Low-dose radiation decreases Lrrk2 levels in the striatum of large mammalian brains: New venues to treat Parkinson's disease?

INTRODUCTION: Among gene mutations and variants linked to an increased risk of PD, mutations of leucine-rich repeat kinase 2 gene (LRRK2) are among the most frequently associated with early- and late-onset PD. Clinical and neuropathological characteristics of idiopathic-PD (iPD) and LRRK2-PD are similar, and these similarities suggest that the pathomechanisms between these two conditions are shared. LRRK2 mutations determine a gain-of-function and yield higher levels of lrrk2 across body tissues, including brain. On another side, recent animal studies supported the potential use of low dose radiation (LDR) to modify the pathomechanisms of diseases such as Alzheimer's disease (AD). METHODS: We assessed if a single total-body LDR (sLDR) exposure in normal swine could alter expression levels of the following PD-associated molecules: alpha-synuclein (α-syn), phosphorylated-α-synuclein (pα-syn), parkin, tyrosine hydroxylase (th), lrrk2, phosphorylated-lrrk2 (pS935-lrrk2), and some LRRK2 substrates (Rab8a, Rab12) across different brain regions. These proteins were measured in frontal cortex, hippocampus, striatum, thalamus/hypothalamus, and cerebellum of 9 radiated (RAD) vs. 6 sham (SH) swine after 28 days from a sLDR of 1.79Gy exposure. RESULTS: Western Blot analyses showed lowered lrrk2 levels in the striatum of RAD vs. SH swine (p < 0.05), with no differences across the remaining brain regions. None of the other protein levels differed between RAD and SH swine in any examined brain regions. No lrrk2 and p-lrrk2 (S935) levels differed in the lungs of RAD vs. SH swine. CONCLUSIONS: These findings show a specific striatal lrrk2 lowering effect due to LDR and support the potential use of LDR to interfere with the pathomechanisms of PD.

Neuroprotective effect of anodal transcranial direct current stimulation on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in mice through modulating mitochondrial dynamics.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the accumulation of protein inclusions and the loss of dopaminergic neurons. Abnormal mitochondrial homeostasis is thought to be important for the pathogenesis of PD. Transcranial direct current stimulation (tDCS), a noninvasive brain stimulation technique, constitutes a promising approach for promoting recovery of various neurological conditions. However, little is known about its mechanism of action. The present study elucidated the neuroprotective effects of tDCS on the mitochondrial quality control pathway in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. We used the MPTP-induced neurotoxicity in vivo model. Mice were stimulated for 5 consecutive days with MPTP treatment. After observation of behavioral alteration using the rotarod test, mice were sacrificed for the measurement of the PD- and mitochondrial quality control-related protein levels in the substantia nigra. tDCS improved the behavioral alterations and changes in tyrosine hydroxylase levels in MPTP-treated mice. Furthermore, tDCS attenuated mitochondrial damage, as indicated by diminished mitochondrial swelling and mitochondrial glutamate dehydrogenase activity in the MPTP-induced PD mouse model. MPTP significantly increased mitophagy and decreased mitochondrial biogenesis-related proteins. These changes were attenuated by tDCS. Furthermore, MPTP significantly increased fission-related protein dynamin-related protein 1 with no effect on fusion-related protein mitofusin-2, and tDCS attenuated these changes. Our findings demonstrated the neuroprotective effect of anodal tDCS on the MPTP-induced neurotoxic mouse model through suppressing excessive mitophagy and balancing mitochondrial dynamics. The neuroprotective effect of anodal tDCS with modulation of mitochondrial dynamics provides a new therapeutic strategy for the treatment of PD.

Decreased dopamine in striatum and difficult locomotor recovery from MPTP insult after exposure to radiofrequency electromagnetic fields.

