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Parkinson's disease (PD) is characterized by a prominent degeneration of nigrostriatal dopamine (DA) neurons with an accompanying neuroinflammation. Despite clinical and preclinical studies of neuroprotective strategies for PD, there is no effective treatment for preventing or slowing the progression of neurodegeneration. The inverse correlation between caffeine consumption and risk of PD suggests that caffeine may exert neuroprotection. Whether caffeine is neuroprotective in a chronic progressive model of PD has not been evaluated nor is it known if delayed caffeine treatment can stop DA neuronal loss. We show that a chronic unilateral intra-cerebroventricular infusion of 1-methyl-4-phenylpyridinium in the rat brain for 28 days produces a progressive loss of DA and tyrosine hydroxylase in the ipsilateral striatum and a loss of DA cell bodies and microglial activation in the ipsilateral substantia nigra. Chronic caffeine consumption prevented the degeneration of DA cell bodies in the substantia nigra. Importantly, neuroprotection was still apparent when caffeine was introduced after the onset of the neurodegenerative process. These results add to the clinical relevance for adenosine receptors as a disease-modifying drug target for PD.

Zonisamide is an FDA-approved antiepileptic drug that blocks voltage-dependent Na(+) channels and T-type Ca(2+) channels and improves clinical outcome in Parkinson's disease (PD) patients when used as an adjunct to other PD therapies. Zonisamide also modifies dopamine (DA) activity, provides protection in ischemia models and influences antioxidant systems. Thus, we tested it for its ability to protect DA neurons in a mouse model of PD and investigated mechanisms underlying its protection. Concurrent treatment of mice with zonisamide and 1-methyl-4-phenyl-1,2,3,6-tetraydropyridine (MPTP) attenuated the reduction in striatal contents of DA, its metabolite DOPAC and tyrosine hydroxylase (TH). We also discovered that zonisamide inhibited monoamine oxidase B (MAO-B) activity in vitro with an IC(50) of 25 muM, a concentration that is well within the therapeutic range used for treating epilepsy in humans. Moreover, the irreversible binding of systemically administered selegiline to MAO-B in mouse brain was attenuated by zonisamide as measured by ex vivo assays. Zonisamide treatment alone did not produce any lasting effects on ex vivo MAO-B activity, indicating that it is a reversible inhibitor of the enzyme. Consistent with the effects of zonisamide on MAO-B, the striatal content of 1-methyl-4-phenylpyridinium (MPP(+)), which is derived from the administered MPTP via MAO-B actions, was substantially reduced in mice treated with MPTP and zonisamide. The potency and reversibility with which zonisamide blocks MAO-B may contribute to the ability of the drug to improve clinical symptoms in PD patients. The results also suggest that caution in its use may be necessary, especially when administered with other drugs, in the treatment of epilepsy or PD.

[3H]Dihydrotetrabenazine ([3H]DTBZ), a specific ligand for the vesicular monoamine transporter (VMAT2), has been used to characterize the integrity of monoaminergic nerve terminals in experimental animals and humans. The purpose of the present studies was to compare the loss of VMAT2 binding with the loss of other neurochemical markers of the dopamine (DA) nerve terminals in mice treated with neurotoxic doses of methamphetamine (METH) or MPTP. Profound decreases (> or =70%) in DA content, tyrosine hydroxylase activity, and PH]carbomethoxy-3-(4-fluorophenyl)tropane binding to the DA transporter were observed in striatal homogenates at both 1 and 6 days after exposure to the neurotoxins. It is surprising that no significant loss of [3H]DTBZ binding in the homogenates was observed at 1 day after exposure, although a significant loss (-50%) was apparent 6 days later. However, in isolated vesicle preparations, [3H]DTBZ binding and active [3H]DA uptake were markedly reduced (>70%) at 1 day. These observations indicate that vesicle function is compromised at an early time point after exposure to neurotoxic insult. Furthermore, the changes in [H]DTBZ binding in homogenates may not be a sensitive indicator of early damage to synaptic vesicles, although homogenate binding reliably identifies a loss of VMAT2 at later times.

Na(+)/H(+) exchanger (NHE) proteins are involved in intracellular pH and volume regulation and may indirectly influence neurotransmission. The abundant NHE isoform 1 (NHE1) has also been linked to brain cell damage during metabolic stress. It is not known, however, whether NHE1 or other NHE isoforms play a role in striatal dopamine (DA) neurotransmission under normal or metabolic stress conditions. Our study tested the hypothesis that NHE inhibition with cariporide mesilate (HOE-642) modifies striatal DA overflow and DAergic terminal damage in mice caused by the mitochondrial inhibitor malonate. We also explored the expression of NHE1-5 in the striatum and substantia nigra. Reverse microdialysis of HOE-642 elicited a transient elevation followed by a reduction in DA overflow accompanied by a decline in striatal DA content. HOE-642 pre-treatment diminished the malonate-induced DA overflow without reducing the intensity of the metabolic stress or subsequent DAergic axonal damage. Although NHE isoforms 1-5 are expressed in the striatum and midbrain, NHE1 protein was not co-located on nigrostriatal DAergic neurons. The absence of NHE1 co-location on DAergic neurons suggests that the effects of HOE-642 on striatal DA overflow are either mediated via NHE1 located on other cell types or that HOE-642 is acting through multiple NHE isoforms.

