Toxic Feedback Loop Involving Iron, Reactive Oxygen Species, α-Synuclein and Neuromelanin in Parkinson's Disease and Intervention with Turmeric.

Parkinson's disease (PD) is a movement disorder associated with severe loss of mainly dopaminergic neurons in the substantia nigra. Pathological hallmarks include Lewy bodies, and loss of neuromelanin, due to degeneration of neuromelanin-containing dopaminergic neurons. Despite being described over 200 years ago, the etiology of PD remains unknown. Here, we highlight the roles of reactive oxygen species (ROS), iron, alpha synuclein (α-syn) and neuromelanin in a toxic feedback loop culminating in neuronal death and spread of the disease. Dopaminergic neurons are particularly vulnerable due to decreased antioxidant concentration with aging, constant exposure to ROS and presence of neurotoxic compounds (e.g. ortho-quinones). ROS and iron increase each other's levels, creating a state of oxidative stress. α-Syn aggregation is influenced by ROS and iron but also increases ROS and iron via its induced mitochondrial dysfunction and ferric-reductase activity. Neuromelanin's binding affinity is affected by increased ROS and iron. Furthermore, during neuronal death, neuromelanin is degraded in the extracellular space, releasing its bound toxins. This cycle of events continues to neighboring neurons in the form of a toxic loop, causing PD pathology. The increase in ROS and iron may be an important target for therapies to disrupt this toxic loop, and therefore diets rich in certain 'nutraceuticals' may be beneficial. Turmeric is an attractive candidate, as it is known to have anti-oxidant and iron chelating properties. More studies are needed to test this theory and if validated, this would be a step towards development of lifestyle-based therapeutic modalities to complement existing PD treatments.

Testosterone enhances mitochondrial complex V function in the substantia nigra of aged male rats.

Deficits in coordinated motor behavior and mitochondrial complex V activity have been observed in aged males. Testosterone supplementation can improve coordinated motor behavior in aged males. We investigated the effects of testosterone supplementation on mitochondrial complex V function in the substantia nigra (a brain region that regulates motor activity) in aged male rats. These rats exhibited diminished ATP levels, attenuated mitochondrial complex V activity, and reduced expression of 3 of the 17 mitochondrial complex V subunits (ATP6, ATP8 and ATP5C1) in the substantia nigra. Testosterone supplementation increased ATP levels, mitochondrial complex V activity, and ATP6, ATP8 and ATP5C1 expression in the substantia nigra of the rats. Conversely, orchiectomy reduced mitochondrial complex V activity, downregulated ATP6 and ATP8 expression, and upregulated ATP5C1, ATP5I and ATP5L expression in the substantia nigra. Testosterone replacement reversed those effects. Thus, testosterone enhanced mitochondrial complex V function in the substantia nigra of aged male rats by upregulating ATP6 and ATP8. As potential testosterone targets, these two subunits may to some degree maintain nigrostriatal dopaminergic function in aged males.

A strategy for iron oxide nanoparticles to adhere to the neuronal membrane in the substantia nigra of mice.

Effective attachment of magnetic nanoparticles to neuronal membranes has far-reaching significance in activating ion channels and treating neurodegenerative diseases. Superparamagnetic iron oxide nanoparticles (SPIONs) synthesized by the polyol pyrolysis method have the advantages of rich surface functional groups, excellent magnetic properties, controllable particle size and water dispersibility. We propose that perfusion of biotin into the targeted brain area should be initially performed because it tends to be adsorbed by cell membranes, followed by injection of streptavidin (SA)-modified SPIONs into the same area of the brain. By means of the strong binding force between SA and biotin, the SPIONs may subsequently adhere to the cell surfaces in the brain area. In this work, fluorescein isothiocyanate-streptavidin (FITC-SA) was modified on the surface of polyethylene imine (PEI)-SPIONs by the EDC-NHS method and stereotaxically injected into the biotin-supplemented substantia nigra of mice. The combination of fluorescence detection with transmission electron microscopy (TEM) confirmed that FITC-SA/PEI-SPIONs adhered to neuronal membranes in the substantia nigra of mice 24 h after injection. The results show that our strategy can promote the attachment of SPIONs to neuronal membranes.