Concern is growing about possible neuronal effects of human exposure to radiofrequency electromagnetic fields because of the increasing usage of cell phones and the close proximity of these devices to the brain when in use. We found that exposure to a radiofrequency electromagnetic field (RF-EMF) of 835 MHz (4.0 W/kg specific absorption rate [SAR] for 5 h/day for 12 weeks) affects striatal neurons in C57BL/6 mice. The number of synaptic vesicles (SVs) in striatal presynaptic boutons was significantly decreased after RF-EMF exposure. The expression levels of synapsin I and II were also significantly decreased in the striatum of the RF-EMF-exposed group. RF-EMF exposure led to a reduction in dopamine concentration in the striatum and also to a decrease in the expression of tyrosine hydroxylase in striatal neurons. Furthermore, in behavioral tests, exposure to RF-EMF impeded the recovery of locomotor activities after repeated treatments with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). These results suggest that the observed decrease in dopamine concentration in the striatum was caused by both a reduction in the number of dopaminergic neurons and a decline in the number of SVs. The decreased dopamine neuron numbers and concentration seen after RF-EMF exposure would have caused the difficult recovery after MPTP treatment. In summary, our results strongly suggest that exposing the brain to RF-EMF can decrease the number of SVs and dopaminergic neurons in the striatum. These primary changes impair the recovery of locomotor activities following MPTP damage to the striatum.

Photobiomodulation Suppresses Alpha-Synuclein-Induced Toxicity in an AAV-Based Rat Genetic Model of Parkinson's Disease.

Converging lines of evidence indicate that near-infrared light treatment, also known as photobiomodulation (PBM), may exert beneficial effects and protect against cellular toxicity and degeneration in several animal models of human pathologies, including neurodegenerative disorders. In the present study, we report that chronic PMB treatment mitigates dopaminergic loss induced by unilateral overexpression of human α-synuclein (α-syn) in the substantia nigra of an AAV-based rat genetic model of Parkinson's disease (PD). In this model, daily exposure of both sides of the rat's head to 808-nm near-infrared light for 28 consecutive days alleviated α-syn-induced motor impairment, as assessed using the cylinder test. This treatment also significantly reduced dopaminergic neuronal loss in the injected substantia nigra and preserved dopaminergic fibers in the ipsilateral striatum. These beneficial effects were sustained for at least 6 weeks after discontinuing the treatment. Together, our data point to PBM as a possible therapeutic strategy for the treatment of PD and other related synucleinopathies.

Neuroprotective effect of EGb761® and low-dose whole-body γ-irradiation in a rat model of Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease. The present study was undertaken to investigate the pretreatment effects of standardized Ginkgo biloba extract (EGb761(®)) and low-dose whole-body γ-irradiation on the neurological dysfunction in the reserpine model of PD. Male Wistar rats were pretreated orally with EGb761 or fractionated low-dose whole-body γ-irradiation or their combination, then subjected to intraperitoneal injection of reserpine (5 mg/kg body weight) 24 h after the final dose of EGb761 or radiation. Reserpine injection resulted in the depletion of striatal dopamine (DA) level, increased catalepsy score, increased oxidative stress indicated via depletion of glutathione (GSH), increased malondialdehyde (MDA) and iron levels, decreased DA metabolites metabolizing enzymes; indicated by inhibition by glutathione-S-transferase, and nicotinamide adenine dinucleotide phosphate (NADPH)-quinone oxidoreductase (NQO) activities, mitochondrial dysfunction; indicated by declined complex I activity, and adenosine triphosphate (ATP) level and increased apoptosis; indicated by decreased mitochondrial B cell lymphoma-2 (Bcl-2) protein level and by transmission electron microscope. EGb761 and low-dose γ-radiation ameliorated the reserpine-induced state of oxidative stress, mitochondrial dysfunction, and apoptosis in brain. It can be concluded that EGb761, a widely used herbal medicine and low dose of γ-irradiation have protective effects for combating Parkinsonism possibly via replenishment of GSH levels.

Optogenetic neuronal stimulation promotes functional recovery after stroke.

