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Background Prolonged inflammation, oxidative stress, and protein aggregation are important factors contributing to Parkinson’s disease (PD) pathology. A known ROS generator, pesticide paraquat (PQ), was indicated as an environmental substance potentially increasing the incidence of PD and is used to model this disease. We investigated if a combination of inflammation and oxidative stress in subthreshold doses would exacerbate the modelled neuropathology. Methods We examined the late effects of acute or repeated peripheral inflammation induced by low dose of LPS (10 μg/kg, ip) on PQ toxicity in the rat nigrostriatal dopaminergic pathway, microglial activation markers and expression of major Lewy bodies proteins, α-synuclein and synphilin-1. Results We observed that LPS increased, while PQ decreased body temperature and microglia CD11b expression in the SN. Single LPS pretreatment, 3 h before repeated weekly PQ injections (4×) slightly aggravated neuronal degeneration in the SN. Moreover, degeneration of dopaminergic neurons after weekly repeated inflammation itself (4×) was observed. Interestingly, repeated LPS administration combined with each PQ dose counteracted such effect. The expression of α-synuclein decreased after repeated LPS injections, while only combined, repeated LPS and PQ treatment lowered the levels of synphilin-1. Therefore, α-synuclein and synphilin-1 expression change was influenced by different mechanisms. Concomitantly, decreased levels of the two proteins correlated with decreased degeneration of dopaminergic neurons and with a normalized microglia activation marker. Conclusions Our results indicate that both oxidative insult triggered by PQ and inflammation caused by peripheral LPS injection can individually induce neurotoxicity. Those factors act through different mechanisms that are not additive and not selective towards dopaminergic neurons, probably implying microglia. Repeated, but small insults from oxidative stress and inflammation when administered in significant time intervals can counteract each other and even act protective as a preconditioning effect. The timing of such repetitive insults is also of essence.

Inflammatory processes and microglia activation accompany most of the pathophysiological diseases in the central nervous system. It is proven that glial pathology precedes and even drives the development of multiple neurodegenerative conditions. A growing number of studies point out the importance of microglia in brain development as well as in physiological functioning. These resident brain immune cells are divergent from the peripherally infiltrated macrophages, but their precise in situ discrimination is surprisingly difficult. Microglial heterogeneity in the brain is especially visible in their morphology and cell density in particular brain structures but also in the expression of cellular markers. This often determines their role in physiology or pathology of brain functioning. The species differences between rodent and human markers add complexity to the whole picture. Furthermore, due to activation, microglia show a broad spectrum of phenotypes ranging from the pro-inflammatory, potentially cytotoxic M1 to the anti-inflammatory, scavenging, and regenerative M2. A precise distinction of specific phenotypes is nowadays essential to study microglial functions and tissue state in such a quickly changing environment. Due to the overwhelming amount of data on multiple sets of markers that is available for such studies, the choice of appropriate markers is a scientific challenge. This review gathers, classifies, and describes known and recently discovered protein markers expressed by microglial cells in their different phenotypes. The presented microglia markers include qualitative and semi-quantitative, general and specific, surface and intracellular proteins, as well as secreted molecules. The information provided here creates a comprehensive and practical guide through the current knowledge and will facilitate the choosing of proper, more specific markers for detailed studies on microglia and neuroinflammatory mechanisms in various physiological as well as pathological conditions. Both basic research and clinical medicine need clearly described and validated molecular markers of microglia phenotype, which are essential in diagnostics, treatment, and prevention of diseases engaging glia activation.

Besides degenerating neurons in Parkinson's disease (PD), astrocytes and microglia also become activated and show pathologic changes. Interestingly, the dopaminergic system has a specific glial environment that makes neurons more vulnerable to inflammation and oxidative stress than in other brain regions. Glia have specific functions in the brain that determine their part in the pathophysiology of PD. Especially, α-synuclein pathology is related to glia functioning in the aging brain. Recent studies have shown alternative activation pathways for microglia and astrocytes, indicating that supportive astrocytes can become toxic for neurons, and activated microglia also can perform regenerative functions. In this chapter we also present animal models to study gliopathy in PD. Importantly, the substantial protective potential of astrocytes in new therapeutic approaches, and new perspectives, are discussed.

Originally believed to primarily affect neurons, Parkinson's disease (PD) has recently been recognized to also affect the functions and integrity of microglia and astroglia, two cell categories of fundamental importance to brain tissue homeostasis, defense, and repair. Both a loss of glial supportive-defensive functions and a toxic gain of glial functions are implicated in the neurodegenerative process. Moreover, the chronic treatment with L-DOPA may cause maladaptive glial plasticity favoring a development of therapy complications. This chapter focuses on the pathophysiology of PD from a glial point of view, presenting this rapidly growing field from the first discoveries made to the most recent developments. We report and compare histopathological and molecular findings from experimental models of PD and human studies. We moreover discuss the important role played by astrocytes in compensatory adaptations taking place during presymptomatic disease stages. We finally describe examples of potential therapeutic applications stemming from an increased understanding of the important roles of glia in PD.