Combined in-situ imaging of structural organization and elemental composition of substantia nigra neurons in the elderly.

Human dopaminergic system in general, and substantia nigra (SN) neurons, in particular, are implicated in the pathologies underlying the human brain aging. The interplay between aberrations in the structural organization and elemental composition of SN neuron bodies has recently gained in importance as selected metals: Fe, Cu, Zn, Ca were found to trigger oxidative-stress-mediated aberration in their molecular assembly due to concomitant protein (alpha-synuclein, tau-protein) aggregation, gliosis and finally oxidative stress. In the present study, we demonstrate an integrated approach to the analysis of the structural organization, assembly, and metals' accumulation in two distinct areas of SN: in the neuromelanin neurons and neuropil. By using the highly brilliant source of PETRA III and the Kirkpatrick-Baez nano-focus, large area histological brain slices are scanned at the sub-neuronal resolution, taking advantage of continuous motor movement and reduced acquisition time. Elemental analysis with synchrotron radiation based X-ray Fluorescence (SRXRF) is combined with X-ray Phase Contrast Imaging (XPCI) to correct for inherent aberrations in the samples' density and thickness, often referred to as the mass thickness effect. Based on the raw SRXRF spectra, we observed the accumulation of P, S, Cl, K, Ca, Fe, Cu and Zn predominantly in the SN neurons. However, upon the mass thickness correction, the distributions of Cl became significantly more uniform. Simultaneously with the fluorescence signal, the Small Angle X-ray Scattering (SAXS) is recorded by a pixel detector positioned in the far-field, enabling fast online computation of the darkfield and differential phase contrast (DPC). The data has demonstrated the SN neurons and neuropil produces excellent contrast which is due to their different mass density and scattering strength, indicative of differences in local structure and assembly therein. In all, the results show that combined SRXRF-XPCI-SAXS experiments can robustly serve as a unique tool for understanding the interplay between the chemical composition and structural organization that may drive the biochemical age-related processes occurring in the human dopaminergic system.

Ginkgo biloba and vitamin E ameliorate haloperidol-induced vacuous chewingmovement and brain-derived neurotrophic factor expression in a rat tardive dyskinesia model.

Neurodegeneration may be involved in the development of tardive dyskinesia (TD), and low levels of brain-derived neurotrophic factor (BDNF) may play a role. Ginkgo biloba (EGb761), a potent antioxidant, may have neuroprotective effects. We hypothesized that there would be decreased BDNF expression in TD, but that treatment with EGb761 would increase BDNF expression and reduce TD manifestations in a rat model. Forty rats were treated with haloperidol (2mg/kg/day via intraperitoneal injections) for 5weeks. EGb761 (50mg/kg/day) and vitamin E (20mg/kg/day) were then administered by oral gavage for another 5weeks, and we compared the effects of treatment with EGb761 or vitamin E on haloperidol-induced vacuous chewing movements (VCMs) and BDNF expression in four brain regions: prefrontal cortex (PFC), striatum (ST), substantia nigra (SNR), and globus pallidus (GP). Our results showed that haloperidol administration led to a progressive increase in VCMs, but both EGb761 and vitamin E significantly decreased VCMs. Haloperidol also decreased BDNF expression in all four brain regions, but both EGb761 and vitamin E administration significantly increased BDNF expression. Our results showed that both EGb761 and VE treatments exerted similar positive effects in a rat model of TD and increased BDNF expression levels in the four tested brain regions, suggesting that both EGb761 and vitamin E improve TD symptoms, possibly by enhancing BDNF in the brain and/or via their free radical-scavenging actions.

Application of laser microdissection ICP-MS for high resolution elemental mapping in mouse brain tissue: a comparative study with laser ablation ICP-MS.