Clinical and research efforts have focused on promoting functional recovery after stroke. Brain stimulation strategies are particularly promising because they allow direct manipulation of the target area's excitability. However, elucidating the cell type and mechanisms mediating recovery has been difficult because existing stimulation techniques nonspecifically target all cell types near the stimulated site. To circumvent these barriers, we used optogenetics to selectively activate neurons that express channelrhodopsin 2 and demonstrated that selective neuronal stimulations in the ipsilesional primary motor cortex (iM1) can promote functional recovery. Stroke mice that received repeated neuronal stimulations exhibited significant improvement in cerebral blood flow and the neurovascular coupling response, as well as increased expression of activity-dependent neurotrophins in the contralesional cortex, including brain-derived neurotrophic factor, nerve growth factor, and neurotrophin 3. Western analysis also indicated that stimulated mice exhibited a significant increase in the expression of a plasticity marker growth-associated protein 43. Moreover, iM1 neuronal stimulations promoted functional recovery, as stimulated stroke mice showed faster weight gain and performed significantly better in sensory-motor behavior tests. Interestingly, stimulations in normal nonstroke mice did not alter motor behavior or neurotrophin expression, suggesting that the prorecovery effect of selective neuronal stimulations is dependent on the poststroke environment. These results demonstrate that stimulation of neurons in the stroke hemisphere is sufficient to promote recovery.

Exposure to extremely low frequency magnetic fields enhances locomotor activity via activation of dopamine D1-like receptors in mice.

We demonstrated that exposure to extremely low frequency magnetic fields (ELF-MF) enhanced dopamine levels in the rat striatum. To extend our understanding, we examined the role of dopaminergic receptors in ELF-MF-induced behavioral changes. Exposure to ELF-MF (2.4 mT, 1 h/day, for one or seven days) enhanced locomotor activity in a time-dependent manner. This hyperlocomotor activity paralleled an increase in c-Fos-like immunoreactivity (c-Fos-IR). Pretreatment with SCH23390, a dopaminergic D(1)-like receptor antagonist, but not with sulpiride, a dopaminergic D(2)-like receptor antagonist, inhibited ELF-MF-induced increased locomotor activity and c-Fos-IR. Thus, our results suggest that ELF-MF-induced behavioral responses are, at least in part, mediated by activation of dopamine D(1)-like receptors.

Gel-based proteomics of unilateral irradiated striatum after gamma knife surgery.

Gamma knife surgery (GKS) is used for the treatment of various brain disorders. The biological effects of focal gamma ray irradiation on targeted or surrounding areas in the brain are not well-known. In the present study, we evaluated protein expression changes in the unilateral irradiated (60 Gy) striatum in rat. Striata of irradiated and control brains were dissected 16 h post-irradiation for analysis by large-format two-dimensional gel electrophoresis (2-DGE). In parallel, we also examined the un-targeted contralateral striatum over the control for potential changes in proteins patterns that may have occurred due to the effects of irradiation to the unilateral striatum. A total of 17 reproducible and differentially expressed silver nitrate-stained protein spots in the irradiated striatum was detected on 2-D gel. Their subsequent analysis by tandem mass spectrometry (nESI-LC-MS/MS) resulted in the identification of 13 nonredundant proteins. Interestingly, out of these 13 changed proteins, 2 proteins were also detected in the contralateral striatum. Some of the significantly changed proteins identified were creatine kinase, protein disulfide isomerase A3 precursor (PDA3), and peroxiredoxin 2 (Prx2). Western analysis with anti-PDA3 and anti-Prx2 antibodies revealed 4 and 2 cross-reacting protein spots on 2-D gel blots. Interestingly, after GKS, in the irradiated and un-irradiated striata, these spots showed a shift toward the acidic side, suggesting post-translational modifications. Taken together, these results indicate that unilateral irradiation during GKS triggers molecular changes in the bilateral striata.

The efficacy of frequency-specific acupuncture stimulation on extracellular dopamine concentration in striatum--a rat model study.