The benefits of a ketogenic diet in childhood epilepsy steered up hope for neuroprotective effects of hyperketonemia in Parkinson’s disease (PD). There are multiple theoretical reasons but very little actual experimental proof or clinical trials. We examined the long-term effects of the ketogenic diet in an animal model of early PD. A progressive, selective dopaminergic medium size lesion was induced by 6-OHDA injection into the medial forebrain bundle. Animals were kept on the stringent ketogenic diet (1% carbohydrates, 8% protein, 70% fat) for 3 weeks prior and 4 weeks after the brain operation. Locomotor activity, neuron count, dopaminergic terminal density, dopamine level, and turnover were analyzed at three time-points post-lesion, up to 4 weeks after the operation. Energy metabolism parameters (glycogen, mitochondrial complex I and IV, lactate, beta-hydroxybutyrate, glucose) were analyzed in the brain and liver or plasma. Protein expression of enzymes essential for gluconeogenesis (PEPCK, G6PC) and glucose utilization (GCK) was analyzed in the liver. Despite long-term hyperketonemia pre- and post-lesion, the ketogenic diet did not protect against 6-OHDA-induced dopaminergic neuron lesions. The ketogenic diet only tended to improve locomotor activity and normalize DA turnover in the striatum. Rats fed 7 weeks in total with a restrictive ketogenic diet maintained normoglycemia, and neither gluconeogenesis nor glycogenolysis in the liver was responsible for this effect. Therefore, potentially, the ketogenic diet could be therapeutically helpful to support the late compensatory mechanisms active via glial cells but does not necessarily act against the oxidative stress-induced parkinsonian neurodegeneration itself. A word of caution is required as the stringent ketogenic diet itself also carries the risk of unwanted side effects, so it is important to study the long-term effects of such treatments. More detailed metabolic long-term studies using unified diet parameters are required, and human vs. animal differences should be taken under consideration.

Progressive degeneration of dopaminergic neurons in the substantia nigra (SN) is the underlying cause of Parkinson’s disease (PD). The disease in early stages is difficult to diagnose, because behavioral deficits are masked by compensatory processes. Astrocytic and microglial pathology precedes motor symptoms. Besides supportive functions of astrocytes in the brain, their role in PD is unrecognized. Prolonged dysfunction of astrocytes could increase the vulnerability of dopaminergic neurons and advance their degeneration during aging. The aim of our studies was to find out whether prolonged dysfunction of astrocytes in the SN is deleterious for neuronal functioning and if it influences their survival after toxic insult or changes the compensatory potential of the remaining neurons. In Wistar rat model, we induced activation, prolonged dysfunction, and death of astrocytes by chronic infusion of fluorocitrate (FC) into the SN, without causing dopaminergic neuron degeneration. Strongly enhanced dopamine turnover in the SN after 7 days of FC infusion was induced probably by microglia activated in response to astrocyte stress. The FC effect was reversible, and astrocyte pool was replenished 3 weeks after the end of infusion. Importantly, the prolonged astrocyte dysfunction and microglia activation accelerated degeneration of dopaminergic neurons induced by 6-hydroxydopamine and blocked the behavioral compensation normally observed after moderate neurodegeneration. Impaired astrocyte functioning, activation of microglia, diminishing compensatory capability of the dopaminergic system, and increasing neuronal vulnerability to external insults could be the underlying causes of PD. This animal model of prolonged astrocyte dysfunction can be useful for in vivo studies of glia–microglia–neuron interaction. Electronic supplementary material The online version of this article (doi:10.1007/s12035-017-0529-z) contains supplementary material, which is available to authorized users.

Glial pathology precedes symptoms of Parkinson's disease and multiple other neurodegenerative diseases. Prolonged impairment of astrocytic functions could increase the vulnerability of dopaminergic neurons in the substantia nigra (SN), accelerate their degeneration and affect ability to compensate for partial degeneration at the presymptomatic stages of the disease. The aim of this study was to investigate the astrocyte depletion in the SN, its impact on the dopaminergic system functioning and multiple markers of energy metabolism during the early stages of neurodegeneration and compensation. We induced death of 30% of astrocytes by chronic infusion of fluorocitrate (FC) into the SN, simultaneously activating microglia response but sparing the dopaminergic neurons. The FC effect was reversible after toxin withdrawal. Dopaminergic neurons were killed by 6-hydroxydopamine causing transient locomotor disability, reversed with time showing compensatory potential. Death of astrocytes diminished the capability of the dopaminergic system to compensate for the degeneration of neurons and caused a local energy deprivation by decreasing lactate and glycogen amount. Studied markers suggest a shift in the usage of energy substrates, via increased glycogenolysis and glycolysis markers, ketone bodies availability and fatty acid transport in remaining cells. Peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1alpha) and AMP-activated protein kinase (AMPK), the energy sensors, showed different regulation between the cell-types. Increased neuronal expression of carnitine palmitoyltransferase 1c could play a role in the adaptation to metabolic stress in response to glia dysfunction. Astrocyte energetic support is one of the essential factors for neuronal compensatory mechanisms of dopaminergic system and might have a leading role in the presymptomatic Parkinson's disease stages. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Background Excessive and hypersynchronous neuronal discharges are key characteristics in the pathophysiology of neurological disorders such as epilepsy. Owing to their ability of regulating neuronal excitability, small conductance calcium-activated potassium (SK) channels have been implicated in several diseases of the brain, and their activation provided neuroprotection in different paradigms of cell death, including oxidative stress and excitotoxicity. Objectives In our study, we investigated the function and regulation of SK channel expression in different models of epilepsy and excessive neuronal firing. Methods As a model for hypersynchronous neuronal firing in vitro, we used primary cortical neurons either challenged with glutamate or deprived of magnesium to increase neuronal firing. In addition, we used perforant pathway stimulation (PPS) to induce hippocampal seizures in vivo. We investigated neuronal firing using multielectrode array recordings, analyze SK channel expression by Western blot, and assess mitochondrial performance by evaluating mitochondrial complex activity. MicroRNA135a-dependent effects on SK3 channels were investigated using a dual-luciferase assay. Results and Conclusions In vitro, analysis of neuronal firing in magnesium deprived primary neuronal cultures revealed that SK channel activation fully blocked the increase in neuronal activity, and restored homeostatic signaling. We found reduced SK3 channel expression following glutamate-induced excitotoxicity in vitro, and following PPS in the rat hippocampus in vivo. Further, PPS in vivo impaired the performance of mitochondrial complex I. Interestingly, we identified miRNA-135a as a key regulator of SK3 channel expression in primary neurons. Thus, we provide strong evidence that SK3 channels are involved in epilepsy(-like) conditions which are characterized by enhanced neuronal firing and an impairment of mitochondrial function, and the miRNA135a-dependent regulation of SK3 channel expression was unraveled as a new regulatory mechanism.