Mapping of elements in biological tissue by laser induced mass spectrometry is a fast growing analytical methodology in life sciences. This method provides a multitude of useful information of metal, nonmetal, metalloid and isotopic distribution at major, minor and trace concentration ranges, usually with a lateral resolution of 12-160 µm. Selected applications in medical research require an improved lateral resolution of laser induced mass spectrometric technique at the low micrometre scale and below. The present work demonstrates the applicability of a recently developed analytical methodology - laser microdissection associated to inductively coupled plasma mass spectrometry (LMD ICP-MS) - to obtain elemental images of different solid biological samples at high lateral resolution. LMD ICP-MS images of mouse brain tissue samples stained with uranium and native are shown, and a direct comparison of LMD and laser ablation (LA) ICP-MS imaging methodologies, in terms of elemental quantification, is performed.

Silibinin attenuates MPP⁺-induced neurotoxicity in the substantia nigra in vivo.

Parkinson's disease (PD) is characterized by degeneration of the nigrostriatal dopaminergic (DA) pathway. The cause of neuronal death in PD is largely unknown, but it is becoming clear that inflammation plays a significant role in the pathophysiology of PD. Silibinin is a major flavonoid in milk thistle which has an anti-inflammatory activity. We investigated whether silibinin could have neuroprotective effects on DA neurons in the 1-methyl-4-phenylpyridinium ion (MPP(+))-treated animal model of PD in vivo. To address this question, animals received intraperitoneal (i.p.) injections 10, 50, or 100 mg/kg of silibinin, starting 1 day before MPP(+) injection and continued daily until 6 days post-lesion for tyrosine hydroxylase (TH) staining, or until 1 hour prior to the MPP(+) injection to examine the expression levels of inflammatory proteins. Finally, their brains were harvested at the indicated time points for the analyses. Silibinin treatment with 10 mg/kg had no significantly neuroprotective effects in the substantia nigra (SN). However, 50 and 100 mg/kg of silibinin ameliorated the MPP(+)-induced neurotoxicity in the SN in a dose-dependent manner, and the increased levels of inflammatory molecules such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß) and inducible nitric oxide synthase (iNOS) by MPP(+) treatment were attenuated by treatment with 100 mg/kg of silibinin. These results indicate that silibinin could be a useful and beneficial natural product offering promise for the prevention of DA neuronal degeneration involved in PD.

Cortical glutamate levels decrease in a non-human primate model of dopamine deficiency.

While Parkinson's disease is the result of dopaminergic dysfunction of the nigrostriatal system, the clinical manifestations of Parkinson's disease are brought about by alterations in multiple neural components, including cortical areas. We examined how 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration affected extracellular cortical glutamate levels by comparing glutamate levels in normal and MPTP-lesioned nonhuman primates (Macaca mulatta). Extracellular glutamate levels were measured using glutamate microelectrode biosensors. Unilateral MPTP-administration rendered the animals with hemiparkinsonian symptoms, including dopaminergic deficiencies in the substantia nigra and the premotor and motor cortices, and with statistically significant decreases in basal glutamate levels in the primary motor cortex on the side ipsilateral to the MPTP-lesion. These results suggest that the functional changes of the glutamatergic system, especially in the motor cortex, in models of Parkinson's disease could provide important insights into the mechanisms of this disease.

Combined treatment with acupuncture reduces effective dose and alleviates adverse effect of L-dopa by normalizing Parkinson's disease-induced neurochemical imbalance.

