, average distances from the mean position of 0.51 mm (SD 0.41 mm) and 0.91 mm (SD 0.48 mm) were measured with the sharp and blunt ear bars, respectively. These results show that a rat brain can be reproducibly positioned using the frame, with a variation of position less than the spatial resolution of modern animal PET scanners. Brain regions of interest defined on one scan and copied across subsequent scans of a frame-repositioned animal resulted in an average coefficient of variation of 5.4% (SD 2.7%) using the sharp ear bars and 6.8% (SD 2.5%) using the blunt ear bars. This methodology has the potential to improve quantitative assessment for serial PET studies.

Serotonin (5-HT) functions are associated with social dominance status in diverse species, but to date the brain regions wherein 5-HT exerts such effects are uncertain. Here, we indexed 5-HT turnover in male Anolis carolinensis as the ratio of 5-HT to its metabolite, 5-hydroxy-indol-acetic acid, and also as the accumulation of the in vivo tracer 14C-alpha-methyl-tryptophan (14C-AMT). After patching one eye, displaying dominant animals increased both measures of 5-HT turnover in the forebrain hemisphere receiving display-evocative visual stimuli, compared to control, contralateral brain, whereas both 5-HT turnover indices were decreased when animals displayed submissively. In contrast, various non-displaying controls showed forebrain symmetry on both measures. Drugs that stimulate 5-HT(2C) receptors in mammals, and have 5-HT(2C)-like binding in A. carolinensis, evoked some elements of dominant display behaviors in non-dominant anole males and also activated dorsolateral basal ganglia as seen in non-medicated dominants when they display [Baxter et al., 2001]. Thus, acute changes in forebrain 5-HT output from baseline equilibrium, acting at 5-HT(2C)-like receptors, might effect some elements of the dominant vs. submissive male anoles' territorial displays. A mechanistic model of how this might occur is offered. Given similarities in 5-HT systems, forebrain functions, and territorial display routines, similar mechanisms might have similar functions in other amniotes, including primates., (Copyright 2001 S. Karger AG, Basel)

Animal and human research suggests that the central serotonin system is involved in the inhibition of impulsive behavior. Two studies were designed to assess this relationship in male vervet monkeys (Cercopithecus aethiops sabaeus) using a standardized test of impulsivity in a social context: the Intruder Challenge. In the first study, an index of impulsivity in response to an unfamiliar adult male intruder (including latency to approach and aggressive and assertive interactions) was inversely correlated with levels of the serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in cisternal cerebrospinal fluid (r = -0.33, p <.01, n = 138). The approach, but not aggressive, component of the Impulsivity Index was the primary contributor to this relationship (partial r = -0.27, p <.01). The second experiment compared responses to the Intruder Challenge after 9 weeks of daily treatment with fluoxetine (2 mg/kg, i.m.) or vehicle. Fluoxetine-treated subjects (n = 6) had significantly lower Impulsivity Index scores than controls (n = 12). The results from these two investigations provide evidence for serotonergic influences on social impulsivity.

Although the behavioral consequences of methamphetamine (METH) abuse have been extensively documented, a more precise and thorough understanding of underlying neurobiological mechanisms still requires the use of animal models. To study these biochemical processes in experimental animals requires consideration for the broad range of human METH abuse patterns and the many factors that have been identified to profoundly influence the behavioral and neurochemical effects of exposure to METH-like stimulants. One potentially critical issue relates to pharmacokinetic differences between the species. In this review, METH plasma pharmacokinetic profiles after single and multiple dose intravenous METH administration are compared for the rat and human. Significant differences in elimination half-life between the two species (t1/2: rat-70 min, human-12 h) result in markedly dissimilar profiles of METH exposure. However, the plasma profile of a human METH binge pattern can be approximated in the rat by increasing METH dose frequency. Consideration of METH pharmacokinetics in animal models should permit a closer simulation of the temporal profile of METH exposure in the human CNS and should provide further insight into the mechanisms contributing to the addiciton and psychopathology associated with METH abuse.