Background The present study sought to further evaluate the role of the serotonergic system especially the postsynaptic 5-HT1A receptors (5-HT1AR) in the mechanism of antidepressant action of zinc. Methods Messenger RNA (mRNA), protein level, and 5-HT1AR density as well as the rate of monoamine (dopamine, DA, and serotonin) metabolism in the prefrontal cortex (PFC) and hippocampus (Hp) of rats subjected to acute and chronic (21 days) zinc (5 mg Zn/kg) treatment were measured. Results Acute or chronic zinc treatment did not induce any changes in 5-HT1AR mRNA levels in the PFC or Hp of rats. However, chronic zinc treatment induced increases in both 5-HT1AR protein levels and density of 5-HT1A receptor binding sites in the Hp of rats. Chronic zinc treatment also increased tissue levels of serotonin metabolite and turnover in the rat Hp. On the other hand, DA, DOPAC, HVA tissue levels increased while DOPAC/DA and 3MT/DA decreased in the PFC of rats after chronic zinc treatment. Acute treatment induced increases only in tissue levels of DOPAC, and DOPAC/DA. Conclusions Our results confirm that the antidepressant effects of zinc are mediated in concert with the modulation of the serotonergic system including postsynaptic 5-HT1ARs and allude to a possible involvement of dopaminergic neurotransmission in this action.

Depression is a frequent comorbid disorder in Parkinson's disease and may antedate its motor symptoms. However, mechanisms underlying Parkinson's disease-associated depression are unknown and its current medication is insufficient. The aim of the present study was to compare antidepressant-like effects of imipramine, fluoxetine and pramipexole in a model of preclinical stages of Parkinson's disease in rats. 6-Hydroxydopamine was bilaterally injected into the ventrolateral region of the caudate-putamen in rats. This treatment induced moderate decreases in the levels of dopamine and its metabolites in the caudate-putamen, nucleus accumbens and frontal cortex and reduced the density of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra pars compacta and ventral tegmental area. The lesion increased immobility measured in the forced swimming test without influencing locomotor activity. Chronic (13 days) administration of pramipexole (1mg/kg sc/twice a day) reversed prolongation of the immobility time in lesioned animals but did not stimulate their locomotion. Chronic pramipexole activated dopaminergic transmission in the brain structures which might contribute to its effectiveness in the forced swimming test. In contrast, the 13-day administration of imipramine (10mg/kg ip/day) and fluoxetine (10mg/kg ip/day) did not shorten the immobility time in lesioned rats but reduced their locomotion. The present study indicates that already a moderate lesion of dopaminergic neurons induces "depressive-like" behaviour in animals which is reversed by chronic administration of the antiparkinsonian drug, pramipexole.

Harmaline induces tremor in animals resembling essential tremor which has been suggested to result from activation of the glutamatergic olivo-cerebellar projection. The aim of the present study was to examine the effects of systemic administration of Lu AF21934, a brain-penetrating positive allosteric modulator of the metabotropic glutamate receptor 4 (mGlu4), on the harmaline-induced tremor and other forms of motor activity in rats using fully automated Force Plate Actimeters. The influence of harmaline on the mGlu4 mRNA expression in the cerebellum and inferior olive was analysed by in situ hybridization. Harmaline at a dose of 15 mg/kg (ip) triggered tremor which was manifested by an increase in the power within 9–15 Hz band and in the tremor index (a difference in power between bands 9–15 Hz and 0–8 Hz). Harmaline induced a biphasic effect on mobility, initially inhibiting the exploratory locomotor activity of rats (0–30 min after administration), followed by an increase in their basic activity. Lu AF21934 (0.5–5 mg/kg sc) did not influence tremor but at doses of 0.5 and 2.5 mg/kg reversed harmaline-induced hyperactivity. MGlu4 mRNA expression was high in the cerebellar cortex and low in the inferior olive. Repeated harmaline (15 mg/kg ip once a day for 5 days] decreased mGlu4 mRNA in the cerebellum and inferior olive. The present study indicates that the mGlu4 stimulation counteracts hyperactivity induced by harmaline which suggests the involvement of cerebellar glutamatergic transmission in this process. In contrast, neuronal mechanisms involved in tremor seem to be insensitive to the stimulation of mGlu4.

