V. S. Kudrin

Modeling of presymptomatic and symptomatic stages of parkinsonism in mice

A degradation of the nigrostriatal dopaminergic (DA-ergic) system is the key component of pathogenesis of Parkinsons disease (PD). Initial clinical symptoms appear 20-30 years after the onset of neurodegeneration, at a 70% DA depletion in the striatum and a 50% loss of nigral DA-ergic neurons. Low efficacy of the therapy might be improved if preclinical diagnostics and preventive therapy are developed. The development of appropriate experimental models should precede clinical trials. This multidisciplinary study first managed to model in mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) all together the following stages of parkinsonism: (a) the early presymptomatic stage manifested by a subthreshold degeneration of axons and DA depletion in the striatum without loss of nigral cell bodies; (b) the advanced presymptomatic stage manifested by a subthreshold degeneration of striatal axons and DA depletion and by a subthreshold loss of nigral cell bodies; (c) the advanced presymptomatic stage characterized by threshold depletion of striatal DA and a loss of DA-ergic axons and nigral cell bodies resulting in motor dysfunction. The degeneration of axons proceeds and prevails that of cell bodies suggesting higher sensitivity to MPTP of the former. Compensatory processes were developed in parallel to neurodegeneration that was manifested by the increase of the DA content in individual nigral cell bodies and DA turnover in the striatum. The developed models might be exploited for: (a) an examination of pathogenetic mechanisms not only in the nigrostriatal system but also in other brain regions and in the periphery; (b) a study of the compensatory mechanisms under DA deficiency; (c) a search of precursors of motor disorders and peripheral biomarkers in presymptomatic parkinsonism; (d) the development of preventive therapy aiming to slow down the neurodegeneration and strengthen compensatory processes. Thus, the models of the early and advanced presymptomaic stages and of the early symptomatic stage of parkinsonism were developed in mice with MPTP.

Dopamine synthesis by non-dopaminergic neurons expressing individual complementary enzymes of the dopamine synthetic pathway in the arcuate nucleus of fetal rats

This study was aimed to test our hypothesis about dopamine (DA) synthesis by non-DAergic neurons expressing individual complementary enzymes of the DA synthetic pathway in cooperation, i.e. L-dihydroxyphenylalanine (L-DOPA) synthesized in tyrosine hydroxylase (TH)-expressing neurons is transported to aromatic L-amino acid decarboxylase (AADC)-expressing neurons for conversion to DA. The mediobasal hypothalamus of rats at the 21st embryonic day was used as an experimental model because it contains mainly monoenzymatic TH neurons and AADC neurons (>99%) whereas the fraction of bienzymatic (DAergic) neurons does not exceed 1%. The fetal substantia nigra containing DAergic neurons served as a control. DA and L-DOPA were measured by high performance liquid chromatography in: (1) cell extracts of the cell suspension prepared ex tempora; (2) cell extracts and incubation medium after the static incubation of the cell suspension with, or without exogenous L-tyrosine; (3) effluents of the incubation medium during perifusion of the cell suspension in the presence, or the absence of L-tyrosine. Total amounts of DA and L-DOPA in the incubation medium and cell extracts after the static incubation were considered as the indexes of the rates of their syntheses. L-Tyrosine administration caused the increased L-DOPA synthesis in the mediobasal hypothalamus and substantia nigra. Moreover, L-tyrosine provoked an increase of DA synthesis in the substantia nigra and its decrease in the mediobasal hypothalamus. This contradiction is most probably explained by the L-tyrosine-induced competitive inhibition of the L-DOPA transport to the monoenzymatic AADC-neurons after its release from the monoenzymatic TH neurons. Thus, this study provides convincing evidence of cooperative DA synthesis by non-DAergic neurons expressing TH or AADC in fetal rats at the end of the intrauterine development.

Presynaptic dopamine and serotonin receptors modulating tyrosine hydroxylase activity in synaptosomes of the nucleus accumbens of rats

Tyrosine hydroxylase (TH) activity was determined by measuring the formation of [3H]DOPA from [3,5-3H]tyrosine in the crude synaptosomal fraction of the nucleus accumbens under conditions preventing dopamine reuptake in 30 mM K+-containing medium. TH seems to be allosterically activated under depolarizing conditions: a 4.4 fold decrease of the Km value for tyrosine of the synaptosomal TH was observed. Synaptosomal TH activity was inhibited concentration dependently by dopamine and apomorphine resulting in IC50 values of 0.4 and 0.25 microM, respectively. The maximal inhibitory effects of dopamine as well as apomorphine were about 50% of the controls. The dopamine-induced inhibition was completely antagonized by neuroleptics. The rank order of antagonistic potencies was haloperidol greater than clozapine greater than sulpiride (with increasing EC50); methiothepine was ineffective. Moreover, synaptosomal TH activity was inhibited by serotonin in a concentration-dependent manner (IC50 = 0.8 microM). This inhibition was completely antagonized by methiothepine while, on the other hand, haloperidol was ineffective. The experimental system demonstrated here appears to be suitable for estimating the presynaptic dopamine and serotonin antagonistic potencies of drugs.