Acupuncture is a practice that has existed in Chinese society for thousands of years. Today, it is gaining greater acceptance and integration into medical practices of the western world. Its mechanism, however, remains elusive. Our study shows that only specific stimulation frequencies at specific acupoints will induce dopamine release in the corpus striatum, as demonstrated by in vivo microdialysis performed on Sprague-Dawley rats. In the first trial, electroacupuncture (EA) stimulation at 15 Hz and 15 mA was conducted at six different points on the upper limbs of the experimental rats. These points mimic acupoints along six different meridians in the human body. Only Point 2 (corresponding to Pericardium 7) induced a response. In the second trial, EA stimulation at varying frequencies of 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30 Hz, and 15 mA were conducted through Point 2. Stimulation at 6 and 15 Hz induced an immediate response; 21 Hz induced a response only after the ceasing of stimulation. All other frequencies failed to induce a response. The data point to the importance of frequency-specific stimulation at specific acupoints for the release of neurotransmitters in the brain. We speculate that each meridian entails a stimulus of a specific frequency and intensity, which induces the release of its associated neurotransmitters or cytokines. This is a concept with far-reaching clinical implications for acupuncture therapy, including the treatment of dopamine-related disorders.

Transcranial magnetic stimulation attenuates cell loss and oxidative damage in the striatum induced in the 3-nitropropionic model of Huntington's disease.

An investigation was conducted on the effect of transcranial magnetic field stimulation (TMS) on the free radical production and neuronal cell loss produced by 3-nitropropionic acid in rats. The effects of 3-nitropropionic acid were evaluated by examining the following changes in: the quantity of hydroperoxides and total radical-trapping antioxidant potential (TRAP), lipid peroxidation products, protein carbonyl groups, reduced glutathione (GSH) content, glutathione peroxidase (GSH-Px), catalase and succinate dehydrogenase (SDH) activities; total nitrite and cell death [morphological changes, quantification of neuronal loss and lactate dehydrogenase (LDH) levels]. Our results reveal that 3-nitropropionic acid induces oxidative and nitrosative stress in the striatum, prompts cell loss and also shows that TMS prevents the harmful effects induced by the acid. In conclusion, the results show the ability of TMS to modify neuronal response to 3-nitropropionic acid.

Endogenous melatonin protects L-DOPA from autoxidation in the striatal extracellular compartment of the freely moving rat: potential implication for long-term L-DOPA therapy in Parkinson's disease.

We previously showed, using microdialysis, that autoxidation of exogenous L-dihydroxyphenylalanine (L-DOPA) occurs in vivo in the extracellular compartment of the freely moving rat, with a consequent formation of L-DOPA semiquinone (L-DOPA-SQ). In the present study, intrastriatal infusion of L-DOPA (1.0 microm for 200 min) increased dialysate L-DOPA concentrations (maximum increases up to 116-fold baseline values); moreover, L-DOPA-SQ was detected in dialysates. Individual dialysate concentrations of L-DOPA were negatively correlated with those of L-DOPA-SQ. Co-infusion of N-acetylcysteine (100 microm) or melatonin (50 microm) increased L-DOPA (up to 151- and 246-fold, respectively) and decreased L-DOPA-SQ (by about 53% and 87%, respectively) dialysate concentrations. Systemic L-DOPA [25 mg/kg intraperitoneally (i.p.) twice in a 12-h interval] significantly increased striatal baseline dialysate concentrations of L-DOPA and decreased dopamine (DA) and ascorbic acid (AsAc) concentrations, when compared with controls. Following systemic L-DOPA, L-DOPA-SQ was detected in dialysates. Endogenous melatonin was depleted in rats maintained on a 24-h light cycle for 1 wk. In melatonin-depleted rats, systemic L-DOPA induced a smaller increase in dialysate L-DOPA, a greater increase in L-DOPA-SQ formation, and a greater reduction in DA and AsAc dialysate concentrations. Co-administration of melatonin (5.0 mg/kg, i.p., twice in a 12-h interval) with L-DOPA, in control as well as in light-exposed rats, significantly increased dialysate L-DOPA concentrations, greatly inhibited L-DOPA-SQ formation, and restored up to the control values dialysate DA and AsAc concentrations. These findings demonstrate that endogenous melatonin protects exogenous L-DOPA from autoxidation in the extracellular compartment of the striatum of freely moving rats; moreover, systemic co-administration of melatonin with L-DOPA markedly increases striatal L-DOPA bioavailability in control as well as in melatonin-depleted rats. These results may be of relevance to the long-term L-DOPA therapy of Parkinson's disease.