A number of studies suggest that the ubiquitin–proteasome system (UPS) impairment may underlie neuronal death in Parkinson’s disease. Celastrol is a neuroprotective agent with anti-inflammatory and antioxidant properties. The aim of this study was to determine whether celastrol may exert neuroprotective effects both in vitro and in vivo under conditions of the lactacystin-induced UPS inhibition. In the in vitro study, mouse primary cortical neurons and neuroblastoma SH-SY5Y cells were incubated with lactacystin for 48 h (2.5 and 10 μg/ml, respectively). The animal study was performed on male Wistar rats injected unilaterally with lactacystin (5 μg/2 μl) into the substantia nigra (SN) pars compacta. In the in vitro study, we did not found any protective effects of celastrol, given either in the pre- or co-treatment mode. Moreover, in the higher concentrations, celastrol itself reduced cell viability, and enhanced the lactacystin-induced cell death in both types of cells. In the in vivo study, none of the celastrol doses (0.3–3 mg/kg) attenuated the lactacystin-induced decrease in the level of dopamine (DA) and its metabolites or protected nigral dopaminergic neurons against the lactacystin-induced degeneration. The highest celastrol dose potentiated the lactacystin-induced decrease in the level of DA and its metabolites in the lesioned striatum, and accelerated the lactacystin-induced increase in the oxidative and total metabolism of DA. Moreover, when given alone, this dose of celastrol bilaterally decreased the number and/or density of dopaminergic neurons in the SN. Our results demonstrate that celastrol does not induce neuroprotective effects under conditions of UPS inhibition.

Cause of Parkinson's disease (PD) is still not understood. Motor symptoms are not observed at early stages of disease due to compensatory processes. Dysfunction of mitochondria was indicated already at preclinical PD. Selective toxin 6-OHDA was applied to kill dopaminergic neurons in substantia nigra and disturb neuronal transmission in striatum. Early phase of active degeneration and later stage, when surviving cells adapted to function normally, were analysed. 2D BN/SDS difference gel electrophoresis (DIGE) of mitochondrial proteome enabled to point out crucial processes involved at both time-points in dopaminergic structures. Marker proteins such as DPYSL2, HSP60, ATP1A3, EAAT2 indicated structural remodelling, cytoskeleton rearrangement, organelle trafficking, axon outgrowth and regeneration. Adaptations in dopaminergic and glutamatergic neurotransmission, recycling of synaptic vesicles, along with enlargement of mitochondria mass were proposed as causative for compensation. Changed expression of carbohydrates metabolism and oxidative phosphorylation proteins were described, including their protein-protein interactions and supercomplex assembly.

To study the influence of the central noradrenergic system on antinociceptive effects mediated by the CB(1)-receptor agonist methanandamide, intact rats were contrasted with rats in which noradrenergic nerves were largely destroyed shortly after birth with the neurotoxin DSP-4 [N-(-2-chloroethyl)-N-ethyl-2-bromobenzylamine (50 mg/kg sc × 2, P1 and P3); zimelidine (10 mg/kg sc, 30 min pretreatment, selective serotonin reuptake inhibitor). When rats attained 10 weeks of age, monoamine and their metabolite concentrations were determined in the frontal cortex, thalamus, and spinal cord by an HPLC/ED method. Antinociceptive effects after methanandamide (10 mg/kg ip) apply were evaluated by a battery of tests. In addition, immunohistochemistry and densitometric analysis of the cannabinoid CB(1) receptor in the rat brain was performed. DSP-4 lesioning was associated with a reduction in norepinephrine content of the frontal cortex (90 %) and spinal cord (80 %) with no changes in the thalamus. Neonatal DSP-4 treatment produced a significant reduction in the antinociceptive effect of methanandamide in the tail-immersion test, hot-plate test and writhing tests. In the paw pressure and formalin hind paw tests results were ambiguous. These findings indicate that the noradrenergic system exerts a prominent influence on analgesia acting via the cannabinoid system in brain, without directly altering CB(1) receptor density in the brain.

The present study was designed to investigate the role of postnatal fluoride intake on [3H]glucose uptake and transport in rat brain and peripheral tissues. Sodium fluoride (NaF) in a concentration of 10 or 50 ppm was added to the drinking water of adult Wistar rats. The control group received distilled water. After 4 weeks, respective plasma fluoride levels were 0.0541 ± 0.0135 μg/ml (control), 0.0596 ± 0.0202 μg/ml (10 ppm), and 0.0823 ± 0.0199 μg/ml (50 ppm). Although plasma glucose levels were not altered in any group, the plasma insulin level in the fluoride (50 ppm) group was elevated (0.72 ± 0.13 μg/ml) versus the control group (0.48 ± 0.24 μg/ml) and fluoride (10 ppm) group. In rats receiving fluoride for 4 weeks at 10 ppm in drinking water, [3H]glucose uptake was unaltered in all tested parts of the brain. However, in rats receiving fluoride at 50 ppm, [3H]glucose uptake in cerebral cortex, hippocampus, and thalamus with hypothalamus was elevated, versus the saline group. Fluoride intake had a negligible effect on [3H]glucose uptake by peripheral tissues (liver, pancreas, stomach, small intestine, atrium, aorta, kidney, visceral tissue, lung, skin, oral mucosa, tongue, salivary gland, incisor, molars, and jawbone). In neither fluoride group was glucose transporter proteins 1 (GLUT 1) or 3 (GLUT 3) altered in frontal cortex and striatum versus control. On the assumption that increased glucose uptake (by neural tissue) reasonably reflects neuronal activity, it appears that fluoride damage to the brain results in a compensatory increase in glucose uptake and utilization without changes in GLUT 1 and GLUT 3 expression.