This study first showed the behavioural benefits of novel combination therapy of L-dopa with acupuncture on Parkinson's disease, and its underlying mechanisms within basal ganglia. The previous study reported that acupuncture may improve the motor function of a Parkinson's disease (PD) mouse model by increasing the dopamine efflux and turnover ratio of dopamine. Hence, we hypothesised that combining L-dopa with acupuncture would have a behavioural benefit for those with PD. We performed unilateral injections of 6-OHDA into the striatum of C57Bl/6 mice to model hemi-Parkinsonian attributes. To test motor function and dyskinetic anomalies, we examined cylinder behaviour and abnormal involuntary movement (AIM), respectively. We found that (1) a 50% reduced dose of L-dopa (7.5 mg/kg) combined with acupuncture showed an improvement in motor function that was comparable to mice given the standard dose of L-dopa treatment (15 mg/kg) only, and that (2) the combination treatment (L-dopa +acupuncture) was significantly superior in reducing AIM scores when equivalent doses of L-dopa were used. The combination treatment also significantly reduces the abnormal increase of GABA contents in the substantia nigra compared to the standard L-dopa treatment. Furthermore, abnormal expression of FosB, the immediate early gene of L-dopa induced dyskinesia (LID), was mitigated in the striatum by the combination treatment. All of these results indicate that acupuncture enhances the benefits of L-dopa on motor function with reduced dose of L-dopa and alleviating LID by normalising neurochemical imbalance within the basal ganglia.

AC-186, a selective nonsteroidal estrogen receptor ß agonist, shows gender specific neuroprotection in a Parkinson's disease rat model.

Drugs that selectively activate estrogen receptor ß (ERß) are potentially safer than the nonselective estrogens currently used in hormonal replacement treatments that activate both ERß and ERα. The selective ERß agonist AC-186 was evaluated in a rat model of Parkinson's disease induced through bilateral 6-hydroxydopamine lesions of the substantia nigra. In this model, AC-186 prevented motor, cognitive, and sensorimotor gating deficits and mitigated the loss of dopamine neurons in the substantia nigra, in males, but not in females. Furthermore, in male rats, 17ß-estradiol, which activates ERß and ERα with equal potency, did not show the same neuroprotective benefits as AC-186. Hence, in addition to a beneficial safety profile for use in both males and females, a selective ERß agonist has a differentiated pharmacological profile compared to 17ß-estradiol in males.

Alcoholic extract of Bacopa monniera Linn. protects against 6-hydroxydopamine-induced changes in behavioral and biochemical aspects: a pilot study.

Parkinson's disease is one of the commonest neurodegenerative diseases, and oxidative stress has been evidenced to play a vital role in its causation. In this study, we evaluated whether alcoholic extract of Bacopa monniera (AEBM), an antioxidant and memory enhancer can slow the neuronal injury in a 6-OHDA-rat model of Parkinson's. Rats were treated with 20 and 40 mg/kg bodyweight of AEBM for 3 weeks. On Day 21, 2 µl of 6-OHDA (12 µg in 0.01 % in ascorbic acid-saline) was infused into the right striatum, while the control group received 2 µl of vehicle. Three weeks after the 6-OHDA injection, the rats were tested for neurobehavioral activity (rotarod, locomotor activity, grip test, forced swim test, radial arm maze) and were killed after 6 weeks for the estimation of lipid peroxidation, reduced glutathione (GSH) content, activities of glutathione-S-transferase, glutathione reductase, glutathione peroxidase, superoxide dismutase (SOD), and catalase (CAT). The deficits in behavioral activity due to 6-OHDA lesioning were significantly and dose dependently restored by AEBM. Lesioning was followed by an increased lipid peroxidation and significant depletion of reduced GSH content in the substantia nigra, which was prevented with AEBM pretreatment. The activities of GSH-dependent enzymes, CAT and SOD in striatum were reduced significantly by lesioning, which were restored significantly and dose dependently by AEBM. This study indicates that the extract of B. monniera might be helpful in attenuating 6-OHDA-induced lesioning in rats.

Topographic trace-elemental analysis in the brain of Wistar rats by X-ray microfluorescence with synchrotron radiation.