Many genes involved in brain development have been associated with human neurodevelopmental disorders, but underlying pathophysiological mechanisms remain undefined. Human genetic and mouse behavioral analyses suggest that ENGRAILED-2 (EN2) contributes to neurodevelopmental disorders, especially autism spectrum disorder. In mouse, En2 exhibits dynamic spatiotemporal expression in embryonic mid-hindbrain regions where monoamine neurons emerge. Considering their importance in neuropsychiatric disorders, we characterized monoamine systems in relation to forebrain neurogenesis in En2-knockout (En2-KO) mice. Transmitter levels of serotonin, dopamine and norepinephrine (NE) were dysregulated from Postnatal day 7 (P7) to P21 in En2-KO, though NE exhibited the greatest abnormalities. While NE levels were reduced ∼35% in forebrain, they were increased 40 -: 75% in hindbrain and cerebellum, and these patterns paralleled changes in locus coeruleus (LC) fiber innervation, respectively. Although En2 promoter was active in Embryonic day 14.5 -: 15.5 LC neurons, expression diminished thereafter and gene deletion did not alter brainstem NE neuron numbers. Significantly, in parallel with reduced NE levels, En2-KO forebrain regions exhibited reduced growth, particularly hippocampus, where P21 dentate gyrus granule neurons were decreased 16%, suggesting abnormal neurogenesis. Indeed, hippocampal neurogenic regions showed increased cell death (+77%) and unexpectedly, increased proliferation. Excess proliferation was restricted to early Sox2/Tbr2 progenitors whereas increased apoptosis occurred in differentiating (Dcx) neuroblasts, accompanied by reduced newborn neuron survival. Abnormal neurogenesis may reflect NE deficits because intra-hippocampal injections of β-adrenergic agonists reversed cell death. These studies suggest that disruption of hindbrain patterning genes can alter monoamine system development and thereby produce forebrain defects that are relevant to human neurodevelopmental disorders.

Mitochondrial dysfunction is observed in sporadic Parkinson's disease (PD) and may contribute to progressive neurodegeneration. While acute models of mitochondrial dysfunction have been used for many years to investigate PD, chronic models may better replicate the cellular disturbances caused by long-standing mitochondrial derangements and may represent a better model for neurotherapeutic testing. This study sought to develop a chronic model of PD that has the advantages of continuous low level toxin delivery, low mortality, unilateral damage to minimize aphagia and adipsia as well as minimal animal handling to reduce stress-related confounds. Infusion by osmotic minipump of the complex I toxin, 1-methyl-4-phenylpyridinium (MPP+), for 28 days into the left cerebral ventricle in rats caused a selective ipsilateral loss of nigral tyrosine hydroxylase immunoreactive somata (35% loss). In animals that were sacrificed 14 days after the chronic MPP+ administration, there was an even greater loss of nigral tyrosine hydroxylase cells (65% loss). Lewy-body-like structures that stained positive for ubiquitin and alpha-synuclein were found in striatal neurons near the infusion site but were not observed in nigral neurons. At the electron microscope level, however, swollen and abnormal mitochondria were observed in the nigral dopamine neurons, which may represent the early formation of an inclusion body. There were no animal deaths with the chronic treatment regimen that was utilized, and the magnitude of nigrostriatal neuronal loss was relatively consistent among the animals. This model of progressive neurodegeneration of nigrostriatal dopamine neurons may be useful for studying neuroprotective therapeutic agents for PD.

Mitochondrial dysfunction may contribute to dopaminergic (DAergic) cell death in Parkinson's disease and GABAergic cell death in Huntington's disease. In the present work, we tested whether blocking A1receptors could enhance the damage to DAergic and GABAergic neurons caused by mitochondrial inhibition, and whether blocking A2areceptors could protect against damage in this model. Animals received an intraperitoneal injection of 8-cyclopentyl-1,3-dipropylxanthine (CPX) (A1antagonist) or 3,7-dimethyl-1-propargylxanthine (DMPX) (A2aantagonist) 30 min before intrastriatal infusion of malonate (mitochondrial complex II inhibitor). Damage was assessed 1 week later by measuring striatal dopamine, tyrosine hydroxylase (TH), and GABA content. In mice and rats, malonate-induced depletion of striatal dopamine, TH, or GABA was potentiated by pretreatment with 1 mg/kg CPX and attenuated by pretreatment with 5 mg/kg DMPX. To determine the location of the A1and A2areceptors mediating these effects, CPX or DMPX was infused directly into the striatum or substantia nigra of rats 30 min before intrastriatal infusion of malonate. When infused into the striatum, CPX (20 ng) potentiated, whereas DMPX (50 ng) prevented malonate-induced GABA loss, but up to 100 ng of CPX or 500 ng of DMPX did not alter malonate-induced striatal dopamine loss. Intranigral infusion of CPX (100 ng) or DMPX (500 ng), however, did exacerbate and protect, respectively, against malonate-induced striatal dopamine loss. Thus, A1receptor blockade enhances and A2areceptor blockade protects against damage to DAergic and GABAergic neurons caused by mitochondrial inhibition. Interestingly, these effects are mediated by A1and A2areceptors located in the substantia nigra for DAergic neurons and in the striatum for GABAergic neurons.

Firing during sensorimotor exam was used to categorize single neurons in the lateral striatum of awake, unrestrained rats. Five rats received unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle to deplete striatal dopamine (DA; >98% depletion, postmortem assay). Three months after treatment, rats exhibited exaggerated rotational behavior induced by L-dihydroxyphenylalanine (L-DOPA) and contralateral sensory neglect. Electrode track “depth profiles” on the DA-depleted side showed fragmented clustering of neurons related to sensorimotor activity of single body parts (SBP neurons). Clusters were smaller than normal, and more SBP neurons were observed in isolation, outside of clusters. More body parts were represented per unit volume. No recovery in these measures was observed up to one year post lesion. Overall distributions of neurons related to different body parts were not altered. The fragmentation of SBP clusters after DA depletion indicates that a percentage of striatal SBP neurons switched responsiveness from one body part to one or more different body parts. Because the specific firing that characterizes striatal SBP neurons is mediated by corticostriatal inputs (Liles and Updyke [1985] Brain Res. 339:245–255), the data indicate that DA depletion resulted in a reorganization of corticostriatal connections, perhaps via unmasking or sprouting of connections to adjacent clusters of striatal neurons. After reorganization, sensory activity in a localized body part activates striatal neurons that have switched to that body part. In turn, switched signals sent from basal ganglia to premotor and motor neurons, which likely retain their original connections, would create mismatches in these normally precise topographic connections. Switched signals could partially explain parkinsonian deficits in motor functions involving somatosensory guidance and their intractability to L-DOPA therapy—particularly if the switching involves sprouting. J. Comp. Neurol. 452:24–37, 2002. © 2002 Wiley-Liss, Inc.