Abnormal synchronous activation of the glutamatergic olivo-cerebellar pathway has been suggested to be crucial for the harmaline-induced tremor. The cerebellum receives two catecholaminergic pathways: the dopaminergic pathway arising from the ventral tegmental area/substantia nigra pars compacta, and the noradrenergic one from the locus coeruleus. The aim of the present study was to examine a contribution of the cerebellar catecholaminergic innervations to the harmaline-induced tremor in rats. Rats were injected bilaterally into the cerebellar vermis with 6-hydroxydopamine (6-OHDA; 8 μg/0.5 μl) either alone or this treatment was preceded (30 min earlier) by desipramine (15 mg/kg ip). Harmaline was administered to animals in doses of 7.5 or 15 mg/kg ip. Tremor of forelimbs was measured as a number of episodes during a 90-min observation. Rats were killed by decapitation 30 or 120 min after harmaline treatment. The levels of dopamine, noradrenaline, serotonin, and their metabolites were measured by HPLC in the cerebellum, substantia nigra, caudate-putamen, and frontal cortex. 6-OHDA injected alone enhanced the harmaline-induced tremor. Furthermore, it decreased the noradrenaline level by ca. 40-80% in the cerebellum and increased the levels of serotonin and 5-HIAA in the caudate-putamen and frontal cortex in untreated and/or harmaline-treated animals. When 6-OHDA treatment was preceded by desipramine, it decreased dopaminergic transmission in some regions of the cerebellum while inducing its compensatory activation in others. The latter lesion did not markedly influence the tremor induced by harmaline. The present study indicates that noradrenergic innervation of the cerebellum interacts with cerebral serotonergic systems and plays an inhibitory role in the harmaline-induced tremor.

In a previous study, a primary culture of midbrain cells was exposed to 9-methyl-beta-carboline for 48 h, which caused an increase in the number of tyrosine hydroxylase-positive cells. Quantitative RT-PCR revealed increased transcription of genes participating in the maturation of dopaminergic neurons. These in vitro findings prompted us to investigate the restorative actions of 9-methyl-beta-carboline in vivo. The compound was delivered for 14 days into the left cerebral ventricle of rats pretreated with the neurotoxin 1-methyl-4-phenyl-pyridinium ion (MPP+) for 28 days applying a dose which lowered dopamine by approximately 50%. Interestingly, 9-methyl-beta-carboline reversed the dopamine-lowering effect of the neurotoxin in the left striatum. Stereological counts of tyrosine hydroxylase-immunoreactive cells in the substantia nigra revealed that the neurotoxin caused a decrease in the number of those cells. However, when treated subsequently with 9-methyl-beta-carboline, the number reached normal values. In search of an explanation for the restorative activity, we analyzed the complexes that compose the respiratory chain in striatal mitochondria by 2-dimension gel electrophoresis followed by MALDI-TOF peptide mass fingerprinting.We found no changes in the overall composition of the complexes. However, the activity of complex I was increased by approximately 80% in mitochondria from rats treated with MPP+ and 9-methyl-beta-carboline compared to MPP+ and saline and to sham-operated rats, as determined by measurements of nicotinamide adenine dinucleotide dehydrogenase activity. Microarray technology and single RT-PCR revealed the induction of neurotrophins: brain-derived neurotrophic factor, conserved dopamine neurotrophic factor, cerebellin 1 precursor protein, and ciliary neurotrophic factor. Selected western blots yielded consistent results. The findings demonstrate restorative effects of 9-methyl-beta-carboline in an animal model of Parkinson's disease that improve the effectiveness of the respiratory chain and promote the transcription and expression of neurotrophin-related genes.

The present study shows that JNJ 16259685-a selective antagonist of glutamate metabotropic receptor subtype 1 (mGluR1) injected in doses of 0.16 and 0.32 mg/kg sc strongly enhances tremor of forelimbs, head and trunk, hypolocomotion, and ataxia induced by harmaline (7.5 mg/kg ip) in rats. JNJ 16259685 inhibited locomotor and exploratory activity per se. These results may suggest an inhibitory influence of mGluR1 on the harmaline-induced motor disturbances.

Our recent study has indicated that a moderate lesion of the mesostriatal and mesolimbic pathways in rats, modelling preclinical stages of Parkinson’s disease, induces a depressive-like behaviour which is reversed by chronic treatment with pramipexole. The purpose of the present study was to examine the role of brain derived neurotrophic factor (BDNF) signalling in the aforementioned model of depression. Therefore, we investigated the influence of 6-hydoxydopamine (6-OHDA) administration into the ventral region of the caudate-putamen on mRNA levels of BDNF and tropomyosin-related kinase B (trkB) receptor. The BDNF and trkB mRNA levels were determined in the nigrostriatal and limbic structures by in situ hybridization 2 weeks after the operation. Pramipexole (1 mg/kg sc twice a day) and imipramine (10 mg/kg ip once a day) were injected for 2 weeks. The lesion lowered the BDNF and trkB mRNA levels in the hippocampus [CA1, CA3 and dentate gyrus (DG)] and amygdala (basolateral/lateral) as well as the BDNF mRNA content in the habenula (medial/lateral). The lesion did not influence BDNF and trkB expression in the caudate-putamen, substantia nigra, nucleus accumbens (shell and core) and ventral tegmental area (VTA). Chronic imipramine reversed the lesion-induced decreases in BDNF mRNA in the DG. Chronic pramipexole increased BDNF mRNA, but decreased trkB mRNA in the VTA in lesioned rats. Furthermore, it reduced BDNF and trkB mRNA expression in the shell and core of the nucleus accumbens, BDNF mRNA in the amygdala and trkB mRNA in the caudate-putamen in these animals. The present study indicates that both the 6-OHDA-induced dopaminergic lesion and chronic pramipexole influence BDNF signalling in limbic structures, which may be related to their pro-depressive and antidepressant activity in rats, respectively.

