Neuroprotective and Immunomodulatory Effects of Probiotics in a Rat Model of Parkinson's Disease.

It is now well recognized that a bidirectional relationship between gut microbiota and the brain, referred to as the gut-brain axis, plays a prominent role in maintaining homeostasis and that a disruption in this axis can result in neuroinflammatory response and neurological disorders such as Parkinson's disease (PD). The protective action of probiotics such as Bifidobacterium animalis ssp. lactis Bb12 and Lactobacillus rhamnosus GG in various animal models of PD has been reported. Therefore, in this study, we used an inflammatory model of PD to assess the effects of a combination of these two probiotics (Microbiot®) on motor behavior as well as on the response of microglia, including microglia morphology, to gain a better understanding of their mechanism of action. Microbiot® (300 µL) was administered orally once daily for 15 days in a lipopolysaccharide-induced PD model using male Wistar rats. Although LPS-induced motor asymmetry in cylinder test was not affected by Microbiot®, impairment of motor coordination in the narrow-beam test was significantly reduced by this probiotic. Moreover, Microbiot® treatment reduced microglial activation suggesting an anti-inflammatory effect. While further mechanistic investigation of Microbiot® in neurodegenerative diseases is warranted, our results support the potential utility of probiotics in PD.

Depression scores correlate with glycosylated hemoglobin a levels in type 2 diabetics from a mayan community / Los puntajes de depresión se correlacionan con los niveles de hemoglobina glucosilada en diabéticos tipo 2 de una comunidad maya

Abstract Objective: To assess whether depression and quality of life scores correlate with glycosylated hemoglobin A (HbA1c) levels in type 2 diabetes mellitus (T2DM) patients of predominant Mayan ethnicity, from a rural community in the state of Yucatán, Mexico. Materials and methods: Instruments: for depression, CES-D (cutoff ≥ 16); for quality of life, D-39; criterion for poor glycemic control, HbA1c ≥ 8%. Results: Depression was detected in 36.3% of 33 diabetic subjects (10 men, 23 women), and their HbA1c levels (mean ± SD) were higher (10.7 ± 2.5%) than for those without depression (8.6 ± 2.2%, p = 0.015; unpaired Student's t-test, two-tailed). Depression occurred in 33.3% of diabetics with HbA1c ≥8%, but only in 3.0% of those with HbA1c < 8% (p = 0.027, Fisher's exact test). HbA1c levels positively correlated with CES-D scores (r2 =0.135; p = 0.035; Pearson). D-39 "Anxiety-Worry" (AW) dimension scores were higher in diabetics with depression (43.3 ± 22.2) than in those without depression (17.7 ± 17.8; p = 0.005, Mann-Whitney U-test). A positive correlation was found between CES-D and AW scores (r2 = 0.304; p = 0.001; Pearson). Conclusions: Considering that depression and anxiety have been related to poor self-care for achieving a good glycemic control, we propose the concomitant use of CES-D and D-39, which are validated and easy-to-apply instruments, as screening tests to detect depression and anxiety in T2DM patients residing in rural communities. Therefore, if patients test positive on one or both instruments, they can be referred to a psychiatrist to confirm the diagnosis and provide appropriate therapy. This would help to promote adherence to diabetes control measures and improve their quality of life.