Previous work has shown that overstimulation of GABA(A) receptors can potentiate neuronal cell damage during excitotoxic or metabolic stress in vitro and that GABA(A) antagonists or GABA transport blockers are neuroprotective under these situations. Malonate, a reversible succinate dehydrogenase/mitochondrial complex II inhibitor, is frequently used in animals to model cell loss in neurodegenerative diseases such as Parkinson's and Huntington's diseases. To determine if GABA transporter blockade during mitochondrial impairment can protect neurons in vivo as compared with in vitro studies, rats received a stereotaxic infusion of malonate (2 micromol) into the left striatum to induce a metabolic stress. The nonsubstrate GABA transport blocker, NO711 (20 nmol) was infused in some rats 30 min before and 3 h following malonate infusion. After 1 week, dopamine and GABA levels in the striata were measured. Malonate caused a significant loss of striatal dopamine and GABA. Blockade of the GABA transporter significantly attenuated GABA, but not dopamine loss. In contrast with several in vitro reports, GABA(A) receptors were not a downstream mediator of protection by NO711. Intrastriatal infusion of malonate (2 micromol) plus or minus the GABA(A) receptor agonist muscimol (1 micromol), the GABA(A) Cl- binding site antagonist picrotoxin (50 nmol) or the GABA(B) receptor antagonist saclofen (33 nmol) did not modify loss of striatal dopamine or GABA when examined 1 week following infusion. These data show that GABA transporter blockade during mitochondrial impairment in the striatum provides protection to GABAergic neurons. GABA transporter blockade, which is currently a pharmacological strategy for the treatment of epilepsy, may thus also be beneficial in the treatment of acute and chronic conditions involving energy inhibition such as stroke/ischemia or Huntington's disease. These findings also point to fundamental differences between immature and adult neurons in the downstream involvement of GABA receptors during metabolic insult.

The vesicular monoamine transporter in the brain can sequester the neurotoxin 1-methyl-4-phenylpyridinium into synaptic vesicles and protect catecholamine-containing neurons from degeneration. Mouse nigrostriatal dopaminergic neurons, and to a lesser extent locus coeruleus noradrenergic neurons, are vulnerable to toxicity produced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. The present study sought to determine whether pharmacological inactivation of the vesicular monoamine transporter in the brain would enhance the degeneration of substantia nigra dopaminergic neurons and locus coeruleus noradrenergic neurons in 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine-treated animals. Mice were treated subacutely with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine alone, or in combination with vesicular monoamine transporter inhibitors (tetrabenazine or Ro4-1284), and 10-24 days later striatal dopamine and cortical norepinephrine levels were measured using chromatographic methods. In the same animals, substantia nigra and locus coeruleus catecholaminergic neurons were counted using tyrosine hydroxylase immunohistochemical staining with computer imaging techniques. Mice in which pharmacological blockage of the vesicular monoamine transporter enhanced the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in the depletion of striatal dopamine concentrations also exhibited enhanced degeneration of substantia nigra dopaminergic neurons. In the same animals, however, vesicular monoamine transporter blockade did not enhance the effects of 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine in the locus coeruleus noradrenergic system. These data are consistent with the hypothesis that the vesicular monoamine transporter can protect catecholamine-containing neurons from 1-methyl-4-phenylpyridinium-induced degeneration by sequestration of the toxin within brain vesicular monoamine transporter-containing synaptic vesicles. Since the amount of vesicular monoamine transporter in locus coeruleus neurons is more than in substantia nigra neurons, and because 1-methyl-4-phenylpyridinium is sequestered within locus coeruleus neurons to a far greater extent than within substantia nigra neurons, it may be that a greater amount of vesicular monoamine transporter inhibition is required for 1-methyl-4-phenylpyridinium to be toxic to locus coeruleus neurons than to substantia nigra dopaminergic neurons.

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine produces a parkinsonian syndrome in man and experimental animals. The toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1-methyl-4-phenylpyridinium, exhibits high-affinity uptake by plasma membrane monoamine transporters and also by the vesicular monoamine transporter. Using autoradiographic and immunohistochemical methods in mice, we demonstrate the accumulation of [ 3 H]1-methyl-4-phenylpyridinium within neurons that contain the vesicular monoamine transporter, following systemic administration of [ 3 H]1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Within 1–24 h following the intraperitoneal administration of 10 μg/kg of [ 3 H]1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, [ 3 H]1-methyl-4-phenylpyridine labelling was found within such regions as the locus coeruleus, dorsal, medial, and pallidal raphe nuclei, substantia nigra pars compacta, ventral tegmental area, and paraventricular nucleus of the hypothalamus. These regions all contain monoaminergic somata as defined by immunohistochemical staining with an antibody against the vesicular monoamine transporter. There was a positive relationship between the density of [ 3 H]1-methyl-4-phenylpyridinium label and the density of vesicular monoamine transporter immunoreactivity: the highest densities of both were found in the locus coeruleus and lowest densities in the midbrain dopaminergic neurons. In addition, [ 3 H]1-methyl-4-phenylpyridinium labelling was detected in the bed nucleus of the stria terminalis and paraventricular nucleus of the thalamus, which also contained vesicular monoamine transporter immunoreactive nerve terminals. The present data indicate that low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine cause a significant accumulation of 1-methyl-4-phenylpyridinium within monoaminergic somata in parallel with theamount of vesicular monoamine transporter in the neuron. Since nuclei with intense labelling are not damaged by doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine that are toxic to midbrain dopaminergic neurons, these data are consistent with the hypothesis that sequestration of 1-methyl-4-phenylpyridinium within monoaminergic synaptic vesicles can protect the neurons from degeneration caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

This study examined whether damage to dopamine (DA) nerve terminals via inhibition of energy metabolism in the striatum would result in the retrograde loss of cell bodies in the substantia nigra. Infusion of 2 micromol malonate into the left striatum of rats resulted in a 67% loss of striatal DA and a 40% loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra. No change in the number of Nissl-positive-TH-negative neurons was observed. These findings demonstrate the retrograde destruction of DA cell bodies in the substantia nigra resulting from energy impairment at their terminal projection site.