Partial mitochondrial inhibition causes striatal dopamine release suppression and medium spiny neuron depolarization via H2O2 elevation, not ATP depletion.

Mitochondrial dysfunction is a potential causal factor in Parkinson's disease. We show here that acute exposure to the mitochondrial complex I inhibitor rotenone (30-100 nM; 30 min) causes concentration-dependent suppression of single-pulse evoked dopamine (DA) release monitored in real time with carbon-fiber microelectrodes in guinea pig striatal slices, with no effect on DA content. Suppression of DA release was prevented by the sulfonylurea glibenclamide, implicating ATP-sensitive K+ (KATP) channels; however, tissue ATP was unaltered. Because KATP channels can be activated by hydrogen peroxide (H2O2), as well as by low ATP, we examined the involvement of rotenone-enhanced H2O2 generation. Confirming an essential role for H2O2, the inhibition of DA release by rotenone was prevented by catalase, a peroxide-scavenging enzyme. Striatal H2O2 generation during rotenone exposure was examined in individual medium spiny neurons using fluorescence imaging with dichlorofluorescein (DCF). An increase in intracellular H2O2 levels followed a similar time course to that of DA release suppression and was accompanied by cell membrane depolarization, decreased input resistance, and increased excitability. Extracellular catalase markedly attenuated the increase in DCF fluorescence and prevented rotenone-induced effects on membrane properties; membrane changes were also largely prevented by flufenamic acid, a blocker of transient receptor potential (TRP) channels. Thus, partial mitochondrial inhibition can cause functional DA denervation via H2O2 and KATP channels, without DA or ATP depletion. Furthermore, amplified H2O2 levels and TRP channel activation in striatal spiny neurons indicate potential sources of damage in these cells. Overall, these novel factors could contribute to parkinsonian motor deficits and neuronal degeneration caused by mitochondrial dysfunction.

Purine level regulation during energy depletion associated with graded excitatory stimulation in brain.

OBJECTIVES: The formation and release of adenosine following graded excitatory stimulation of the brain may serve important physiological functions such as sleep regulation, as well as an early resistance mechanism against excitotoxicity. However, adenosine at high levels may reflect merely the results of obstructed energy metabolism. METHODS: We examined the extent to which levels of adenosine and adenylate energy charge are affected in vivo by graded excitatory stimulations of brain using unilateral intrastriatal injections of glutamatergic agents and head-focused high energy microwaving for accurate and precise measures of purines. RESULTS: Our results confirmed that adenosine levels rise when adenylate energy charge decreases and showed that these increases occurred in three distinct phases with the rate of adenosine formation in each phase increasing as tissue adenylate energy charge was further depleted. In addition, we observed that, in most cases, the effects of focal excitatory stimulation on changes in tissue purine levels were restricted spatially within the immediate vicinity of the injection site; however, when strongly depolarizing stimuli were used, changes in purine levels could be observed in adjacent and, occasionally, even in contralateral brain regions. DISCUSSION: These results provide new insight into purine regulation that occurs under physiologically relevant conditions, such as sleep and during the early stages of brain insults that induce excitotoxicity.

Absence of hematopoiesis from transplanted olfactory bulb neural stem cells.