Resumen Objetivo: Evaluar si las puntuaciones de depresión y calidad de vida se correlacionan con los niveles de hemoglobina glucosilada (HbA1c) en pacientes con diabetes mellitus tipo 2 (DM2) de etnia maya predominante, de una comunidad rural en el estado de Yucatán, México. Materiales y Métodos: Instrumentos: para depresión, CES-D (punto de corte ≥ 16); para calidad de vida, D-39; criterio de mal control glucémico, HbA1c ≥ 8%. Resultados: Se detectó depresión en el 36.3% de 33 sujetos diabéticos (10 hombres, 23 mujeres), y sus niveles de HbA1c (media ± DE) fueron más altos (10.7 ± 2.5%) que para los que no tenían depresión (8.6 ± 2.2%, p = 0.015; prueba t de Student, no pareada). La depresión se presentó en el 33.3% de los diabéticos con HbA1c ≥ 8%, pero solo en el 3.0% de aquellos con HbA1c <8% (p = 0.027, prueba exacta de Fisher). Los niveles de HbA1c se correlacionaron positivamente con las puntuaciones CES-D (r2 = 0.135; p = 0.035; Pearson). Los puntajes de la dimensión "Ansiedad-Preocupación" (AW) del D-39 fueron más altos en diabéticos con depresión (43.3 ± 22.2) que en aquellos sin depresión (17.7 ± 17.8; p = 0.005, prueba U de Mann-Whitney). Se encontró una correlación positiva entre las puntuaciones CES-D y AW (r2 = 0.304; p = 0.001; Pearson). Conclusiones: Considerando que la depresión y la ansiedad han sido asociadas a autocuidados inadecuados para alcanzar un buen control glicémico, proponemos el uso concomitante de CES-D y D-39, que son instrumentos validados y de fácil aplicación, como pruebas de cribado para detectar depresión y ansiedad en pacientes con DM2 residentes en comunidades rurales. Por lo tanto, si los pacientes dan positivo en uno o ambos instrumentos, se les puede derivar a un psiquiatra para confirmar el diagnóstico y proporcionar la terapia adecuada. Esto ayudaría a promover el cumplimiento de las medidas de control de la diabetes y a mejorar su calidad de vida.

Chronic Treatment with the Probiotics Lacticaseibacillus rhamnosus GG and Bifidobacterium lactis BB12 Attenuates Motor Impairment, Striatal Microglial Activation, and Dopaminergic Loss in Rats with 6-Hydroxydopamine-induced Hemiparkinsonism.

Gut dysbiosis is considered a risk factor for Parkinson's disease (PD), and chronic treatment with probiotics could prevent it. Here we report the assessment of a probiotic mixture [Lacticaseibacillus rhamnosus GG (LGG), and Bifidobacterium animalis lactis BB-12 (BB-12)] administered to male rats 2 weeks before and 3 weeks after injecting 6-hydroxydopamine (6-OHDA) into the right striatum, a model that mimics the early stages of PD. Before and after lesion, animals were subjected to behavioral tests: narrow beam, cylinder test, and apomorphine (APO)-induced rotations. Dopaminergic (DA) denervation and microglia recruitment were assessed with tyrosine hydroxylase (TH+) and ionized calcium-binding protein-1 adapter (Iba1+) immunostaining, respectively. Post 6-OHDA injury, rats treated with sunflower oil (probiotics vehicle) developed significant decrease in crossing speed and increases in contralateral paw slips (narrow beam), forepaw use asymmetry (cylinder), and APO-induced rotations. In striatum, 6-OHDA eliminated ≈2/3 of TH+ area and caused significant increase of Iba1+ microglia population. Retrograde axonal degeneration suppressed ≈2/5 of TH+ neurons in the substantia nigra pars compacta (SNpc). In hemiparkinsonian rats, probiotics treatment significantly improved the crossing speed, and also reduced paw slips (postlesion days 14 and 21), the loss of TH+ neurons in SNpc, and the loss of TH+ area and of Iba1+ microglia count in striatum, without affecting the proportion of microglia morphological phenotypes. Probiotics treatment did not attenuate forepaw use asymmetry nor APO-induced rotations. These results indicate that the mixture of probiotics LGG and BB-12 protects nigrostriatal DA neurons against 6-OHDA-induced damage, supporting their potential as preventive treatment of PD.

Chronic feeding with 3% dried raw blueberries (V. corymbosum) reduces apomorphine-induced rotations and striatal dopaminergic loss in hemiparkinsonian rats.