Consumption of coffee is associated with reduced risk of Parkinson's disease (PD), an effect that has largely been attributed to caffeine. However, coffee contains numerous components that may also be neuroprotective. One of these compounds is eicosanoyl-5-hydroxytryptamide (EHT), which ameliorates the phenotype of α-synuclein transgenic mice associated with decreased protein aggregation and phosphorylation, improved neuronal integrity and reduced neuroinflammation. Here, we sought to investigate if EHT has an effect in the MPTP model of PD. Mice fed a diet containing EHT for four weeks exhibited dose-dependent preservation of nigral dopaminergic neurons following MPTP challenge compared to animals given control feed. Reductions in striatal dopamine and tyrosine hydroxylase content were also less pronounced with EHT treatment. The neuroinflammatory response to MPTP was markedly attenuated, and indices of oxidative stress and JNK activation were significantly prevented with EHT. In cultured primary microglia and astrocytes, EHT had a direct anti-inflammatory effect demonstrated by repression of lipopolysaccharide-induced NFκB activation, iNOS induction, and nitric oxide production. EHT also exhibited a robust anti-oxidant activity in vitro. Additionally, in SH-SY5Y cells, MPP(+)-induced demethylation of phosphoprotein phosphatase 2A (PP2A), the master regulator of the cellular phosphoregulatory network, and cytotoxicity were ameliorated by EHT. These findings indicate that the neuroprotective effect of EHT against MPTP is through several mechanisms including its anti-inflammatory and antioxidant activities as well as its ability to modulate the methylation and hence activity of PP2A. Our data, therefore, reveal a strong beneficial effect of a novel component of coffee in multiple endpoints relevant to PD.

The calcium-binding protein calbindin-D28k (CB) is located in midbrain dopaminergic (DA) neurons that are less vulnerable to degeneration in Parkinson's disease and in an animal model of the disorder, the MPTP-treated monkey. The present study sought to determine whether CB-containing DA neurons are also less vulnerable to degeneration in the MPTP-treated mouse. Double-labelling immunocytochemical staining and computer imaging techniques were employed to map and quantify the tyrosine hydroxylase-, CB- and CB-containing tyrosine hydroxylase neurons in portions of nucleus A9 and nucleus A10 (ventral tegmental area and central linear nucleus) following MPTP treatment in the C57BL/6 mouse. A cumulative dose of 140 mg/kg MPTP produced a significantly greater loss of DA neurons that lack CB in both nucleus A9 (71 +/- 4%) and the ventral tegmental area (70 +/- 4%), compared to the loss of DA neurons that contain CB (44 +/- 6% and 25 +/- 14%, respectively). In the central linear nucleus there was no loss of CB-containing DA neurons. These data demonstrate that the presence of CB in midbrain DA neurons identifies a population of cells in the mouse that are less vulnerable to MPTP-induced degeneration. The mouse, therefore, can serve as a useful model in which to investigate the putative neuroprotective effects of CB in an animal model of Parkinson's disease.

The neurotoxin MPTP has been used to create an animal model of Parkinson's disease in the mouse, in part, because it causes a significant loss of dopaminergic neurons in the substantia nigra (nucleus A9). The purpose of the present study was to determine whether MPTP also causes degeneration of midbrain dopaminergic neurons in nuclei A8 and A10 in the mouse, as occurs in humans with Parkinson's disease. Two commonly used strains of mice were used: FVB/N and C57BL/6. MPTP was administered in cumulative doses of 50—300 mg/kg. Seven days later, dopamine concentrations were measured in the striatum using high performance liquid chromatography, and midbrain dopaminergic neurons were identified using an antibody against tyrosine hydroxylase. The cell locations were mapped with a computer imaging system. In the FVB/N strain, there was a dose-dependent decrease in striatal dopamine concentrations. Although the highest dose (300 mg/kg) caused an 86% reduction in striatal dopamine concentrations, there was only a moderate and non-significant loss of midbrain dopaminergic neurons. In the C57BL/6 strain, however, a high dose of MPTP (240 mg/kg) caused a significant reduction in both striatal dopamine concentrations (95%), and midbrain dopaminergic cells; 69% loss of nucleus A8 cells, 75% loss of nucleus A9 cells, and in nucleus A10 subnuclei there was 42% loss of ventral tegmental area cells, 55% loss of interfascicular nucleus cells, and no loss of cells in the central linear nucleus. These data (1) provide further evidence for differential susceptibility to MPTP toxicity among different mouse strains, (2) indicate that a significant depletion of striatal dopamine is not necessarily due to degeneration of midbrain dopaminergic neurons, (3) provide the precise locations of midbrain dopaminergic cells that are vulnerable to MPTP, which will aid future studies that seek to determine the mechanism/s by which MPTP selectively destroys only certain midbrain dopaminergic neurons, and (4) indicate that MPTP produces midbrain dopaminergic neuronal degeneration in the same nuclei in the C57BL16 mouse that degenerate in humans with Parkinson's disease..

The Brandel Superfusion 600 is an automated system designed for the simultaneous measurement of neurotransmitter release from tissue slices from six chambers. In optimizing a technique for the measurement of endogenous dopamine (DA) with this system, a number of factors were identified which affected adversely both the accuracy and the precision of within and between experiments. These factors included bubbles in the perfusion chamber, an apparent loss of tissue viability during preparation of the slices, depletion of DA stores after multiple stimulations and degradation of DA in the perfusate after collection. The method was substantially improved with modifications in the Superfusion 600 system and by the addition of high Mg2+ to the preparation buffer. The addition of tyrosine to the perfusion buffer and the prevention of analyte degradation with antioxidant agents also improved accuracy and precision of measurements.

Studies were conducted to determine if treatment of mice with methamphetamine (METH) would produce a loss of dopaminergic cells in the substantia nigra. The number of TH+/Nissl-stained was significantly decreased in both Swiss-Webster (S-W) and C57bl mice (approx. cell loss of 40% and 45%, respectively) 5-8 days after treatment with METH. In these same mice there was a corresponding decrease in neostriatal dopamine (DA) content (90% and 92%, respectively). In parallel studies, treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produced similar neuropathological effects. The finding that nigral cell loss occurs after METH treatment indicates that the METH-treated mouse may be a very relevant model of Parkinson's disease (PD).