After administration of methamphetamine (METH) (2x2 mg/kg, 6 h apart) to vervet monkeys, long term but reversible dopaminergic deficits were observed in both in vivo and post-mortem studies. Longitudinal studies using positron emission tomography (PET) with the dopamine transporter (DAT)-binding ligand, [11C]WIN 35,428 (WIN), were used to show decreases in striatal WIN binding of 80% at 1 week and only 10% at 1.5 years. A post-mortem characterization of other METH subjects at 1 month showed extensive decreases in immunoreactivity (IR) profiles of tyrosine hydroxylase (TH), DAT and vesicular monoamine transporter-2 (VMAT) in the striatum, medial forebrain bundle and the ventral midbrain dopamine (VMD) cell region. These IR deficits were not associated with a loss of VMD cell number when assessed at 1.5 years by stereological methods. Further, at 1.5 years, IR profiles of METH subjects throughout the nigrostriatal dopamine system appeared similar to controls although some regional deficits persisted. Collectively, the magnitude and extent of the dopaminergic deficits, and the subsequent recovery were not suggestive of extensive axonal degeneration followed by regeneration. Alternatively, this apparent reversibility of the METH-induced neuroadaptations may be related primarily to long-term decreases in expression of VMD-related proteins that recover over time.

This paper summarizes the present status of the researches conducted with [(18)F]4-(2'-methoxyphenyl)-1-[2'-[N-(2"-pyridinyl)-p-fluorobenzamido ]ethyl]-piperazine known as [(18)F]p-MPPF, a new 5-HT(1A) antagonist for the study of the serotonergic neurotransmission with positron emission tomography (PET). This includes chemistry, radiochemistry, animal data (rats, cats, and monkeys) with autoradiography and PET, human data with PET, toxicity, and metabolism.

The effects of glial cell line-derived neurotrophic factor (GDNF) pretreatment on methamphetamine (METH)-induced striatal dopamine system deficits in the vervet monkey were characterized with [(11)C]WIN 35,428 (WIN)-positron emission tomography (PET). WIN, a cocaine analog that binds to the dopamine transporter (DAT), was used to provide an index of striatal dopamine terminal integrity. In two subjects, GDNF (200 microg/40 microl) was injected into the caudate and putamen unilaterally vs. saline contralaterally. After 1-2 weeks, + and -GDNF striatal WIN-PET binding values were equivalent as calculated by multiple time graphic analysis, suggestive of an absence of unilateral DAT up-regulation. Three other subjects (n = 3) received GDNF injections into the caudate and putamen unilaterally and one week later, were administered METH HCl (2 x 2 mg/kg; i.m., 24 hours apart; a neurotoxic dosage for this species). At 1 week post-METH, WIN-PET studies showed that mean WIN binding was decreased by 72% in the +GDNF and by 92% in the -GDNF striatum relative to pre-drug assessment values. Thus, GDNF pretreatment reduced the extent of METH-induced decreases in WIN binding. Subsequent WIN-PET studies (1.5-9-month range) showed a protracted recovery of WIN binding in each striatum, indicative of long-term but partially reversible METH neurotoxicity. Further, at each time point, WIN binding remained relatively higher in the +GDNF vs. -GDNF striatum. These results provide further evidence that the adult non-human primate brain remains responsive to exogenously administered GDNF and that this pharmacotherapy approach can counteract aspects of neurotoxic actions associated with methamphetamine., (Copyright 2000 Wiley-Liss, Inc.)

We compared the behavioral and caudate-putamen extracellular dopamine responses following intravenous (3.6 mg/kg) and subcutaneous (8 mg/kg) amphetamine administration using 2-min microdialysate sampling intervals, and doses of the drug selected to achieve comparable maximal brain concentrations. Following intravenous amphetamine, dopamine peaked within the first 2 min, then declined with a first-order decay rate of 0.018+/-0.007 min(-1). Following subcutaneous amphetamine, dopamine achieved maximum concentrations at 9 min and remained near peak levels for about 30 min before declining with a first-order decay rate of 0.019+/-0.008 min(-1). Maximal brain amphetamine levels and peak dopamine concentrations were equivalent following either route of drug administration. In contrast to the short latency to maximal extracellular dopamine, the onset of oral stereotypies was delayed until about 30 min following both routes of drug administration. Furthermore, in contrast to the behavioral response to amphetamine, apomorphine administration resulted in the rapid appearance of oral stereotypies within 5-10 min after drug administration. These results suggest that although caudate-putamen dopamine receptor activation may be a critical factor in the expression of focused oral stereotypies, other effects of amphetamine may interfere with the ability of animals to exhibit these behaviors.