Knowledge about the spatial distribution and the local concentration of trace elements in tissues is of great importance, since trace elements are involved in many biological functions of living organisms. However, there are few methods available to measure the spatial (two (three)-dimensional) elemental distribution in animal brain. X-ray microfluorescence with synchrotron radiation is a multielemental mapping technique, which was used in this work to determine the topographic of iron, zinc and copper in coronal sections of female Wistar rats of different ages. Young (14 days old) and middle-aged (20 months old) rats (n = 8) were analyzed. The measurements were carried out at the XRF beam line at the Synchrotron Light National Laboratory (Campinas, Brazil). Two-dimensional scanning was performed in order to study the tendency of elemental concentration variation. The acquisition time for each pixel was 10 s/step and the step size was 300 microm/step in both directions. It was observed that the iron distribution was more conspicuous in the cortical area, thalamus and bellow the thalamus. On the other hand, the zinc distribution was more pronounced in the hippocampus. The iron, copper and zinc levels increased with advancing age. Therefore, this study reinforces the idea that these elements are involved in the chemical mechanisms of the brain that induce some neurological diseases, since they are also present in high levels in specific areas of the brain, such as the hippocampus and the substantia nigra of patients with these disorders.

Hypocretin (orexin) cell loss in Parkinson's disease.

It has recently been reported that Parkinson's disease (PD) is preceded and accompanied by daytime sleep attacks, nocturnal insomnia, REM sleep behaviour disorder, hallucinations and depression, symptoms which are frequently as troublesome as the motor symptoms of PD. All these symptoms are present in narcolepsy, which is linked to a selective loss of hypocretin (Hcrt) neurons. In this study, the Hcrt system was examined to determine if Hcrt cells are damaged in PD. The hypothalamus of 11 PD (mean age 79 +/- 4) and 5 normal (mean age 77 +/- 3) brains was examined. Sections were immunostained for Hcrt-1, melanin concentrating hormone (MCH) and alpha synuclein and glial fibrillary acidic protein (GFAP). The substantia nigra of 10 PD brains and 7 normal brains were used for a study of neuromelanin pigmented cell loss. The severity of PD was assessed using the Hoehn and Yahr scale and the level of neuropathology was assessed using the Braak staging criteria. Cell number, distribution and size were determined with stereologic techniques on a one in eight series. We found an increasing loss of hypocretin cells with disease progression. Similarly, there was an increased loss of MCH cells with disease severity. Hcrt and MCH cells were lost throughout the anterior to posterior extent of their hypothalamic distributions. The percentage loss of Hcrt cells was minimal in stage I (23%) and was maximal in stage V (62%). Similarly, the percentage loss of MCH cells was lowest in stage I (12%) and was highest in stage V (74%). There was a significant increase (P = 0.0006, t = 4.25, df = 15) in the size of neuromelanin containing cells in PD patients, but no difference in the size of surviving Hcrt (P = 0.18, t = 1.39, df = 14) and MCH (P = 0.28, t = 1.39, df = 14) cells relative to controls. In summary, we found that PD is characterized by a massive loss of Hcrt neurons. Thus, the loss of Hcrt cells may be a cause of the narcolepsy-like symptoms of PD and may be ameliorated by treatments aimed at reversing the Hcrt deficit. We also saw a substantial loss of hypothalamic MCH neurons. The losses of Hcrt and MCH neurons are significantly correlated with the clinical stage of PD, not disease duration, whereas the loss of neuromelanin cells is significantly correlated only with disease duration. The significant correlations that we found between the loss of Hcrt and MCH neurons and the clinical stage of PD, in contrast to the lack of a relationship of similar strength between loss of neuromelanin containing cells and the clinical symptoms of PD, suggests a previously unappreciated relationship between hypothalamic dysfunction and the time course of the overall clinical picture of PD.

The nociceptin/orphanin FQ receptor antagonist J-113397 and L-DOPA additively attenuate experimental parkinsonism through overinhibition of the nigrothalamic pathway.