Blueberries (BB) are rich in antioxidant polyphenols, and their intake could prevent Parkinson's disease (PD). Here we assessed whether rats chronically fed dried raw BB develop resistance to dopaminergic denervation and motor disorders caused by unilateral intrastriatal injection of 6-hydroxydopamine (6-OHDA), a dopaminergic neurotoxin acting mainly by inducing oxidative stress. Male rats were fed either with LabDiet® alone or supplemented with 3% lyophilized raw BB for 2 weeks before and 3 weeks after injecting 6-OHDA (day 0) or vehicle (mock lesion) into the right striatum. The cylinder test was performed on days -14, -7, -1, +7, +14, and +21; the percentage of ipsilateral forepaw (IF) use asymmetry was determined by counting the wall contacts made with either forepaw or with both. Apomorphine (0.25 mg/kg, s.c.)-induced rotation was performed on days -1, +7, +14, and +21. Full contralateral rotations were counted in 3-min periods, every 15 min, up to 90 min. Striatal slices were immunostained for tyrosine hydroxylase (TH) and the ionized calcium-binding protein-1 adapter (Iba1) [immunoreactive area or microglia count in right striatum expressed as % of the left striatum]. Antioxidants in BB methanolic extracts neutralized the free radical 2,2-diphenyl-1-picrylhydrazyl in a concentration-dependent manner. Anthocyanins have been reported as the most abundant polyphenols in BB. Using the pH differential method, the total anthocyanin content (malvidin-3-glucoside equivalents) in raw BB averaged 21.04 mg/g dry weight. The range of anthocyanin intake by rats throughout the study varied from 37.7 to 72.2 mg/kg body weight. The time and food type factors, as well as their interaction were significant according to two-way RM-ANOVA in both the apomorphine-induced rotations and the cylinder test. Compared with LabDiet® alone, chronic supplementation with 3% dried raw BB decreased apomorphine-induced rotations on days +14 and +21 (p < 0.001) and produced a 46% reduction in total rotations post-surgery (p < 0.05), but only caused a partial, non-significant, decrease of IF asymmetry. BB supplementation reduced TH loss in the striatum (p < 0.05) but did not attenuate the increase of Iba1+ microglia. The consumption of 3% dried raw blueberries attenuates dopaminergic denervation and partially reverses motor disorders in the 6-OHDA-induced PD model in rats. The phytochemicals of raw blueberries that contribute to the observed neuroprotective effect are yet to be identified.

Synergistic antidepressant-like effect of capsaicin and citalopram reduces the side effects of citalopram on anxiety and working memory in rats.

RATIONALE: We have previously shown that in rats, capsaicin (Cap) has antidepressant-like properties when assessed using the forced swimming test (FST) and that a sub-threshold dose of amitriptyline potentiates the effects of Cap. However, synergistic antidepressant-like effects of the joint administration of Cap and the selective serotonin reuptake inhibitor citalopram (Cit) have not been reported. OBJECTIVES: To assess whether combined administration of Cap and Cit has synergistic effects in the FST and to determine whether this combination prevents the side effects of Cit. METHODS: Cap, Cit, and the co-administration of both substances were evaluated in a modified version of the FST (30-cm water depth) conducted in rats, as well as in the open field test (OFT), elevated plus maze (EPM), and Morris water maze (MWM). RESULTS: In line with previous studies, independent administration of Cap and Cit displayed antidepressant-like properties in the FST, while the combined injection had synergistic effects. In the OFT, neither treatment caused significant increments in locomotion. In the EPM, the time spent in the closed arms was lower in groups administered either only Cap or a combination of Cap and Cit than in groups treated with Cit alone. In the MWM, both Cap and the joint treatment (Cap and Cit) improved the working memory of rats in comparison with animals treated only with Cit. CONCLUSION: Combined administration of Cap and Cit produces a synergistic antidepressant-like effect in the FST and reduces the detrimental effects of Cit on anxiety and working memory.

Neurotensin-polyplex-mediated brain-derived neurotrophic factor gene delivery into nigral dopamine neurons prevents nigrostriatal degeneration in a rat model of early Parkinson's disease.

BACKGROUND: The neurotrophin Brain-Derived Neurotrophic Factor (BDNF) influences nigral dopaminergic neurons via autocrine and paracrine mechanisms. The reduction of BDNF expression in Parkinson's disease substantia nigra (SN) might contribute to the death of dopaminergic neurons because inhibiting BDNF expression in the SN causes parkinsonism in the rat. This study aimed to demonstrate that increasing BDNF expression in dopaminergic neurons of rats with one week of 6-hydroxydopamine lesion recovers from parkinsonism. The plasmids phDAT-BDNF-flag and phDAT-EGFP, coding for enhanced green fluorescent protein, were transfected using neurotensin (NTS)-polyplex, which enables delivery of genes into the dopaminergic neurons via neurotensin-receptor type 1 (NTSR1) internalization. RESULTS: Two weeks after transfections, RT-PCR and immunofluorescence techniques showed that the residual dopaminergic neurons retain NTSR1 expression and susceptibility to be transfected by the NTS-polyplex. phDAT-BDNF-flag transfection did not increase dopaminergic neurons, but caused 7-fold increase in dopamine fibers within the SN and 5-fold increase in innervation and dopamine levels in the striatum. These neurotrophic effects were accompanied by a significant improvement in motor behavior. CONCLUSIONS: NTS-polyplex-mediated BDNF overexpression in dopaminergic neurons has proven to be effective to remit hemiparkinsonism in the rat. This BDNF gene therapy might be helpful in the early stage of Parkinson's disease.