Neural stem cells giving rise to neurons and glia cells have been isolated from the embryonic and adult central nervous system. The extent to which they are able to differentiate into cells of non-neural lineages, such as the hematopoietic lineage, is nonetheless unclear. We previously reported the isolation of stem cells from the mouse olfactory bulb neuroepithelium. In the present study, we analysed whether olfactory bulb stem cells (OBSC) can generate cells with hematopoietic features. Cells were prepared from the olfactory bulbs of transgenic mice expressing enhanced green fluorescent protein (EGFP). In culture, transgenic cells proliferated with the same kinetics as wild-type cells. Following mitogen removal, both cell types gave rise to similar numbers of neurons, astrocytes and oligodendrocytes, indicating that EGFP overexpression does not alter OBSC proliferation and differentiation patterns. When these cells were injected into the tail vein of irradiated mice, no hematopoietic cells derived from the OBSC could be recovered in their peripheral blood, spleen or bone marrow. By contrast, when OBSC were transplanted into the adult brain, EGFP-positive cells were found in the striatum and corpus callosum; differentiated cells expressed antigenic markers of neurons and astrocytes. These results suggest that embryonic olfactory bulb stem cells are not endowed with the potential to produce hematopoiesis.

Magnesium activation of GTP hydrolysis or incubation in S-adenosyl-l-methionine reverses iron-56-particle-induced decrements in oxotremorine enhancement of K+-evoked striatal release of dopamine.

Previous research has determined that the deficits in motor behavior seen in aged animals irradiated with (56)Fe particles involved alterations in muscarinic receptor sensitivity. In the present experiments, we determined whether increasing either membrane fluidity by exposure of striatal slices from irradiated ((56)Fe particles) animals to S-adenosyl-l-methionine (SAM) or GTP hydrolysis with Mg(2+) would reverse this (56)Fe-particle-induced loss of muscarinic receptor sensitivity, as has been observed in aged animals. Results indicated that, while increasing Mg(2+) concentrations in the incubation medium was effective in reducing the radiation effects, SAM was able to effect some reversal of the radiation effects only at the lower concentration (200 microM). These results suggest that similar mechanisms may be involved in the deficits in signal transduction seen after (56)Fe-particle irradiation to those seen in aging, and that these may include changes in the membrane structure or composition that could alter subsequent responsiveness of transduction pathways. The results further suggest that, as has been reported previously, (56)Fe-particle irradiation may accelerate brain aging, and that since these HZE particles contribute at least 1% of the dose that astronauts would receive from cosmic rays, long-term exposure on extended space flights (e.g. to Mars) may produce similar deficits that could have immediate or delayed effects on behavior.

Effect of neutron-gamma radiation on dopamine and serotonin metabolism in the rat brain: a regional analysis.

The concentrations of dopamine (DA) and its metabolites and the levels of 5-hydroxyindoleacetic acid (5-HIAA), the metabolite of serotonin, were determined in discrete cerebral areas of rats 3 hr after (neutron-gamma) irradiation at 4 and 7 Gy. After the 7 Gy irradiation, no significant effect was observed. After the 4 Gy exposure, the most marked difference between irradiated and control rats was in the levels of DA and its metabolites in the striatum. We observed a decrease of DA, HVA, and DOPAC levels in the striatum and an opposite pattern in the substantia nigra. Whatever the brain area observed, an increase of 5-HIAA levels was noted.

Effect of ionizing radiation on in vivo striatal release of dopamine in the rat.

The time-course effect of ionizing radiation on the levels of basal and KCl-stimulated striatal release of dopamine (DA) was examined in vivo using microdialysis techniques. The basal level of extracellular DA in sham-irradiated controls was 0.172 +/- 0.042 pmol/sample (n = 9), and it increased 7.1-fold after the stimulation by 30 mM KCl (20microliters). However, the release of dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), two metabolites of DA, was reduced significantly by 30 mM KCl (P < 0.05). In the presence of 10 microM forskolin, an activator of adenylate cyclase, a second stimulation by 30 mM KCl increased the release of DA 6.9-fold. Radiation exposure, at a dose of 10 Gy at 10 Gy/min, had no significant effect on the levels of either basal or KCl-stimulated release of DA or on the release of DOPAC and HVA. Striatal DA release increased in response to two consecutive challenges of KCl. However, the release of DA in response to the second challenge of KCl was significantly smaller than that after the first challenge (543 +/- 110% compared to 794 +/- 164%, P < 0.05; Student's paired t test). Pretreatment with 10 muM forskolin, which by itself had no significant effect on the level of basal release of DA, prevented the decreased response of DA to the second challenge of KCl. Our results suggest that radiation exposure at the dose we used has no significant effect on the level of the basal release of DA or the release of DA stimulated by 30mM KCl in the rat striatum, and that a reduced release of DA in response to repeated KCl stimulation might involve the cAMP effector system.