Disrupted Serotonergic and Sympathoadrenal Systems in Patients with Chronic Heart Failure May Serve as New Therapeutic Targets and Novel Biomarkers to Assess Severity, Progression and Response to Treatment

Background: It is well established that the serotonergic system (SS) plays important roles in the pathogenesis of cardiovascular diseases. However, the impact of serotonin and its inter-relation with the sympathoadrenal system (SAS) in chronic heart failure (CHF) is poorly understood. Methods: Utilizing high-performance liquid chromatography with electrochemical detection, we determined blood plasma levels of serotonin (5-hydroxy-triptamine, [5-HT]p), 5- hydroxy-indole-acetic acid ([5-HIAA]p), epinephrine ([E]p), norepinephrine ([NE]p), 3,4-dihydroxy-L-phenyl-alanine ([DOPA]p), dopamine ([DA]p) and the platelet concentration of serotonin ([5-HT]pt) in CHF patients with different morphofunctional alterations of myocardium. The morphofunctional alterations included diastolic dysfunction (DD), diastolic dysfunction with left ventricular hypertrophy (DD&LVH), and diastolic and systolic dysfunction (D&SD). Results: All CHF groups showed significant rises of [5-HT]p and [5-HT]pt. DD&LVH and D&SD individuals also had increased [5-HIAA]p. Levels of SAS blood biomarkers were also significantly changed. The correlation between SS and SAS was increased in CHF and corresponded with disease severity. Conclusions: These results clearly demonstrate that in CHF patients significant changes in SS and SAS occur, which are thought to relate to the morphofunctional alterations of myocardium. The observed changes in the levels of these biomarkers may serve as potential surrogates to monitor severity of disease, to evaluate response to drug treatment, and as a rational basis for new therapeutic approaches.

Strain differences in profiles of dopaminergic neurotransmission in the prefrontal cortex of the BALB/C vs. C57Bl/6 mice: consequences of stress and afobazole.

We found that in mice the basal activity of monoamine oxidase B (MAO-B) in the medial prefrontal cortex (mPFC) is lower in BALB/C than in C57Bl/6J mice, whereas activity of MAO-A is similar between strains. BALB/C mice, in comparison to C57Bl/6N mice, have higher basal content of dopamine in the mPFC, in both microdialysates and tissue content. Novelty stress (open field test) elicits a further increase in the microdialysate levels of dopamine in BALB/C, but not in C57Bl/6N mice; a subsequent accumulation of extracellular 3,4-dioxyphenylacetic acid (DOPAC) reaffirms the difference in catabolic capacity of monoaminergic systems between the strains. We demonstrated that in stress-susceptible BALB/C mice the novel anxiolytic afobazole, 5mg/kg, selectively mitigates trait anxiety; however it does not change the behavioral response in stress-resilient C57Bl/6N mice. Afobazole inhibits MAO-A in in vitro; it also lowers the microdialysate DOPAC levels in both strains (which testifies to its MAO-A inhibiting activity in vivo) and slightly suppresses dopamine release when elevated. Therefore, it is likely that the drug may mediate its anxiolytic activity via modulation of volume dopaminergic transmission at level of the mPFC.

Lamotrigine and Carbamazepine Affect Differently the Release of D-[3H]Aspartate from Mouse Cerebral Cortex Slices: Involvement of NO

The effects of lamotrigine and carbamazepine on the release of preloaded D-[3H]aspartate and the involvement of nitric oxide were studied with mouse cerebral cortical slices in a superfusion system. Lamotrigine inhibited the veratridine-evoked release, whereas the K+-stimulated release was attenuated more strongly by carbamazepine than by lamotrigine. These effects were accentuated by the N-methyl-D-aspartate receptor antagonist L-2-amino-5-phosphonovalerate and the nitric oxide synthase inhibitor L-nitroarginine, but diminished by the nitric oxide donor sodium nitroprusside. The results show that in addition to the blockade of voltage-sensitive Na+ (and Ca2+) channels, NO-mediated mechanisms are probably involved in the anticonvulsant actions of carbamazepine and, in particular, those of lamotrigine.