ß-Adrenergic blockade protects BALB/c mice against infection with a small inoculum of Leishmania mexicana mexicana (LV4).

In order to test the influence of the sympathetic nervous system on Leishmania mexicana infection, groups of female BALB/c mice were treated (i.p.) with the non-selective ß-adrenergic receptor (ß-AR) antagonist (S)-propranolol (5mg/kg thrice a day), the ß2-AR agonist clenbuterol (1mg/kg once a day) or the α2-AR antagonist yohimbine (2mg/kg twice a day) during 5days. During the second day of treatments, mice were inoculated in the footpad with 1×10(6) or 1×10(3) metacyclic promastigotes of L. mexicana mexicana (LV4). The lesion size was measured weekly, and parasite burden on week 12. In mice treated with (S)-propranolol, the percentage of splenic T lymphocytes producing IFN-γ after antigen challenge was determined by flow cytometry. In mice infected with 1×10(6) parasites, only (S)-propranolol caused a reduction of footpad swelling (p<0.05, weeks 11-12), without effects on parasite burden, or in the percentage of IFN-γ-immunopositive CD4(+) or CD8(+) T lymphocytes. In mice infected with 1×10(3) parasites, the effects of treatments vs. control group were as follows: (a) inhibition of footpad swelling by (S)-propranolol (p<0.01, weeks 3-12), clenbuterol (p<0.05, weeks 7-10), and yohimbine (p<0.01, week 7); (b) a decrease of the parasite burden by (S)-propranolol (p<0.01) and yohimbine (p<0.05); (c) in control mice the percentage of CD4(+) T-cells producing IFN-γ was 6.2±0.5%, while in those treated with (S)-propranolol it increased to 8.7±0.6% (p<0.01); (d) in control mice the percentage of CD8(+) T-cells producing IFN-γ was 3.1±0.4%, while in those treated with (S)-propranolol it increased to 10.4±0.2% (p<0.01). These results indicate that the blockade of ß-ARs during infection of BALB/c mice with an inoculum of L. mexicana mexicana similar to that delivered by the bite of a sand fly produces a Th1 bias in the immune response, favoring an increment of T lymphocytes secreting IFN-γ, which correlated with a reduced parasite burden (p<0.05, Spearman's test). We suggest that ß-AR antagonists could be of therapeutic value, either as treatment or as adjuvant of vaccines for L. mexicana.

The potency and efficacy of anticholinergics to inhibit haloperidol-induced catalepsy in rats correlates with their rank order of affinities for the muscarinic receptor subtypes.

Extrapyramidal syndromes (EPS) caused by antipsychotic therapy are currently treated with anticholinergics that lack selectivity for the five muscarinic receptor subtypes. Since these receptors are heterogeneously expressed among the different classes of striatal neurons and their afferents, it can be expected that their simultaneous blockade will cause distinct, sometimes opposed, effects within the striatal circuitry. In order to test the hypothesis that the differential blockade of the muscarinic receptor subtypes would influence their potency and efficacy to prevent EPS, here we tested four anticholinergics with varying order of affinities for the muscarinic receptor subtypes, and compared their dose-response curves to inhibit haloperidol-induced catalepsy in male rats. Drugs were applied into the lateral ventricle 15 min before haloperidol (2 mg/kg, s.c.). Catalepsy was measured in the bar test at 15 min intervals during 5 h. The preferential M1/M4 antagonist pirenzepine (3, 10, 30, 100, and 300 nmol) caused a dose-dependent inhibition of catalepsy intensity: ED50 = 5.6 nmol [95% CI, 3.9-8.1], and latency: ED50 = 5.6 nmol [95% CI, 3.7-8.6]. Pirenzepine had the steepest dose-response curve, producing maximal inhibition (84 ± 5%) at the dose of 10 nmol, while its effect tended to reverse at higher doses (62 ± 11%). The purported M1/M3 antagonist 4-DAMP (30, 100, and 300 nmol) also caused a dose-dependent inhibition of catalepsy intensity: ED50 = 29.5 nmol [95% CI, 7.0 to 123.0], and latency: ED50 = 28.5 nmol [95% CI, 2.2 to 362.0]. However, the curve for 4-DAMP had a less pronounced slope, reaching its maximal effect (63 ± 14%) at the dose of 300 nmol. The M2/M4 antagonist AF-DX 116 (10, 30, and 300 nmol) only caused a partial inhibition of catalepsy (30 ± 11%) at the dose of 30 nmol, but this changed to a non-significant increment (15 ± 10%) at the dose of 100 nmol. The alleged M4 antagonist tropicamide (30, 100, 300, and 600 nmol) produced a partial inhibition of catalepsy (36 ± 12%) at the dose of 300 nmol, but lacked effect at higher or lower doses. Concurrent treatment with pirenzepine (10 nmol) and tropicamide (300 nmol) produced an effect similar to that of tropicamide alone. The greater potency and efficacy of pirenzepine for catalepsy inhibition could be due to its higher affinity for M1 receptors and, to a lesser extent, for M4 receptors. It is suggested that selective M1 antagonists would be more effective than M2, M3 or M4 antagonists to prevent EPS caused by antipsychotic drugs.