Regarding the pathogenesis of Parkinson's disease, a neurotoxin hypothesis was proposed following the discovery that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces a Parkinson-like syndrome in humans and primates. Since then, researchers have searched for endogenous and exogenous compounds that are structurally similar to this neurotoxin. Such compounds include beta-carbolines, formed from tryptophan and its derivatives. beta-carbolines are present naturally in the human brain and cerebrospinal fluid. The present study examined the effect of bilateral, intranigral administration of 2,9-dimethyl-beta-carbolinium ion on muscle tone, electromyographic activity, dopamine metabolism in the striatum, and the number of tyrosine hydroxylase-immunoreactive neurons and volume of the substantia nigra in rats. We found that the beta-carbolinium ion (15 or 40 nmol per side) caused a significant decrease in the striatal levels of dopamine and its metabolites, which was accompanied by an enhancement of muscle tone and electromyographic activity. Stereological counting revealed that the beta-carbolinium caused a significant decrease in the total number of tyrosine hydroxylase-immunoreactive neurons and shrinkage of the substantia nigra. The findings suggest that the methylated beta-carbolinium ion produces a dose-dependent degeneration of nigrostriatal neurons, leading to deficits in dopaminergic neurotransmission and an increase of muscle resistance and electromyographic activity, a syndrome equivalent to muscle rigidity in Parkinson's disease.

Several data indicate that inhibition of glutamatergic transmission may be important to alleviate of parkinsonian symptoms. Therefore, the aim of the present paper is to review recent studies on the search for putative antiparkinsonian-like effects of mGluR ligands and their brain targets. In order to inhibit glutamatergic transmission, the group I mGluRs (mGluR1 and mGluR5) were blocked, and group II (mGluR2/3) or III (mGluR4/7/8) mGluRs were activated. Systemic or intrastriatal administration of group I mGluR antagonists (mGluR5 - MPEP, MTEP; mGluR1 - AIDA) was found to inhibit parkinsonian-like symptoms (catalepsy, muscle rigidity) in rats. MPEP administered systemically and mGluR1 antagonists (AIDA, CPCCOEt, LY367385) injected intrastriatally reversed also the haloperidol-increased proenkephalin (PENK) mRNA expression in the striatopallidal pathway. Similarly, ACPT-1, a group III mGluR agonist, administered into the striatum, globus pallidus or substantia nigra inhibited the catalepsy. Intrastriatal injection of this compound reduced the striatal PENK expression induced by haloperidol. In contrast, a group II mGluR agonist (2R,4R-APDC) administered intrastriatally reduced neither PENK expression nor the above-mentioned parkinsonian-like symptoms. Moreover, a mixed mGluR8 agonist/AMPA antagonist, (R,S)-3,4-DCPG, administered systemically evoked catalepsy and enhanced both the catalepsy and PENK expression induced by haloperidol. The results reviewed in this article seem to indicate that group I mGluR antagonists or some agonists of group III may possess antiparkinsonian properties, and point at the striatopallidal pathway as a potential target of therapeutic intervention.

A deficiency of the dopaminergic transmission in the mesocortical system has been suggested to contribute to cognitive disturbances in Parkinson's disease. Therefore, the aim of the present study was to examine whether the long-term administration of a commonly used herbicide, paraquat, which has already been found to induce a slowly progressing degeneration of the nigrostriatal neurons, influences mesocortical dopaminergic neurons in rats. Paraquat at a dose of 10 mg/kg i.p. was injected either acutely or once a week for 4, 8, 12 and 24 weeks. Acute treatment with this pesticide increased the level of homovanillic acid (HVA) and HVA/dopamine ratio in the prefrontal cortex. After 8 weeks of administration paraquat increased the number of stereologically counted tyrosine hydroxylase-immunoreactive (TH-ir) neurons and their staining intensity in the ventral tegmental area (VTA), which is a source of the mesocortical dopaminergic projection. At the same time, few TH-ir neurons appeared in different regions of the cerebral cortex: in the frontal, cingulate, retrosplenial and parietal cortices. Chronic paraquat administration did not influence the level of dopamine in the prefrontal cortex but increased the levels of its metabolites: 3,4-dihydroxyphenylacetic acid (after 8-12 weeks), HVA (after 4 and 12 weeks) and HVA/dopamine ratio (4 weeks). After 24 weeks this pesticide reduced the number of TH-ir neurons in the VTA by 42% and of the Nissl-stained neurons by 26%, and induced shrinkage of this structure by ca. 25%. Moreover, TH-ir neurons in the cortex were no more visible after such a long period of administration and levels of dopamine metabolites returned to control values. The present results suggest that the long-term paraquat administration destroys dopaminergic neurons of the VTA. However, compensatory activation of the VTA neurons and cortex overcomes progressing degeneration and maintains cortical dopaminergic transmission.

The aim of the present study was to examine the influence of the long-term paraquat administration on the dopaminergic nigrostriatal system in rats. Paraquat was injected at a dose of 10 mg/kg i.p. for 4-24 weeks. We found that this pesticide reduced the number of tyrosine hydroxylase-immunoreactive neurons of the substantia nigra; after the 4-week treatment the reduction (17%, nonsignificant) was confined to the rostrocentral region of this structure but, after 24 weeks, had spread along its whole length and was approximately 37%. Moreover, it induced a biphasic effect on dopaminergic transmission. First, levels of dopamine, its metabolites and turnover were elevated (4-8 weeks) in the caudate-putamen, then all these parameters returned to control values (12 weeks) and dropped by 25-30% after 24 weeks. The binding of [3H]GBR 12,935 to dopamine transporter in the caudate-putamen was decreased after 4-8 weeks, then returned to control values after 12 weeks but was again decreased after 24 weeks. Twenty-four-week paraquat administration also decreased the level of tyrosine hydroxylase (Western blot) in the caudate-putamen. In addition, paraquat activated serotonin and noradrenaline transmission during the first 12 weeks of treatment but no decreases in levels of these neurotransmitters were observed after 24 weeks. The above results seem to suggest that long-term paraquat administration produces a slowly progressing degeneration of nigrostriatal neurons, leading to delayed deficits in dopaminergic transmission, which may resemble early, presymptomatic, stages of Parkinson's disease.