Behavioral alteration in the adult rats prenatally exposed to para-chlorophenylalanine

In the present work, effects of maternal administration of para-chlorophenylalanine (PCPA), a serotonin synthesis inhibitor, on behavior of adult offspring were studied. Pregnant rats were injected intraperitoneally with PCPA (200/100/100/50 mg/kg) either on the gestational days (GD) 8-11 or 14-17, or with vehicle at the same days. Behavioral parameters, in an open field, the Porsolt forced swim test and the Morris water maze test were evaluated at the age of 3-3.5 months in the male and female offspring. The prenatal PCPA increased activity in an open field in the offspring treated on either GD 8-11 or 14-17. The highest levels of the activity were revealed in the male and female offspring treated on GD 14-17. Besides, the PCPA treatment on GD 8-11 or 14-17 facilitated the intersession habituation of activity to repeated exposures to an open field in the male offspring. Both male and female offspring treated on GD 14-17 showed an increased immobility in the Porsolt forced swim test and a significant learning impairment in the Morris water maze. Thus, it has been shown that administration of PCPA to pregnant rats might cause significant changes in the adult offspring behavior. These results provide further evidence that unfavorable influence may have more adverse effects on the behavioral development of rats when exposed during the final trimester of pregnancy than during the second trimester.

Ethanol and Δ-sleep-inducing peptide: Effects on brain monoamines

The brain content of dopamine (DA) and its metabolites [dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)] were the same in rats with different immobilization times in forced swimming test, while the serotonin (5-HT) concentration was higher in high active (HA, immobilization 5 min) animals. Ethanol (2 g/kg, PO) tended to increase the DA level in the striatum and nucleus accumbens in LA rats and decrease the 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) concentration in HA rats. delta-Sleep-inducing peptide (DSIP) injection reduced the level of 5-HT in the medial prefrontal cortex (MFC) in both groups, did not affect the concentration of DA or DOPAC, but increased HVA in the striatum of HA rats. DSIP injected before ethanol administration augmented the ethanol effects on 5-HT in the MFC and attenuated the action of ethanol on 5-HIAA in the nucleus accumbens. A relationship between the different levels of voluntary alcohol consumption and sensitivity to stress among LA and HA rats and the differences in DA and 5-HT concentrations is suggested. The use of LA and HA rats in developing models for testing of stress-shielding compounds is also described.

The effects of irradiation by 12C carbon ions on monoamine exchange in several rat brain structures

Rats were irradiated with carbon ions (12C) in a Nuklotron accelerator. The irradiation dose was 1 Gy, the energy of the ions was 500 MeV/nuclon, and the linear energy transmission (LET) was 10.6 keV/micron. The animals were decapitated 1 day after irradiation. We isolated the prefrontal cortex, nucleus accumbens, hypothalamus, hippocampus, and striatum, where we determined the concentrations of monoamines and their metabolites. Strong changes were observed in three structures, viz., the prefrontal cortex, nucleus accumbens, and hippocampus. However, significant changes were found in the prefrontal cortex and weaker changes were seen in the nucleus accumbens, whereas changes were insignificant in the hippocampus. This reaction may be related to the fact that the animals were examined on the 2nd day after irradiation. It was shown that an increase in the interval between irradiation and examination of animals results in enhancement of the effects of radiation treatment. The experiments revealed the high sensitivity and reactivity of the prefrontal cortex, which we relate to the key role of this structure in vitally critical processes of behavior.

Effects of amphetamine and sydnocarb on dopamine release and free radical generation in rat striatum.

Microdialysis technique was used to compare the effects of four repeated intraperitoneal administrations of two psychostimulant drugs, D-amphetamine and sydnocarb, at the equimolar doses 5 and 23.8 mg/kg, respectively, on the extracellular level of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and hydroxyl radicals (.OH) in the dorsal striatum of freely moving 3-month-old male Wistar rats 250-300 g in weight. D-amphetamine caused immediate increase of DA concentration up to 950% with quick decline towards baseline values thereafter, followed by much less increase after further injections. Sydnocarb elicited moderate elevation in DA level achieving 400% after the fourth injection. D-amphetamine induced deep decrease in DOPAC concentration, while sydnocarb caused its increase after the first and second dosing. Both drugs enhanced generation of .OH, the effect of D-amphetamine was more pronounced. D-Amphetamine induced more intensive stereotyped behavior in rats compare to sydnocarb. It is concluded that the psychostimulant action of sydnocarb is accompanied by facilitation of the central dopaminergic transmission in rat neostriatum and followed by less pronounced neurotoxic effect than that of D-amphetamine.