Caffeine has greater potency and efficacy than theophylline to reverse the motor impairment caused by chronic but not acute interruption of striatal dopaminergic transmission in rats.

In order to assess whether caffeine and theophylline have the same potency and efficacy to reverse the impairment of motor function caused by acute or chronic interruption of striatal dopamine transmission, a comparison of their dose-response relationship was made in the acute model of haloperidol-induced catalepsy, and the chronic model of unilateral lesion of the dopamine nigrostriatal pathway with 6-hydroxydopamine. At equimolar doses, both drugs reduced catalepsy intensity and increased its onset latency in a dose-dependent fashion, showing comparable potencies and attaining the maximal effect at similar doses. Catalepsy intensity: caffeine ED50 = 24.1 µmol/kg [95% CI, 18.4-31.5]; theophylline ED50 = 22.0 µmol/kg [95% CI, 17.0-28.4]. Catalepsy latency: caffeine ED50 = 27.0 µmol/kg [95% CI, 21.1-34.6]; theophylline ED50 = 28.8 µmol/kg [95% CI, 22.5-36.7]. In one group of hemiparkinsonian rats (n = 5), caffeine caused a dose-dependent recovery of the contralateral forepaw stepping: ED50 = 2.4 µmol/kg/day [95% CI, 1.9-3.1]), reaching its maximum at the dose of 5.15 µmol/kg/day. When the treatment of these same rats was switched to 5.15 µmol/kg/day of theophylline, the stepping recovery was only 51 ± 12% of that induced by caffeine. Assessing the dose-response relationship of theophylline in another group of hemiparkinsonian rats (n = 7) revealed that it caused stepping recovery in an all-or-none fashion. Thus, the three lower doses had no effect, but at the dose of 5.15 µmol/kg/day theophylline suddenly increased the stepping to 56 ± 5% of the maximal effect observed when the treatment of these same rats was switched to an equimolar dose of caffeine. Increasing the dose of theophylline up to 15.45 µmol/kg/day caused no further stepping improvement since it was only 41 ± 6% of the maximal effect produced by caffeine at the dose of 5.15 µmol/kg/day. Given that theophylline showed less potency and efficacy than caffeine to reverse the motor impairment caused by chronic, but not acute, interruption of striatal dopaminergic transmission in rats, it is suggested that caffeine would provide more benefits than theophylline to improve the motor function in patients with Parkinson's disease.

A system for automatic recording and analysis of motor activity in rats.