Radiation inactivation studies of the dopamine reuptake transporter protein.

Using radiation inactivation, we have estimated the target size for the neuronal dopamine transporter protein. The specific binding of several radioligands previously shown to label the dopamine transporter was determined in an irradiated striatal membrane preparation. The apparent target size of the 1-[1-(2-[3H]benzo[b]thienyl)cyclohexyl]piperidine site was approximately 98 kDa. However, the apparent target size of the "cocaine binding site," as measured with the cocaine analogue 2 beta-[3H]carbomethoxy-3 beta-(4-fluorophenyl)tropane in the same assays, was approximately 140 kDa. Radiation inactivation of the binding of other ligands (GBR-12935 and mazindol) led to target size estimates in the same range (94 kDa and 133 kDa, respectively). All of these target sizes are significantly larger than the estimate of 70 kDa derived from the deduced amino acid sequence for the cloned dopamine reuptake transporter cDNA. Larger target sizes than expected have also been reported for ligand binding to the sodium-dependent serotonin transporter and glucose transporter. The estimated sizes for the ligand binding site(s) associated with the dopamine transporter protein are difficult to reconcile with a single transporter protein of 70 kDa. We conclude that the dopamine transporter protein is a homo- or hetero-oligomer when occupied in situ by uptake-blocking drugs like cocaine.

Deficits in the sensitivity of striatal muscarinic receptors induced by 56Fe heavy-particle irradiation: further "age-radiation" parallels.

We had previously shown that there was a loss of sensitivity of muscarinic receptors (mAChR) to stimulation by cholinergic agonists (as assessed by examining oxotremorine enhancement of K(+)-evoked release of dopamine from neostriatal slices) in animals that had been exposed to energetic particles (56Fe, 600 MeV/n), an important component of cosmic rays. This loss of mAChR sensitivity was postulated to be the result of radiation-induced alterations in phosphoinositide-mediated signal transduction. The present experiments were undertaken as a first step toward determining the locus of these radiation-induced deficits in signal transduction by examining K+ enhancement of release of dopamine in 56Fe-exposed animals (0, 0.1, and 1.0 Gy) with agents [A23187, a potent Ca2+ ionophore, or 1,4,5-inositol trisphosphate (IP3)] that "bypass" the mAChR-G protein interface and by comparing the response to oxotremorine-enhanced K(+)-evoked release of dopamine. Results showed that although oxotremorine-enhanced K(+)-evoked release of dopamine was reduced significantly in the radiation groups, no radiation effects were seen when A23187 or IP3 was used to enhance K(+)-evoked release of dopamine. Since similar findings have been observed in aging, the results are discussed in terms of the parallels between aging and radiation effects in signal transduction that might exist in the neostriatum.

[The effect of ionizing radiation at superhigh doses on the processes of dopamine release and reuptake by nerve endings in different sections of the brain]. / Vliianie ioniziruiushchego izlucheniia v sverkhvysokikh dozakh na protsessy vysvobozhdeniia i obratnogo zakhvata dofamina nervnymi terminaliami razlichnykh otdelov golovnogo mozga.

Rats exposed to fast 24 MeV electrons (100 Gy) at the state of early transient incapacity (ETI) exhibited active release and reuptake of dopamine in nerve terminals of the striatum. No changes in the indices under study were found in rats exposed to 25 Gy radiation that did not cause the ETI development. The in vitro irradiation of the isolated synaptosomes (100 Gy) inhibited dopamine reuptake and increased the number of sites of 3H-spiperone binding to D2-receptors in a membrane fraction isolated from the striatum.