This study evaluated whether the caudate-putamen dopamine response that has been observed after deprenyl administration could be attributed exclusively to metabolically generated l-methamphetamine (l-MeAmp). Brain and plasma levels of deprenyl and l-MeAmp were measured after deprenyl (10 mg/kg s.c.) from 10 to 60 min in conscious rats. Peak caudate-putamen levels were observed for deprenyl (15 nmol/g) at 10 min and for l-MeAmp (3 nmol/g) at 30 min. In a parallel study, l-MeAmp metabolism was evaluated. After l-MeAmp (20 mg/kg s.c.), metabolite levels remained low relative to those of the parent compound: l-amphetamine, approximately 5 to 12%; and para-hydroxy-l-methamphetamine (OH-MeAmp), approximately 0.25%. Accordingly, l-MeAmp was considered to be the primary pharmacologically active deprenyl metabolite. A pharmacokinetic-pharmacodynamic analysis was then used to relate these pharmacokinetic data to the results of previous microdialysis studies in which increases in extracellular dopamine were measured in the caudate-putamen after l-MeAmp (3-18 mg/kg) and after deprenyl (10 mg/kg). Dopamine response-area under curve versus dose plots were generated and used to show that an administered dose of 4 mg/kg l-MeAmp would be necessary to effect a dopamine response-area under curve comparable to that observed after the deprenyl dose. However, the present pharmacokinetic results indicated that l-MeAmp brain levels after deprenyl corresponded to those that would be obtained from 0.4 mg/kg l-MeAmp (i.e., one tenth of the required dose). Collectively, these results suggest that the acute increases in extracellular dopamine observed after deprenyl are not due uniquely to metabolically generated l-MeAmp but also to other actions of deprenyl at the dopamine terminal.

[11C]WIN 35,428 (WIN), a cocaine analog that binds to the dopamine transporter (DAT), and positron emission tomography (PET) were used to evaluate the potential neuroprotective effects of dizocilpine (MK-801) on methamphetamine (MeAmp) induced neurotoxicity in the striatal dopamine system of the vervet monkey. MK-801 (1 mg/kg, i.m.) was administered 30 min prior to a neurotoxic MeAmp dosage for this species (2 x 2 mg/kg, 4 h apart); control subjects received MeAmp. MK-801 treated subjects were anesthetized by the drug for 6-8 h; throughout that period, a 2-3 degrees C decrease in body temperature was measured. At 1-2 weeks post-MeAmp, decreases of approximately 75% in striatal WIN binding were observed for both MK-801/MeAmp and MeAmp subjects. Thus, in this non-human primate species, the combination of MK-801 pretreatment and reduced body temperature did not provide protection from the MeAmp-induced loss of DAT. Further, the absence of an elevated body temperature during the acute MeAmp exposure period indicated that hyperthermia, per se, was not a necessary concomitant of the MeAmp neurotoxicity profile as has been previously demonstrated in rodents. These results provide evidence that different regulatory factors maintain the integrity of the rodent and primate striatal dopamine systems.

6-[F-18]Fluoro-L-3,4-dihydroxyphenylalanine (FDOPA) has been used to measure the central dopaminergic function in many species, including humans and monkeys. For transport across the blood brain barrier (BBB), FDOPA competes with plasma large neutral amino acids (LNAA). In this article we evaluate the effects of normal physiological LNAA concentration variation on BBB transport (K1) and the FDOPA uptake measurement, Ki. We also investigate a method for reducing the dependency of FDOPA quantitation on LNAA. Adult vervet monkeys (Cercopithecus aethiops sabaeus, n = 19) were fasted overnight before FDOPA positron emission tomography scans. Blood samples were drawn for LNAA determination, metabolite analysis, and compartmental modeling. The estimated K1 and Ki were both negatively correlated with LNAA concentrations (r2 = 0.51 and 0.62, respectively). Using an adjustment to K1 and Ki based on these correlations, the LNAA dependency was reduced (SD of the data for K1 was reduced by 33%, for Ki by 40%). Experiments with amino acid loading on an additional six animals indicate that BBB transport can be described using Michaelis-Menten kinetics. Results show a clear dependence of FDOPA uptake on plasma LNAA concentrations, which can be removed to increase the precision of FDOPA quantitation.