The calcium-binding protein calbindin-D28k (CALB) has been localized in high concentrations in several neuronal populations within the central nervous system (CNS) and is believed to act as an intracellular calcium (Ca2+) buffer. There has been much interest and speculation concerning its potential neuroprotective function. However, there is little direct evidence linking CALB content of individual neurons to Ca2+ buffering ability, resistance to Ca(2+)-mediated excitotoxicity, or vulnerability to Ca(2+)-mediated degeneration. It is necessary to demonstrate these relationships on a cellular level so that more definitive conclusions can be made. We have utilized immunocytochemical and Western blot techniques to determine whether cellular CALB content is altered in the nucleus A10 dopaminergic region of the midbrain following administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our data demonstrate a significant increase in the CALB content of nucleus A10 neurons (up to 227 +/- 23% above control) 3 and 6 h after MPTP treatment. CALB elevation demonstrated both time and dosage dependence as 6-h groups exhibited larger increases than 3-h groups, and a 60 mg/kg dosage induced a larger increase than a 20 mg/kg dosage. These data support the hypothesis that MPTP is neurotoxic by causing increases in free intracellular Ca2+ and that increased CALB in the midbrain dopaminergic neurons is a protective response to elevated intracellular free Ca2+.

Endogenous striatal dopamine (DA) overflow has been associated with neuropathological conditions resulting from ischemia, psychostimulants, and metabolic inhibition. Malonate, a reversible inhibitor of succinate dehydrogenase, models the effects of energy impairment in neurodegenerative disorders. We have previously reported that the striatal DA efflux and damage to DA nerve terminals resulting from intrastriatal malonate infusions is prevented by prior DA depletion, suggesting that DA plays a role in the neuronal damage. We presently report that the malonate-induced DA efflux is partially mediated by reverse transport of DA from the cytosol to the extracellular space via the DA transporter (DAT). Pharmacological blockade of the DAT with a series of structurally different inhibitors [cocaine, mazindol, 1-(2-(bis(4-fluophenyl methoxy) ethyl)-4-(3-(4-fluorophenyl)-propyl)piperazine) dimethane sulfonate (GBR 13098) and methyl(-)-3beta-(p-fluorophenyl)-1alphaH,5alphaH-tropane-2beta-carboxylate1,5-naphthalene (Win 35,428)] attenuated malonate-induced DA overflow in vivo and protected mice against subsequent damage to DA nerve terminals. Consistent with these findings, the DAT inhibitors prevented malonate-induced damage to DA neurons in mesencephalic cultures and also protected against the loss of GABA neurons in this system. The DAT inhibitors did not modify malonate-induced formation of reactive oxygen species or lactate production, indicating that the DAT inhibitors neither exert antioxidant effects nor interfere with the actions of malonate. Taken together, these findings provide direct evidence that mitochondrial impairment and metabolic stress cause striatal DA efflux via the DAT and suggest that disruptions in DA homeostasis resulting from energy impairment may contribute to the pathogenesis of neurodegenerative diseases.

Caffeine and more specific antagonists of the adenosine A(2A) receptor recently have been found to be neuroprotective in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease. Here we show that 8-(3-chlorostyryl)caffeine (CSC), a specific A(2A) antagonist closely related to caffeine, also attenuates MPTP-induced neurotoxicity. Because the neurotoxicity of MPTP relies on its oxidative metabolism to the mitochondrial toxin MPP(+), we investigated the actions of CSC on striatal MPTP metabolism in vivo. CSC elevated striatal levels of MPTP but lowered levels of the oxidative intermediate MPDP(+) and of MPP(+), suggesting that CSC blocks the conversion of MPTP to MPDP(+) in vivo. In assessing the direct effects of CSC and A(2A) receptors on monoamine oxidase (MAO) activity, we found that CSC potently and specifically inhibited mouse brain mitochondrial MAO-B activity in vitro with a K(i) value of 100 nm, whereas caffeine and another relatively specific A(2A) antagonist produced little or no inhibition. The A(2A) receptor independence of MAO-B inhibition by CSC was further supported by the similarity of brain MAO activities derived from A(2A) receptor knockout and wild-type mice and was confirmed by demonstrating potent inhibition of A(2A) receptor knockout-derived MAO-B by CSC. Together, these data indicate that CSC possesses dual actions of MAO-B inhibition and A(2A) receptor antagonism, a unique combination suggesting a new class of compounds with the potential for enhanced neuroprotective properties.

The vesicular monoamine transporter 2 (VMAT2) has sequence homology with bacterial multidrug transporters which in turn share homology with mammalian P-glycoprotein (P-GP). Both VMAT2 and P-GP can detoxify cells. 1-Methyl-4-phenylpyridinium (MPP + ), the toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is a substrate for VMAT2 that has several structural features in common with P-GP substrates and inhibitors. The present studies investigated whether P-GP is responsible for the elimination of MPP + from the brain. Additionally, VMAT2 and P-GP are inhibited by many of the same compounds. Thus we also investigated whether VMAT2 inhibitors could block P-GP in vitro and vice versa whether P-GP inhibitors could block VMAT2 mediated transport of [ 3 H]-DA into synaptic vesicles. In mice treated with MPTP and a P-GP inhibitor (quinidine, trans -flupentixol or cyclosporine A), the elimination of MPP + from the striatum was significantly delayed. However, in experiments using various cell lines expressing either mouse or human P-GP, MPP + did not reverse the P-GP mediated resistance to vincristine, suggesting that MPP + is a poor substrate for P-GP. Additional experiments were performed using mdr1a/b double knockout mice which lack functional P-GP encoded by these two genes. Data from mdr1a/b knockout mice treated with MPTP also suggest that MPP + is not extruded from the brain by P-GP. In other studies, we demonstrated that the VMAT2 inhibitors tetrabenazine and Ro 4-1284 inhibit P-GP and that the P-GP inhibitors trans -flupentixol and quinidine inhibit VMAT2. Thus, several new drugs can be added to the list of compounds that are able to inhibit both VMAT2 and P-GP, providing further evidence of the similarity between these two transporters.