We have examined the pattern of striatal messenger RNA expression of over 8000 genes in a rat model of levodopa (L-DOPA)-induced dyskinesia and Parkinson disease (PD). 6-Hydroxydopamine (6-OHDA)-lesioned rats were treated with L-DOPA or physiological saline for 22 days and repeatedly tested for antiakinetic response to L-DOPA and the development of abnormal involuntary movements (AIMs). In a comparison of rats that developed a dyskinetic motor response to rats that did not, we found striking differences in gene expression patterns. In rats that developed dyskinesia, GABA neurons had an increased transcriptional activity, and genes involved in Ca2+ homeostasis, in Ca2+-dependent signaling, and in structural and synaptic plasticity were upregulated. The gene expression patterns implied that the dyskinetic striatum had increased transcriptional, as well as synaptic activity, and decreased capacity for energy production. Some basic maintenance chores such as ribosome protein biosynthesis were downregulated, possibly a response to expended of ATP levels.

The aim of the present study was to examine the influence of a unilateral 6-hydroxydopamine (6-OHDA)-induced partial lesion of both the substantia nigra pars compacta (SNc, A9) and retrorubral field (RRF, A8) on the tremor evoked by harmaline. 6-OHDA (8μg/2μl) was injected unilaterally into the region of the posterior part of the SNc and RRF. Harmaline was administered in a dose of 7.5mg/kg ip on the eighth day after the operation and tremor of forelimbs, head and trunk was measured. We found that the lesion increased intensity of the tremor induced by harmaline but did not influence its character. Stereological examination of the lesion extent revealed losses of dopaminergic (tyrosine hydroxylase-immunoreactive) neurons in the anterior (30%) and posterior (72%) SNc, as well as in RRF (72% on the average). Levels of dopamine and all its metabolites, as well as noradrenaline concentrations, were ipsilaterally moderately decreased in the caudate-putamen in the lesioned animals, however, dopamine and DOPAC in the anterior cerebellum were increased. In the caudate-putamen, the ipsi/contra ratio of dopamine level correlated negatively, while that of dopamine turnover positively with the tremor intensity. However, in the anterior cerebellum an inverse relationship was found. Moreover, this symptom correlated positively with the serotonin level and negatively with the 5-HIAA/serotonin ratio on the contralateral side of the posterior cerebellum. The present results seem to indicate that the modulation of dopaminergic and serotonergic transmissions by the lesion modelling early stages of Parkinson's disease may influence tremor triggered in the cerebellum.

Systemic exposure of rodents to the herbicide paraquat (PQ) was suggested to reproduce pathological features of Parkinson's disease. Our recent data showed that long-term PQ administration influenced levels of glycogen synthase kinase 3β (GSK-3β) and its active form phosphorylated on tyrosine 216 in the nigrostriatal system, which may be related to its vulnerability to PQ toxicity. The aim of this study was to analyse selectivity of the toxic effect of PQ after its systemic administration in rats. PQ was administered for 37 weeks and the protein level of total GSK-3β and its active GSK-3β (pY216) form in subcellular fractions of hippocampus, brain cortex and cerebellum was examined. Our data indicated that the long-term administration of PQ significantly decreased the level of both GSK-3β forms in nuclear and cytosolic fractions of hippocampus in rats. In the brain cortex and cerebellum PQ decreased the level of both forms of GSK-3β in the nuclear fraction but increased their levels in mitochondria and in some cases also in the cytosol. The results of the present study indicate that PQ influenced levels and activation of GSK-3β in different brain structures, which may contribute to its toxicity, but on the other hand may suggest development of adaptive, protective mechanisms.

Parkinson's disease is a slowly progressing disease, due to a lesion of dopaminergic neurons in the substantia nigra and a dramatic loss of dopamine in the striatum. It is now accepted that several environmental agents including the herbicide paraquat (PQ) may contribute to its pathogenesis. However, till now nothing is known about the role of glycogen synthase kinase-3β (GSK-3β) in the PQ toxicity. Therefore, the aim of this study was to examine the influence of 37-week administration of PQ in rats on the immunohistochemically measured levels of the total GSK-3β and its active, tyrosine 216 (pY216)-phosphorylated form in subcellular fractions of the midbrain with pons, as well as of the striatum. The present results revealed that the long-term PQ administration increased the levels of total and active forms of GSK-3β in the midbrain with pons, whereas decreased them in the striatum. Examination of the lesion extent showed a decrease in the number of tyrosine-immunoreactive neurons in the substantia nigra pars compacta, ventral tegmental area, and locus coeruleus, as well as lower DOPAC/dopamine ratio and noradrenaline level in the striatum in rats treated with PQ. The long-term PQ administration disturbed also motor activity of rats. Summarizing, the present data indicate that the long-term exposure of rats to PQ, a commonly used herbicide, diversely alters levels of GSK-3β in different brain structures, which may be associated with their vulnerability to its toxicity.