In six vervet monkeys, presynaptic striatal dopamine function was assessed longitudinally by [18F]fluoro-L-DOPA (FDOPA)-positron emission tomography (PET) after administration (2 x 2 mg/kg, i.m., 4 h apart) of either amphetamine (Amp), n = 3, or methamphetamine (MeAmp), n = 3. At 1-2 weeks postdrug, both Amp and MeAmp exposure effected similar decreases (60-70%) in the FDOPA influx rate constant (FDOPA Ki), an index of striatal dopamine synthesis capacity. Subsequent studies in these subjects showed that FDOPA Ki values were decreased by 45-67% at 3-6 weeks, by 25% at 10-12 weeks and by 16% in one Amp-treated subject at 32 weeks. Biochemical analysis showed that striatal dopamine concentrations were decreased by 75% at 3-4 weeks and by 55% at 10-12 weeks. These results indicate that in vervet monkey striatum, an acute Amp or MeAmp drug dosage produces extensive striatal dopamine system neurotoxicity. However, these effects were reversible; observed time-dependent recovery in both FDOPA Ki and dopamine concentrations indicates that neurochemical plasticity remains active in the adult primate striatum. At 3-4 and 10-12 weeks postdrug, the concurrent characterization of the striatal FDOPA Ki and dopamine concentrations for individual subjects showed that Ki decreases between 24 and 67% corresponded to dopamine depletions of 55-85%. These relatively larger postdrug decrements in steady-state striatal dopamine concentrations suggest that compensatory increases in dopamine synthesis capacity develop in the partially lesioned striatum. In contrast to the dopamine depletion in striatum, substantia nigra concentrations remained unchanged from referent values at both 3-4 and 10-12 weeks postdrug. Thus, the integrity of the substantia nigra could not be inferred from decreases in the striatal FDOPA Ki parameter. This disparity between striatum and substantia nigra reactivity to systemic administration of amphetamines suggests that each has unique dopamine system regulatory mechanisms.

To further delineate amphetamine-dopamine pharmacokinetic-pharmacodynamic relationships, we examined extracellular levels of dopamine and amphetamine in caudate-putamen after the s.c. administration of 8 mg/kg amphetamine. In a parallel group of animals, we also assessed caudate-putamen tissue levels of the drug. Extracellular concentrations of the transmitter and the drug exhibited similar temporal profiles, each achieving maximum concentrations within 30 min of drug administration. Tissue levels of amphetamine exhibited a similar, although slightly earlier time to maximum levels. The concentrations of amphetamine and dopamine in the extracellular fluid and amphetamine in tissue rapidly declined with similar rates of elimination. In contrast to the temporal profiles for both dopamine and amphetamine, stereotyped behaviors achieved maximum intensity at about 60 min. In addition, although transmitter and drug declined almost 10-fold from maximum values over the 4-hr interval after amphetamine administration, stereotyped behaviors persisted for at least 3 hr before abating. The results of these studies confirm our previous observation that the temporal profiles for stereotyped behaviors and extracellular dopamine are dissociated, and also extend this dissociation to extracellular amphetamine. In addition, although there was a close correspondence between dopamine and amphetamine within each experimental animal, individual animals exhibited a broad range of maximal dopamine responses, suggesting a differential responsiveness to amphetamine.