Recent epidemiological studies have established an association between the common consumption of coffee or other caffeinated beverages and a reduced risk of developing Parkinson's disease (PD). To explore the possibility that caffeine helps prevent the dopaminergic deficits characteristic of PD, we investigated the effects of caffeine and the adenosine receptor subtypes through which it may act in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin model of PD. Caffeine, at doses comparable to those of typical human exposure, attenuated MPTP-induced loss of striatal dopamine and dopamine transporter binding sites. The effects of caffeine were mimicked by several A(2A) antagonists (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH 58261), 3,7-dimethyl-1-propargylxanthine, and (E)-1,3-diethyl-8 (KW-6002)-(3,4-dimethoxystyryl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione) (KW-6002) and by genetic inactivation of the A(2A) receptor, but not by A(1) receptor blockade with 8-cyclopentyl-1,3-dipropylxanthine, suggesting that caffeine attenuates MPTP toxicity by A(2A) receptor blockade. These data establish a potential neural basis for the inverse association of caffeine with the development of PD, and they enhance the potential of A(2A) antagonists as a novel treatment for this neurodegenerative disease.

Defects in mitochondrial energy metabolism have been implicated in the pathology of several neurodegenerative disorders. In addition, the reactive metabolites generated from the metabolism and oxidation of the neurotransmitter dopamine (DA) are thought to contribute to the damage to neurons of the basal ganglia. We have previously demonstrated that infusions of the metabolic inhibitor malonate into the striata of mice or rats produce degeneration of DA nerve terminals. In the present studies, we demonstrate that an intrastriatal infusion of malonate induces a substantial increase in DA efflux in awake, behaving mice as measured by in vivo microdialysis. Furthermore, pretreatment of mice with tetrabenazine (TBZ) or the TBZ analogue Ro 4-1284 (Ro-4), compounds that reversibly inhibit the vesicular storage of DA, attenuates the malonate-induced DA efflux as well as the damage to DA nerve terminals. Consistent with these findings, the damage to both DA and GABA neurons in mesencephalic cultures by malonate exposure was attenuated by pretreatment with TBZ or Ro-4. Treatment with these compounds did not affect the formation of free radicals or the inhibition of oxidative phosphorylation resulting from malonate exposure alone. Our data suggest that DA plays an important role in the neurotoxicity produced by malonate. These findings provide direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular DA levels and indicate that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through a mechanism involving DA.

Defects in energy metabolism have been detected in patients with Parkinson's disease and have been proposed as a contributing factor in the disease. Previous in vitro studies showed that NMDA receptors contribute to the loss of dopamine neurons caused by the metabolic inhibitor malonate. In vivo, it is not known whether this interaction occurs through a postsynaptic action on the cell body in the substantia nigra or through a presynaptic action at the dopamine terminal in the striatum. So we could discern the anatomical level of NMDA receptor involvement, rats were infused with malonate, either into the left striatum or into the left substantia nigra. NMDA receptors were locally blocked by an intranigral or intrastriatal coinfusion of malonate plus MK-801 followed by a second infusion of MK-801 3 h later. Animals were examined at 1 week for striatal and nigral dopamine and GABA levels. Intranigral infusion of malonate (0.5 micromol) produced an approximate 50% loss of both nigral dopamine and GABA. MK-801 (0.1 micromol) provided significant protection against both nigral dopamine and GABA loss and against anterograde damage to dopamine terminals in the striatum. Intrastriatal administration of malonate (2 micromol) produced a 68 and 35% loss of striatal dopamine and GABA, respectively. In contrast to intranigral administration, intrastriatal blockade of NMDA receptors did not protect against striatal dopamine loss, although GABA loss was significantly attenuated. Core body temperature monitored several hours throughout the experiment was unchanged. Consistent with a lack of effect of NMDA antagonists on malonate-induced toxicity to dopamine neurons in striatum, intrastriatal infusion of NMDA had a pronounced effect on long-term GABA toxicity with little effect of dopamine loss. These findings are consistent with a postsynaptic action of NMDA receptors on mediating toxicity to dopamine neurons during impaired energy metabolism.

Ten patients with Parkinson's disease and severe motor fluctuations were given Sinemet (25/100) for 4 weeks followed by 4 weeks of Sinemet (CR-4). After each drug preparation was optimized, patients were rated by neurological examination and plasma levodopa (LD) measured at hourly intervals (9 a.m.-4 p.m.). For the group as a whole, variations throughout the day of plasma LD and clinical state were no different on the 2 formulations. Three patients whose fluctuations responded well to CR-4 had either much less variable plasma LD levels on CR-4 or were able to maintain plasma LD above a minimum threshold. In severe fluctuators, a major benefit from CR-4 can be expected only in those patients who can maintain steady plasma LD levels above the threshold for achieving the 'on' state.

In vitro studies indicate that mesencephalic dopamine neurons are more vulnerable than other neurons to impairment of energy metabolism. Such findings may have bearing on the loss of dopamine neurons in Parkinson's disease, in which mitochondrial deficiencies have been identified, but would only be relevant if the selective vulnerability were maintained in vivo. To examine this, rats were stereotaxically administered various concentrations of the succinate dehydrogenase inhibitor, malonate (0.25–4 μmol), either into the left substantia nigra or striatum. One week following injection, dopamine and γ-aminobutyric acid (GABA) levels in the mesencephalon and striatum were measured. Intranigral injection of malonate caused nigral dopamine and GABA to be comparably reduced at all doses tested. The 50% dose level for malonate vs. dopamine and GABA loss was 0.39 and 0.42 μmol, respectively. Tyrosine hydroxylase immunocytochemistry of the midbrains of rats which received an intranigral injection of malonate showed normal staining with 0.25 μmol malonate, but almost complete loss of tyrosine hydroxylase positive nigral pars compacta cells with 1 μmol malonate. Intrastriatal injection of malonate produced a loss of both tyrosine hydroxylase activity and dopamine. In contrast to what was seen in substantia nigra, there was a greater loss of dopamine than GABA in striatal regions nearest the injection site. In striatal regions most distal to the injection site, and which received the lowest concentration of malonate due to diffusion, dopamine levels were significantly reduced with all doses of malonate (0.5–4 μmol), whereas GABA levels were unaffected. Intrastriatal coinfusion of succinate along with malonate completely prevented the loss of dopamine and GABA indicating that succinate dehydrogenase inhibition was the cause of toxicity. These findings indicate that dopamine terminals in the striatum of adult rats are selectively more vulnerable than are the GABA neurons to a mild energy impairment.