We describe the design and evaluation of an electronic system for the automatic recording of motor activity in rats. The device continually locates the position of a rat inside a transparent acrylic cube (50 cm/side) with infrared sensors arranged on its walls so as to correspond to the x-, y-, and z-axes. The system is governed by two microcontrollers. The raw data are saved in a text file within a secure digital memory card, and offline analyses are performed with a library of programs that automatically compute several parameters based on the sequence of coordinates and the time of occurrence of each movement. Four analyses can be made at specified time intervals: traveled distance (cm), movement speed (cm/s), time spent in vertical exploration (s), and thigmotaxis (%). In addition, three analyses are made for the total duration of the experiment: time spent at each x-y coordinate pair (min), time spent on vertical exploration at each x-y coordinate pair (s), and frequency distribution of vertical exploration episodes of distinct durations. User profiles of frequently analyzed parameters may be created and saved for future experimental analyses, thus obtaining a full set of analyses for a group of rats in a short time. The performance of the developed system was assessed by recording the spontaneous motor activity of six rats, while their behaviors were simultaneously videotaped for manual analysis by two trained observers. A high and significant correlation was found between the values measured by the electronic system and by the observers.

Quinolinic acid lesions of the pedunculopontine nucleus impair sleep architecture, but not locomotion, exploration, emotionality or working memory in the rat.

Anatomical and functional studies have shown that the NADPH-diaphorase-positive cholinergic neurons of the pedunculopontine nucleus (PPN) send projections to several areas in the brain. The purpose of this work was to investigate whether bilateral lesions with quinolinic acid, a neurotoxin with greater selectivity for NADPH-diaphorase-positive neurons, aimed at the compact portion of the PPN would affect the performance of adaptive behaviors, such as sleep, locomotion, and spontaneous alternation. Lesioned animals were divided in a low lesion group (LL, <50% neuron loss) and a high lesion group (HL, ≥50% neuron loss). The LL animals did not show any significant changes in sleep patterns, as compared to controls. In contrast, the HL group showed a significant increase in the number of REM sleep periods, and a reduction of REM sleep average duration, but did not differ in the total time spent in REM sleep. HL animals also showed an increase in the number of SWS periods, though wakefulness parameters did not show significant alterations. The duration and number of both REM and SWS sleep episodes were significantly correlated with the number of NADPH-diaphorase-positive neurons in the PPN. The short-term habituation pattern of locomotion, the vertical exploratory activity, as well as the thigmotaxis (an index of emotionality), displayed by LL and HL rats in a novel environment were similar to those of control animals. Likewise, there were no significant differences in spontaneous alternation among the groups. Our results indicate that quinolinic acid lesions of NADPH-diaphorase-positive cholinergic neurons localized in the posterior region of the PPN disrupt normal sleep structure, while motor activity and spontaneous alternation remain unaffected.

Long-lasting resistance to haloperidol-induced catalepsy in male rats chronically treated with caffeine.

Chronic caffeine consumption has been inversely associated with the risk of developing Parkinson's disease. Here we assessed whether chronic caffeine treatment increases the resistance of male Wistar rats to haloperidol (1mg/kg, s.c.)-induced catalepsy, measured in the bar test at 15 min intervals during 3h. Caffeine (5mg/kg/day) was delivered for 6 months via drinking water. Control rats received only tap water. Treatments began when animals were 3-4 months old. In order to unveil long-lasting catalepsy refractoriness not attributable to the presence of caffeine in the brains of rats, they were evaluated from day 18 to day 27 after caffeine withdrawal, a time that is far in excess for the full excretion of a caffeine dose in this species. The average cataleptic immobility measured in caffeine-treated rats (n=23) was 1148+/-140 s, a value 34+/-8% lower than that recorded in control animals (n=20), whose mean immobility was 1736+/-137 s (P=0.0026, t-test). The percentage of catalepsy reduction measured in caffeine-treated rats evaluated on days 18-20 after caffeine discontinuation (-32+/-13%, n=12, P<0.05) was comparable to the catalepsy decrease recorded in those animals tested on days 21-27 (-36+/-10%, n=11, P<0.02), a finding compatible with the notion that the effect was indeed mediated by enduring changes of brain functioning and not by the physical presence of caffeine or its metabolites. Caffeine-treated rats also had higher catalepsy latency scores compared with control rats (P<0.01, U-test). The present findings show that chronic consumption of caffeine produces perdurable resistance to catalepsy induced by dopamine receptor blockade, possibly through enhancement of dopamine transmission, giving further support to the epidemiological results indicating that prolonged caffeine consumption affords neuroprotection against Parkinson's disease.

A digital programmable telemetric system for recording extracellular action potentials.