A chronic 10-day amphetamine (Amp) protocol was used to induce significant long-term decrements of the striatal [18F]fluoro-L-DOPA influx rate constant (FDOPA Ki) in the vervet monkey. Longitudinal FDOPA-positron emission tomography (PET) assessment in Amp-treated subjects subsequently revealed a gradual recovery of striatal dopamine function: FDOPA Ki values were decreased by approximately 70% at 1 month, approximately 45% at 6 months, approximately 20% at 12 months and were similar to pre-Amp values at 24 months. Motoric and social behavioral measures were obtained on all subjects within a species-typical group setting. Behavioral observations were conducted during both basal and stressor-challenge conditions, the latter being created by placing a potential intruder-animal in an individual cage adjacent to the subject's group enclosure. During basal conditions, post-Amp stereotypies were present at 2 weeks and locomotor behaviors were increased throughout 1 month; both alterations occurred while FDOPA Ki values were significantly decreased. Social behaviors were also significantly affected; affiliative behavior was decreased up to 6 months while aggressive behavior was increased for 12 months. However, a different pattern of behavioral changes emerged under stressor-challenge conditions. Motoric and social changes were of greater magnitude and persisted longer than in basal settings while aggressive behavior remained elevated at 24 months. These results indicate that chronic Amp-induced decreases in FDOPA Ki values and behavioral alterations are reversible. Changes in striatal dopamine function as indexed with FDOPA-PET are not correlated with post-Amp alterations in behaviors and moreover, expression of those behaviors is context-dependent.

Biochemical and behavioral criteria were established to determine the long-term stability of a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced unilateral striatal dopamine deficiency in the vervet monkey. At time points over a 12-month period, post-MPTP striatal dopamine synthesis capacity was indexed with 6-[18F]fluoro-L-DOPA (FDOPA)-positron emission tomography. For the MPTP-treated subjects (n = 4), an intrasubject FDOPA influx rate constant (Ki) ratio method of right (lesioned) striatum/left (unlesioned) striatum values was used to assess changes in striatal activity. Striatal FDOPA Ki ratios differed less than 5% between studies conducted at 1-2, 5-7, and 9-11 months post-MPTP; these results indicated a stable MPTP-induced striatal lesion over this time period. At the 5-7 and 9-11 month time points, behavioral indices of the MPTP-induced deficits were obtained within a species-typical group setting. For three of the four subjects, persistent decrements in motoric, affiliative, and vigilance behavior were observed while the frequency of aggression toward group members was increased. At the 9-11 month time point, one subject showed a 30% improvement in the social measures, indicative of a partial recovery from the MPTP-induced behavioral decrements although its striatal FDOPAKi ratio remained unchanged. Thus, behavioral and noninvasive biochemical methods can provide complementary indices to assess individual differences in sensitivity to MPTP-induced deficits. Both types of data are required to determine lesion stability and, subsequently, the efficacy of interventions designed to restore normal function in this primate Parkinsonian model.

In this work, we introduce 6-[18F]fluoro-L-m-tyrosine (6-FMT) and compare its in-vivo kinetic and bio-chemical behaviors in monkeys and rodents with those of 4-FMT and 6-[18F]fluoro-L-3, 4-dihydroxyphenylalanine (DOPA) (FDOPA). These radiofluorinated m-tyrosine presynaptic dopaminergic probes, resistant to peripheral 3-O-methylation, offer a nonpharmacological alternative to the use of catechol-O-methyltransferase inhibitors. Like FDOPA, 4-FMT and 6-FMT are analogs that essentially follow the L-DOPA pathway of central metabolism. After i.v. administration in nonhuman primates and rodents, these new radiofluorinated m-tyrosine analogs accumulate selectively in striatal structures and allow for the detection of additional innervation sites (e.g., brain stem) rich in aromatic amino acid decarboxylase. Bio-chemical analyses in rodents and monkeys revealed the specificity of their central and peripheral metabolism. Molecular and enzymatic mechanisms involved in their retention in central brain structures are consistent with involvement of dopaminergic neurons. The high signal-to-noise ratios observed make these radiofluorinated m-tyrosine analogs outstanding candidates for probing the integrity of central dopaminergic mechanisms in humans.