The mechanisms involved in methamphetamine (METH)-induced damage to nigrostriatal dopaminergic neurons in experimental animals are unknown. We have examined the possibility that perturbations in energy metabolism contribute to METH-induced toxicity by investigating the effects of systemic METH treatment in mice which received a unilateral intrastriatal infusion of malonate, a metabolic inhibitor which decreases ATP levels. Malonate (1–4 μmol) produced a dose-dependent decrease in striatal dopamine (DA). The combined treatment of intrastriatal malonate with systemic METH resulted in greater damage to dopaminergic neurons than by METH or malonate treatment alone. In parallel studies, MPTP was administered to mice which received intrastriatal infusions of saline or malonate. Similar to results obtained with METH, decreases in striatal DA content and tyrosine hydroxlase (TH) activity were greatest in MPTP-treated mice infused with malonate. The present results lend credence to the hypothesis that METH-induced increases in energy utilization create a state of metabolic stress for DA neurons which may ultimately contribute to the neurodegenerative effects of METH. Moreover, the fording that combined malonate and MPTP treatment produced greater damage than either substance alone is consistent with the hypothesis that perturbations in energy metabolism contribute to the neuronal death produced by MPP+.

Although it has been known for several decades that the administration of amphetamines to experimental animals produces damage to monoaminergic neurons, the mechanism(s) underlying this neuropathology is unknown. In recent years, it has been demonstrated that various N-methyl- d -aspartate (NMDA) receptor antagonists can prevent the damage produced by the amphetamines. The purpose of this communication is to review the evidence which demonstrates the role of NMDA receptors in the neuropathology of neostriatal dopaminergic neurons produced by the amphetamines and to discuss how the action of the amphetamines may potentially affect NMDA receptor function.

Previous studies from this laboratory demonstrated that (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801), an N-methyl-D-aspartate (NMDA) receptor antagonist, did not prevent neurotoxicity to dopaminergic neurons in mice produced by systemic treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). However, Turski et al. [Nature 349, 414-418 (1991)] reported that extended treatment of rats with NMDA receptor antagonists (six injections at 4-h intervals) did prevent the loss of nigral dopaminergic neurons resulting from an intranigral infusion of 1-methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of MPTP. The present studies examined if a similar extended treatment with MK-801 would protect mice from the neurotoxicity of systemically administered MPTP. Six intraperitoneal injections of MK-801 given at 4-h intervals did not protect mice against the MPTP-induced neostriatal dopamine loss measured 1 week after treatment. In other experiments, designed to replicate and expand on the results of Turski et al. (1991), the extended treatment of rats with MK-801 did not prevent MPP(+)-induced cell loss in the infused substantia nigra pars compacta or the dopamine depletion in the ipsilateral neostriatum at 7-11 days after MPP+ infusion. These results do not support the hypothesis that NMDA receptors are involved with MPTP/MPP(+)-induced neurodegeneration.

The administration to mice of 1-methyl-4-(2′-methylphenyl)-1,2,3,6-tetrahydropyridine (2′-CH3-MPTP), a substituted analog of the dopaminergic neurotoxin MPTP caused even more dopaminergic toxicity than MPTP itself. Under conditions in which MPTP was relatively ineffective (i.e. two injections per day of 0.113 mmol/kg at an interval of 6 h for one or two days), 2′-CH3-MPTP caused a very large decrement in the neostriatal content of dopamine and its metabolites and a corresponding decrement in the capacity of a neostriatal synaptosomal preparation to take up [3H]dopamine. Moreover, 2′-CH3-MPTP administration (as few as four injections) caused a virtually complete loss of nerve cells in the zona compacta of the substantia nigra. This compound, like MPTP, may prove to be a valuable research tool.

The effects of different dosing paradigms for the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were investigated in C57-black and CF-W albino mice. Several groups of mice received a total dose of 80 mg/kg of MPTP administered at different doses per injection and/or at different time intervals. In C57-black mice, the effects of MPTP administration on the neostriatal content of dopamine ranged from a 91% depletion (4 injections of 20 mg/kg per injection at 1 h intervals) to a non-significant effect (4 injections of 10 mg/kg per injection at 2 h intervals on each of 2 successive days). There was also considerable influence of the MPTP dose per injection and the dosing interval in CF-W mice, although the extent of neostriatal dopamine depletion in CF-W mice was not as great as that observed in C57-black mice. In addition, MPTP produced variable effects on neostriatal dopamine levels in different strains of mice as well as in Swiss-Webster mice obtained from different sources. Some of the strains were affected to a great extent while others were only marginally affected.

Multiple administrations of methamphetamine to rats produced elevated concentrations of substance P-like immunoreactivity within the substantia nigra, an effect which is believed to be mediated by increases in dopaminergic activity induced by methamphetamine. The results reported here demonstrate that the effect of methamphetamine is mediated specifically by the dopamine pathway in the nigrostriatum. Thus, extensive destruction of dopamine neurons in the nigrostriatum by lesions induced by 6-hydroxydopamine or blockade of synthesis of dopamine with α -methyl- p -tyrosine prevented the increases in substance P-like immunoreactivity in the nigra, induced by methamphetamine. Similarly, the concurrent administration of the D 2 receptor antagonist, sulpiride, also prevented the effects of methamphetamine on substance P-like immunoreactivity in a dose-dependent way. In addition, multiple administrations of the D 2 agonist, RU24926, elevated, whereas the D 1 agonist, SKF38393, decreased the content of substance P-like immunoreactivity in the nigra. The effects of methamphetamine were not altered in rats with lesions induced by 5,7-dihydroxytryptamine. Thus, these findings suggest that the effect of methamphetamine on the striatonigral substance P pathway is mediated by nigrostriatal dopaminergic actions on D 2 receptors.