By using a battery of behavioral tests, we showed that nociceptin/orphanin FQ receptor (NOP receptor) antagonists attenuated parkinsonian-like symptoms in 6-hydroxydopamine hemilesioned rats (Marti et al., 2005). We now present evidence that coadministration of the NOP receptor antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H benzimidazol-2-one (J-113397) and L-DOPA to 6-hydroxydopamine hemilesioned rats produced an additive attenuation of parkinsonism. To investigate the neurobiological substrates underlying this interaction, in vivo microdialysis was used in combination with behavioral measurements (bar test). J-113397 and L-DOPA alone reduced the time on bars (i.e., attenuated akinesia) and elevated GABA release selectively in the lesioned substantia nigra reticulata. J-113397 also reduced nigral glutamate levels, whereas L-DOPA was ineffective. J-113397 and L-DOPA coadministration produced additive antiakinetic effect, which was associated with additive increase in nigral GABA release but no additional reductions in glutamate levels. To investigate whether the increase in nigral GABA release could translate to changes in nigrothalamic transmission, GABA release was monitored in the ventromedial thalamus (one of the main target areas of the nigrothalamic projections). J-113397 and L-DOPA decreased thalamic GABA release and attenuated akinesia, their combination resulting in a more profound effect. These actions were prevented by perfusing the voltage-dependent Na+ channel blocker tetrodotoxin or the GABA(A) receptor antagonist bicuculline in the substantia nigra reticulata. These data demonstrate that J-113397 and L-DOPA exert their antiparkinsonian action through overinhibition of nigrothalamic transmission and suggest that NOP receptor antagonists may be useful as an adjunct to L-DOPA therapy for Parkinson's disease.

Distribution of serotonin 5-HT(2A) receptors in afferents of the rat striatum.

Treatment with conventional antipsychotic drugs (APDs) is accompanied by extrapyramidal side effects (EPS), which are thought to be due to striatal dopamine D(2) receptor blockade. In contrast, treatment with atypical APDs is marked by a low incidence or absence of EPS. The reduced motor side effect liability of atypical APDs has been attributed to a high serotonin 5-HT(2A) receptor affinity coupled with a relatively low D(2) affinity. Despite the high density of 5-HT(2A) binding sites in the striatum, there are few detectable 5-HT(2A) mRNA-expressing neurons in the striatum. This suggests that most striatal 5-HT(2A) receptors are heteroceptors located on afferent axons. A combined retrograde tracer-immunohistochemistry method was used to determine the sites of origin of striatal 5-HT(2A)-like immunoreactive axons. 5-HT(2A)-like immunoreactive neurons in both the cortex and globus pallidus were retrogradely labeled from the striatum; very few nigrostriatal or thalamostriatal neurons expressed 5-HT(2A)-like immunoreactivity. Within the striatum, parvalbumin-containing interneurons displayed 5-HT(2A) immunolabeling; these neurons are the targets of cortical and pallidal projections. Our data indicate that cortico- and pallido-striatal neurons are the major source of 5-HT(2A) receptor binding in the striatum, and suggest that cortico- and pallido-striatal neurons are strategically positioned to reduce the motor side effects that accompany striatal D(2) receptor blockade or are seen in parkinsonism.

Immunohistochemical detection of tyrosine hydroxylase and concentrations of monoamines in the substantia nigra and hypothalamus of hereditary microphthalmic rats.

We compared tyrosine hydroxylase immunoreactivity in the substantia nigra and hypothalamus of hereditary microphthalmic rats with that of normal rats. A considerable number of neuronal cell bodies expressing tyrosine hydroxylase were present in the substantia nigra of the microphthalmic mutant as well as normal rats. Neuronal cells positive for tyrosine hydroxylase in the hypothalamus were fewer than in the substantia nigra in both rats. The concentrations of monoamines (dopamine, noradrenaline, adrenaline, and serotonin) in the substantia nigra and hypothalamus in the microphthalmic mutant were approximately the same as those of normal rats, although the diurnal fluctuation of a few monoamines was observed in normal rats. These results suggest that the metabolic aspects of catecholamine in the substantia nigra and hypothalamus of the microphthalmic mutant rat do not markedly differ from those of normal rats.

Region-specific alterations in the concentrations of catecholamines and indoleamines in the brains of young and old F344 rats after L-deprenyl treatment.