The acute and long-term effects of chronic amphetamine administration on the striatal dopamine system in monkeys were assessed with 6-[18F]fluoro-L-DOPA (FDOPA) and positron emission tomography (PET). Vervet monkeys (Cerecopithecus aethiops) were administered amphetamine doses, i.m., that increased from 4 mg/kg/d to 18 mg/kg/d over a 10 day period. Post-amphetamine FDOPA-PET scans at 1-2, 3-4, and 6 week time points in individual subjects showed persistent decrements in dopamine synthesis capacity as reflected by FDOPA influx rate constant (Ki) values being approximately 30% that of pre-drug assessment. In other animals that were administered the same drug regimen, biochemical analysis of striatal regions at 1-2 weeks post-drug indicated that dopamine concentrations were decreased by approximately 95% throughout caudate and putamen regions, while the homovanillic acid/dopamine level ratio was increased 3-10-fold. Post-drug FDOPA-PET Ki values remained consistently low up to 6 weeks; however, at the 5-6 month time point, relative increases in FDOPA-Ki values (approximately 53% of pre-drug values) were observed for all subjects, indicative of partial recovery of striatal dopamine synthesis capacity. These results demonstrate that FDOPA-PET can reveal temporal activity changes within the striatal dopamine system of individual subjects. The apparent, partial reversibility of amphetamine's neurotoxic effects suggests a plasticity of dopaminergic function that may include regeneration of dopaminergic terminals and compensatory increases in residual dopamine synthesis rates. The persistence of the partial decrement in dopamine synthesis capacity, however, may indicate a long term component of amphetamine's toxic effects.

To establish whether the actions of D-amphetamine (Amp) and D-methamphetamine (MeAmp) on the striatal dopamine system were equipotent, pharmacokinetic profiles of each drug were applied to an analysis of their respective induced dopamine efflux profiles. Amp or MeAmp (1 and 5 mg/kg i.v.) was administered to chloral hydrate-anesthetized rats; plasma and brain kinetics were then assessed from 5 to 60 min. Dose-dependent increases in Amp and MeAmp plasma levels resulted in proportional increases in striatum levels that were equivalent for both drugs; elimination rates also were similar and were characterized by a first-order decay process. After MeAmp administration, low levels of brain MeAmp metabolites were detected throughout the 1-hr time period; relative to MeAmp, Amp and p-hydroxy-MeAmp levels were less than 10 and 1%, respectively. The drug-induced dopamine efflux profiles in the striatum were characterized by microdialysis; Amp and MeAmp (1, 2.5 and 5 mg/kg i.v.) effected equivalent, dose-dependent increases in extracellular dopamine levels. For both drugs at 5- and 10-min postinjection, increases in drug striatum levels preceded increases in dopamine efflux. In contrast, from the time of the peak dopamine responses observed at 10 to 20 min until the end of the study at 90 min, changes in striatal drug levels were correlated with extracellular dopamine levels; this correlation was similar for both drugs. These results indicate that Amp and MeAmp pharmacokinetics and their subsequent dopamine responses in the striatum are equivalent. The pharmacokinetic analysis can be extended to the interpretation of other comparative studies that assess effects of Amp and MeAmp.(ABSTRACT TRUNCATED AT 250 WORDS)

To estimate the striatal uptake constant of [18F]-L-6-fluorodopa (FDOPA) in humans, we studied two methods that can account for the image resolution and the background level of FDOPA. These two methods utilize information obtained from multiple ROIs varying in size around the putamen and from profiles crossing the middle of the putamen. The estimation of the uptake constant was based on a model of one-dimensional activity variation. Simulated data were used to evaluate the adequacy of these two methods. Parametric images of FDOPA uptake constants were generated using Patlak analysis for five studies in normals and were then analyzed with the two methods. Results from the simulated data indicated a good agreement between the estimated values and the true simulated values. Results from studies in normals show stable estimates of the FDOPA uptake constant that are not affected by image resolution. The two methods were not sensitive to the misplacement of ROIs and profiles.