Animals treated with high doses of amphetamines have been used as a model of schizophrenia due to the similarities between the psychosis associated with this mental disorder and that induced by chronic amphetamine abuse. When administered to naive rats in high doses, the amphetamine-like CNS stimulant methamphet-amine produces drastic alterations in the neurochemical parameters of the neostriatal monoaminergic systems. These alterations are characterized by a decrease in the activities of the rate-limiting enzymes for dopamine and serotonin synthesis, as well as a decrease in the concentrations of both neurotransmitters and their metabolites. However, tolerance develops to these neurochemical effects when drug administration occurs in a pattern similar to that encountered during chronic amphetamine abuse. The results indicate that the neurochemical alterations produced by amphetamines in naive and tolerant animals differ widely. This suggests that the administration of high doses of amphetamine-like central stimulants to naive rats may not be an appropriate model for studying the neurochemical changes associated with psychosis and amphetamine abuse.

Several analogs of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were synthesized and screened for their capacity to be oxidized by monoamine oxidase (MAO-A or MAO-B) and their capacity to produce nigrostriatal dopaminergic neurotoxicity in mice. All of the compounds were relatively weak substrates for MAO-A but many of the compounds were found to be good substrates for MAO-B. Only three of the compounds, in addition to MPTP itself, were found to be neurotoxic. These were 1-methyl-4-cyclohexyl-1,2,3,6-tetrahydropyridine, 1-methyl-4-(2'-methylphenyl)-1,2,3,6-tetrahydropyridine and 1-methyl-4-(3'-methoxyphenyl)-1,2,3,6-tetrahydropyridine. All three of these neurotoxic compounds were found to be substrates for MAO-B; in contrast no compound was found to be neurotoxic that was not oxidized by MAO-B. The capacity of the compounds studied to be oxidized by MAO-B appears to be an important aspect of the neurotoxic process.

We have reported that the ability of high doses of methamphetamine to impair dopamine and serotonin synthesis in the rat brain is attenuated when animals are pretreated with gradually increasing doses of methamphetamine. To examine the mechanism of this tolerance phenomenon, the effect of methamphetamine on several neurochemical parameters was determined in naive and methamphetamine-pretreated rats. The elevation of nigral substance P concentrations by methamphetamine was attenuated in pretreated compared to naive rats. The methamphetamine-induced reduction in [3H]sulpiride binding in the rat neostriatum and nucleus accumbens was similarly attenuated in animals pretreated with methamphetamine. Determination of brain concentrations of methamphetamine and amphetamine revealed significantly lower concentrations of both compounds in the brains of pretreated compared to naive animals. The results indicate a reduction in the ability of methamphetamine to increase dopamine transmission in the brains of methamphetamine-pretreated rats. Furthermore, this effect appears to be due, at least in part, to a change in the disposition of methamphetamine in pretreated animals.

We report the long-term clinical responses of 2 parkinsonian patients with severe "on-off" phenomena who have been receiving continuous duodenal levodopa (LD) infusions. The rate of LD infusion needed to keep the patients just "on" declined gradually during the initial 60 days of infusion and then remained constant. Total LD intake declined rapidly during the first several days and gradually for another 60 days, after which it remained stable. Motor fluctuations virtually disappeared in both patients during the time of the day when the pump was in use. This was accompanied by a decline in the plasma LD concentration needed to just maintain the "on" stage in one patient.

The MPTP-treated mouse has proven to be a valuable model of parkinsonism. For example, C57 black mice treated with MPTP exhibit a large decrement in the neostriatal content of dopamine and its metabolites, a marked reduction in the capacity of neostriatal synaptosomal preparations to accumulate [3H]dopamine, a large decrease in neostriatal tyrosine hydroxylase activity, a marked loss of nerve cells in the zona compacta of the substantia nigra, and pronounced behavioral deficits. These biochemical, pathological and behavioral deficits are similarly observed in MPTP-treated primates and in humans with idiopathic parkinsonism. A great deal of our current knowledge concerning MPTP has come from experimentation carried out in the mouse.

We report the clinical spectrum of 3 patients with Parkinson9s disease who experienced complex patterns of levodopa-related dystonia. Dystonia was unrelieved by multiple medication regimens but responded well to continuous, duodenal levodopa infusions. Patients were able to remain mobile without severe dystonia despite a very narrow window of benefit between the levodopa concentration necessary to achieve the “on” state and that which caused the onset of dystonic spasms.

The nigrostriatal dopaminergic neurotoxicity of MPTP was prevented in mice in a dose-dependent manner by the monoamine oxidase-B (MAO-B) inhibitor deprenyl. This finding, combined with other observations, points out the important role of MAO-B in the bioactivation of MPTP. In the present study, some comparisons between MPTP and several of its structural analogs will be presented.

We previously reported that C57 black mice were more sensitive than several other strains of mice to the neurotoxic actions of MPTP. We now report that a significantly higher amount of the 1-methyl-4-phenylpyridinium species, a very toxic metabolite of MPTP, was found in the brains of C57 black mice than in the brains of CF-W or CD-1 mice, two strains of mice which are less sensitive to MPTP. In addition, we also found that there were strain differences in the response of mice to the neurotoxic actions of methamphetamine with CF-W mice being more sensitive to methamphetamine than C57 black mice.

The roles of different dopamine receptors in mediating the responses of the striato-nigral substance P (SP) feedback loop were investigated. Subacute treatment of rats with the D1 agonist, SK&F 38393, produced a significant decrease in SP content in the substantia nigra whereas treatment with the D2 agonist, RU 24926, elevated nigral SP concentrations. Consequently, the elevation in nigral SP content which occurs following treatment with methamphetamine is likely a D2-mediated event. This conclusion is supported by our observation that the D2 antagonist, sulpiride, totally blocked the methamphetamine effect. These findings suggest that D1 and D2 receptors mediate opposing influences on the SP feedback system.