The effects of L-deprenyl, a monoamine oxidase-B (MAO-B) inhibitor, on the concentrations of norepinephrine (NE), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5-hydroxytryptamine), and 5-hydroxyindoleacetic acid (5-HIAA) in medial basal hypothalamus (MBH), substantia nigra (SN), striatum (Str), and nucleus accumbens (NAc) of young (3 month) and old (21 month) male F344 rats were examined after a 7-day wash-out period following 1, 15, or 30 days of deprenyl treatment in young rats and a 9-day wash-out period after a 10-week deprenyl treatment in old rats. The brain areas were microdissected and the concentrations of neurotransmitters were measured by High Performance liquid chromatography with electrochemical detection (HPLC-EC). Deprenyl administration following the drug wash-out period increased the concentrations of DOPAC in the SN, Str, and in the NAc of young rats but it was decreased in the NAc of old rats. The concentration of HVA was lower in the Str of young deprenyl-treated rats, and in the Str and NAc of old deprenyl-treated rats, but it was higher in the SN of young deprenyl-treated rats. The concentration of 5-HIAA was increased in the MBH, SN, and in the NAc of young deprenyl-treated rats, but it was decreased in the Str and NAc of old deprenyl-treated rats. The concentration of NE was increased in the MBH, SN, Str, and in the NAc of young rats treated with deprenyl and in the MBH of old deprenyl-treated rats. The concentration of 5-HT was increased in the SN of young deprenyl-treated rats. The concentration of DA increased in the Str of both young and old deprenyl-treated rats. We concluded that a drug wash-out period after deprenyl treatment differentially affects the metabolism of catecholamines and indoleamine depending on the region of the brain and that this effect may be due to variation in the kinetics of MAO inhibition.

MR imaging of human brain at 3.0 T: preliminary report on transverse relaxation rates and relation to estimated iron content.

PURPOSE: To determine the transverse relaxation rates R2 and R2' from several gray matter regions and from frontal cortical white matter in healthy human brains in vivo and to determine the relationship between relaxation rates and iron concentration [Fe]. MATERIALS AND METHODS: Six healthy adults aged 19-42 years underwent thin-section gradient-echo sampling of free induction decay and echo magnetic resonance (MR) imaging at 3.0 T. Imaging covered the mesencephalon and basal ganglia. RESULTS: Relaxation rates (mean +/- SD) were highest in globus pallidus (R2 = 25.8 seconds-1 +/- 1.1, R2' = 12.0 seconds-1 +/- 2.1) and lowest in prefrontal cortex (R2 = 14.4 seconds-1 +/- 1.8, R2' = 3.4 seconds-1 +/- 1.1). Frontal white matter measurements were as follows: R2 = 18.0 seconds-1 +/- 1.2 and R2' = 3.9 seconds-1 +/- 1.2. For gray matter, both R2 and R2' showed a strong correlation (r = 0.92, P < .001 and r = 0.90, P < .001, respectively) with [Fe]. Although the slopes of the regression lines for R2' versus [Fe] and for R2 versus [Fe] were similar, the iron-independent component of R2' (2.2 seconds-1 +/- 0.6), the value when [Fe] = 0, was much less than that of R2 (12.7 seconds-1 +/- 0.7). CONCLUSION: The small iron-independent component R2', as compared with that of R2, is consistent with the hypothesis that R2' has higher iron-related specificity.

Acute stress induces a differential increase of 5-HT-moduline (LSAL) tissue content in various rat brain areas.