Carbidopa (L-alpha-hydrazino-alpha-methyl-b-(3,4-dihydroxyphenyl) propionic acid is a known inhibitor of aromatic amino acid decarboxylase. In both humans and monkeys, we studied the effects of carbidopa on plasma and brain kinetics of 6-[18F]fluoro-L-DOPA (FDOPA), an analog of L-DOPA used for PET studies of the central dopaminergic system. Pretreatment with carbidopa resulted in increases in the plasma levels of FDOPA and 3-O-methyl-6-[18F]fluoro-L-DOPA (3-OMFD). Total striatal and cerebellar activities measured with PET were also increased. Furthermore, increases observed in the specific striatal activity (striatum minus cerebellum total activity) were correlated with increases in the plasma FDOPA curve. Carbidopa pretreatment did not affect the influx rate constant (K) for FDOPA from plasma to striatum in humans as determined by Patlak graphical analysis. Thus, an increase in measured striatal tomographic activity was secondary to the increase in plasma FDOPA levels rather than as a result of changes in the FDOPA influx rate constant.

This report critically appraises methods for the synthesis of 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine (6-FDOPA) that are based on labelling by non-regioselective electrophilic fluorination, regioselective fluorodemetalation or nucleophilic substitution. Recommendations for the standardization of labelling procedures, the optimization of radiochemical yield and the assurance of product quality and safety are given. Studies of the metabolism of 6-FDOPA in vivo are also reviewed to emphasize the importance of the biochemical component of the development of this tracer for positron emission tomography (PET).

Kinetics of L-3,4-dihydroxy-6-[18F]fluorophenylalanine (FDOPA) in striatum and cerebellum were measured in 10 normal human subjects with positron emission tomography (PET) from 0 to 120 min after an intravenous bolus injection of the tracer. The time course of the arterial plasma concentrations of the tracer and its metabolites was also assayed biochemically. FDOPA compartmental models that are based on biochemical information were investigated for their consistency with the measured striatal and cerebellar tissue kinetics. A modeling approach was also developed for separating plasma FDOPA and metabolite time-activity curves from the measured total 18F time-activity curve in plasma. Results showed that a model consisting of three separate compartments for tissue FDOPA, tissue 6-[18F]fluorodopamine (FDA) and its metabolites, and tissue L-3,4-dihydroxy-6-[18F]fluoro-3-O-methylphenylalanine (3-OMFD) could describe adequately the striatal kinetics in humans. Based on this model, the FDOPA transport constant across the blood-brain barrier (BBB) (K1), the FDOPA decarboxylation rate constant (k3), and the turn-over rate constant of FDA and its metabolites (k4) could be estimated by model fitting to the tissue kinetics and were found for the normal subjects to be 0.031 +/- 0.006 ml/min/g (mean +/- SD), 0.041 +/- 0.015/min, and 0.004 +/- 0.002/min, respectively. About 50% of the FDOPA that crossed the BBB from plasma to striatum was decarboxylated. The decarboxylation constant with respect to plasma FDOPA (K3) was 0.015 +/- 0.003 ml/min/g. The BBB transport corresponded to a permeability-surface area product of 0.032 ml/min/g for FDOPA. For 3-OMFD, the BBB transport was 1.7 times faster. The effects of tissue heterogeneity on the FDOPA kinetics and on the estimated model parameters were also investigated. The usefulness and implications of these findings for interpretation of PET FDOPA studies are discussed.

Characterization of peripheral and cerebral L-3,4-dihydroxy-6-[18F]fluorophenylalanine (FDOPA) metabolism in humans and monkeys has shown FDOPA to be an analogue of L-DOPA for the study of the dopaminergic system with positron emission tomography (PET). In human studies with carbidopa pretreatment, L-3,4-dihydroxy-6-[18F]fluoro-3-O-methylphenylalanine (3-OMFD) was the only FDOPA metabolite detected in plasma. FDOPA administration in monkeys resulted in selective accumulation of FDOPA metabolites in central dopaminergic regions, whereas 3-OMFD of peripheral origin was uniformly distributed among putamen, caudate, frontal cortex, and cerebellum. At 60 min, 3-OMFD and 6-[18F]fluorodopamine (FDA) each represented approximately 35% of the total activity, the remainder being FDOPA and FDA metabolites. These data on monkey and human FDOPA metabolism provide the basis for the configuration of an FDOPA tracer kinetic model with PET.