The aim of the present study was to examine the expression of synphilin-1, alpha-synuclein, and tyrosine hydroxylase in human elderly brains and the incidence of Marinesco bodies (MBs, intranuclear inclusions) in the neuromelanin-containing substantia nigra neurons. The brains of twenty-two individuals without clinical signs and symptoms of parkinsonism and dementia and an additional two parkinsonian patients were dissected and subjected to histopathological examination and western blotting. Ubiquitin-positive and agr;-synuclein-negative MBs were found in 0.84-9.45% of the nigral neurons from brains of 15 healthy individuals and both parkinsonian patients. The frequency of pigmented nigral neurons containing MBs was positively correlated with age. The levels of tyrosine hydroxylase in the caudate nucleus and putamen decreased with age, and were inversely correlated with the MB frequency. The level of synphilin-1 in the caudate nucleus was positively correlated both with age and the MBs. Additionally, the MB appearance was correlated with synphilin-1 level in the substantia nigra. No significant correlation between alpha-synuclein expression and age or MBs was found. Our results suggest that synphilin-1 expression increases with aging. Further studies on expression of this protein in elderly brains are warranted.

Paraquat is a toxin suggested to contribute to pathogenesis of Parkinson’s disease. The aim of the present study was to examine toxic influence of subchronic treatment with this pesticide (5 days, one injection per day, 2–3 days of withdrawal) on dopaminergic, serotonergic, noradrenergic and GABAergic neurons. Paraquat decreased the number of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the substantia nigra by 22% (measured 3 days after withdrawal). Two days after withdrawal the levels of the dopamine metabolites and dopamine turnover in the caudate–putamen, substantia nigra and prefrontal cortex were reduced by ca. 20–60%, and the binding of [ 3 H]GBR 12,935 to dopamine transporter dropped by 25–40% in the caudate–putamen. Three days after paraquat withdrawal, the level of dopamine in the caudate–putamen was significantly increased, and earlier decreases in DOPAC and HVA in the substantia nigra, as well as [ 3 H]GBR 12,935 binding in the caudate–putamen were reversed. Moreover, an increase in serotonin turnover in the caudate–putamen and prefrontal cortex, and noradrenaline level in the former structure was observed 2–3 days after paraquat withdrawal. Three days after the last paraquat injection 24–35% decreases in the proenkephalin mRNA levels and 5–7% reduction in glutamic acid decarboxylase (GAD)67 mRNA were found in the caudate–putamen. The present study suggests that subchronic paraquat administration triggers processes characteristic of early stages of dopaminergic neuron degeneration, and activates compensatory mechanisms involving dopaminergic, noradrenergic, serotonergic and GABAergic transmissions.

Group III metabotropic glutamate receptors (mGluRs) are widely distributed in the basal ganglia, especially on the terminals of pathways which seem to be overactive in Parkinson's disease. The aim of the present study was to determine whether (1S,3R,4S)-1-aminocyclo-pentane-1,3,4-tricarboxylic acid (ACPT-1), an agonist of group III mGluRs, injected bilaterally into the globus pallidus (GP), striatum or substantia nigra pars reticulata (SNr), can attenuate the haloperidol-induced catalepsy in rats, and whether that effect was related to modulation of proenkephalin (PENK) or prodynorphin (PDYN) mRNA expression in the striatum. Administration of ACPT-1 (0.05-1.6 microg/0.5 microl/side) caused a dose-and-structure-dependent decrease in the haloperidol (0.5 mg/kg i.p. or 1.5 mg/kg s.c.)-induced catalepsy whose order was as follows: GP>striatum>SNr. ACPT-1, given alone to any of those structures, induced no catalepsy in rats. Haloperidol (3 x 1.5 mg/kg s.c.) significantly increased PENK mRNA expression in the striatum, while PDYN mRNA levels were not affected by that treatment. ACPT-1 (3 x 1.6 microg/0.5 microl/side) injected into the striatum significantly attenuated the haloperidol-increased PENK mRNA expression, whereas administration of that compound into the GP or SNr did not influence the haloperidol-increased striatal PENK mRNA levels. Our results demonstrate that stimulation of group III mGluRs in the striatum, GP or SNr exerts antiparkinsonian-like effects in rats. The anticataleptic effect of intrastriatally injected ACPT-1 seems to correlate with diminished striatal PENK mRNA expression. However, since the anticataleptic effect produced by intrapallidal and intranigral injection of ACPT-1 is not related to a simultaneous decrease in striatal PENK mRNA levels, it is likely that a decrease in enkephalin biosynthesis is not a necessary condition to obtain an antiparkinsonian effect.

Selective toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a parkinsonism inducing compound, is well known to be related to an uptake of its active metabolite MPP+ into dopaminergic neurons by dopamine transporter (DAT). The aim of the present study was to examine whether paraquat, a commonly used herbicide, which is an 1-methyl-4-phenyl-pyridinium ion (MPP+) analogue, affects DAT in vivo in rats. Paraquat administered at a dose of 10 mg/kg ip decreased the binding of [3H]GBR 12,935 to DAT measured by quantitative autoradiography in the dorsal and ventral caudate-putamen, but not in the substantia nigra pars compacta. Moreover, this compound increased the level of 3-methoxytyramine (3-MT) and 3-MT/dopamine ratio in the anterior and posterior caudate-putamen measured by HPLC with electrochemical detection. No other alterations in the levels of dopamine and its metabolites were found in the caudate-putamen and substantia nigra. The present study seems to suggest that systemic paraquat administration affects striatal DAT and dopamine metabolism in the nigrostriatal neurons in rats which may be crucial for its neurotoxic effects on dopaminergic neurons.