5-HT-moduline is an endogenous cerebral tetrapeptide (LSAL) which specifically interacts as an allosteric modulator with 5-HT1B receptors controlling serotonergic activity [O. Massot, J.C. Rousselle, M.P. Fillion, B. Grimaldi, I. Cloez-Tayarani, A. Fugelli, N. Prudhomme, L. Seguin, B. Rousseau, M. Plantefol, R. Hen, G. Fillion, 5-Hydroxytryptamine-moduline, a new endogenous cerebral peptide, controls the serotonergic activity via its specific interaction with 5-hydroxytryptamine1B/1D receptors, Mol. Pharmacol. 50 (1996) 752-762; J.C. Rousselle, O. Massot, M. Delepierre, E. Zifa, G. Fillion, Isolation and characterization of an endogenous peptide from rat brain interacting specifically with the serotonergic1B receptor subtypes, J. Biol. Chem. 271 (1996) 726-735; J.C. Rousselle, M. Plantefol, M.P. Fillion, O. Massot, P.J. Pauwels, G. Fillion, Specific interaction of 5-HT-moduline with human 5-HT1b as well as 5-HT1d receptors expressed in transfected cultured cells, Naunyn-Schmiedeberg's Arch. Pharmacol. 358 (1998) 279-286]. Cerebral tissue contents of 5-HT-moduline were determined in various rat brain areas after an acute restraint stress, and after repetition of this stress, to examine whether or not mechanisms involving this peptide could be affected by stress situations. The measurement of the peptide was carried out using specific polyclonal antibodies [B. Grimaldi, M.P. Fillion, A. Bonnin, J.C. Rousselle, O. Massot, G. Fillion, Immunocytochemical localization of neurons expressing 5-HT-moduline in the mouse brain, Neuropharmacology 36 (1997) 1079-1087] in a dot-ELISA (enzyme-linked-immunosorbent assay) assay in cortex, hippocampus, hypothalamus, substantia nigra, striatum and in adrenal glands. Tissue contents of 5-HT-moduline progressively and transiently increased in most studied brain regions and reached a maximal value 20 min after the beginning of the restraint stress. The increase in 5-HT-moduline tissue contents represented 323% of the value observed in unstressed control animals in the cortex, 207% in the hippocampus, 149% in the hypothalamus and 156% in the substantia nigra. Thereafter, the peptide content of the latter tissues diminished during the last 20 min of restraint and returned to control values within 1 h after the end of the stress period. The striatum did not show any significant variation of 5-HT-moduline content during restraint stress. In adrenal glands, the 5-HT-moduline content rapidly decreased (60% of controls) after the beginning of the restraint stress, the effect of this stress being progressively less pronounced, still representing 80% of controls after 40 min. Repetition of the restraint stress daily for 3 weeks totally abolished the effect of the stress on variations of 5-HT-moduline tissue content in all the studied brain regions. These results show that an acute restraint stress induces a rapid and significant increase in the amount of 5-HT-moduline contained in various brain areas. This phenomenon is likely to be related to the stress-induced 5-HT1B receptor desensitization which was previously demonstrated.

Effect of streptozotocin-induced diabetes on rat brain sulfonylurea binding sites.

Both high and low affinity sulfonylurea receptors (SURs) reside on glucose responsive neurons where they influence cell firing and neurotransmitter release via the adenosinetriphosphate (ATP)-sensitive K+ (katp) channel. Here, the effect of diabetes on [3H] glyburide binding to SURs was assessed in male obesity-resistant Sprague-Dawley rats rendered diabetic with streptozotocin (65 mg/kg, i.p.). Additional streptozotocin-treated rats were supplemented with insulin (1.5 U/kg/ day). Streptozotocin reduced plasma insulin to 13% of control associated with hyperglycemia (25.3 +/- 1.7 mmol/l), while insulin lowered plasma glucose (9.56 +/- 1.78 mmol/l) to near control levels (7.65 +/- 0.22 mmol/l). Over 7 days, all streptozotocin-treated rats lost 12% of their initial body wt. while controls gained 1%. Despite equivalent wt. loss, streptozotocin-induced diabetes selectively increased high affinity [3H] glyburide binding in the hypothalamic dorsomedial nuclei (DMN) and ventromedial nuclei (VMN) and lateral area (LH). This was prevented by insulin injections. Low affinity binding was similarly increased in the DMN and VMN, as well as two amygdalar subnuclei but decreased in the substantia nigra, pars compacta. Insulin fully prevented these changes only in the DMN and one amygdalar nucleus and the substantia nigra. Therefore, binding to (SURs) appears to be generally upregulated in the face of hypoinsulinemia with hyperglycemia and this is prevented by insulin treatment. These and other data suggest that this combination of abnormalities in diabetes should have an adverse effect on the glucose sensing capacity of the brain.