This article describes the design and preliminary evaluation of a small-sized and low energy consumption wearable wireless telemetry system for the recording of extracellular neuronal activity, with the possibility of selecting one of four channels. The system comprises four radio frequency (RF) transceivers, three microcontrollers, and a digital amplifier and filter. This constitutes an innovative distributed processing approach. Gain, cutoff frequencies, and channel selection are remotely adjusted. Digital data transmission is used for both the bioelectrical signals and the control commands. This feature offers superior immunity to external RF interference. Real-time viewing of the acquired data allows the researcher to select only relevant data for storage. Simultaneous recordings of neuronal activity from the striatum of a freely moving rat, both with the wireless device and with a wired data acquisition system, are shown.

Differential effects of caffeine on the antidepressant-like effect of amitriptyline in female rat subpopulations with low and high immobility in the forced swimming test.

The interaction of caffeine (1 mg/kg) and amitriptyline (15 mg/kg) on the immobility time (IT) during Porsolt's forced swimming test (FST) was investigated in female Wistar rats. Akaike's Information Criterion indicated that the ITs recorded from 142 rats during the first day of the FST followed a bimodal distribution. Hence, the median (125.5 s) was used to classify the animals in subpopulations with low (<125.5 s, LI-rats) or high (>125.5 s, HI-rats) immobility. The paired t-test was used to compare the change of ITs between the first and second swimming sessions. Vehicle-treated animals had a significant increase of ITs during the second day of the test, either in LI-rats (77+/-12 s vs. 196+/-8 s, P<0.0001, n=6) or HI-rats (150+/-8 s vs. 201+/-10 s, P<0.02, n=6). In LI-rats amitriptyline only prevented the increase of ITs during the second session (74+/-27 s vs. 97+/-42 s, n=12), whereas in HI-rats the antidepressant produced a significant decrease of ITs during the second session (161+/-22 s vs. 118+/-32 s, n=7, P<0.02). While caffeine alone prevented the increase of ITs in both groups, the methylxanthine abolished the effect of amitriptyline in HI-rats (165+/-23 s vs. 165+/-46 s, n=9), leaving the antidepressant action unaffected in LI-rats (87+/-23 s vs. 96+/-58 s, n=9). These results suggest that the anti-immobility effect of amitriptyline in HI-rats is mediated in part by endogenous adenosine.

Sustained improvement of motor function in hemiparkinsonian rats chronically treated with low doses of caffeine or trihexyphenidyl.

The effects of chronic oral treatment with low doses of caffeine (1-3 mg/kg) and trihexyphenidyl (0.1-0.2 mg/kg) were tested on hemiparkinsonian rats, which received the following treatments in a counterbalanced order: vehicle, caffeine, trihexyphenidyl, and caffeine plus trihexyphenidyl. Three preclinical models were used: the stepping test, the cylinder test, and the staircase test. Compared to pre-lesion values, the forepaw contralateral to the dopamine-denervated side showed impaired stepping, fewer wall contacts in the cylinder test, and fewer pellets retrieved in the staircase test. In the stepping test both doses of caffeine produced a complete recovery of motor function (100%), whereas the effect of trihexyphenidyl was less intense (77-80%). In this same test the maximal effect of drugs did not develop tolerance during 2-3 weeks, and was completely reversible after drug cessation. In the cylinder test only the wall contacts performed simultaneously with both forepaws were significantly increased by caffeine (3 mg/kg) and trihexyphenidyl (0.2 mg/kg), and this effect was also reversible. In the staircase test none of the treatments improved food pellet retrieval with the contralateral forepaw. Altogether, these results show that chronic treatment with caffeine, at doses similar to daily human consumption, produces a sustained improvement in the use of the contralateral forelimb in unilaterally 6-hydroxydopamine denervated rats, without the development of tolerance. Although the combined administration of caffeine plus trihexyphenidyl showed no synergism in these models, the results suggest that low doses of caffeine (1-3 mg/kg/day) could be of therapeutic value for the reversal of motor symptoms in parkinsonian patients.

Treatment with subthreshold doses of caffeine plus trihexyphenidyl fully restores locomotion and exploratory activity in reserpinized rats.

Trihexyphenidyl (THP) is a drug commonly used to reduce parkinsonian symptoms. An important side effect of this agent is memory impairment. Since caffeine enhances the potency of THP to inhibit haloperidol-induced catalepsy, caffeine may be used as an adjuvant of lower doses of THP, in order to improve its antiparkinsonian effects without causing memory disruption. To further assess the synergism between caffeine and THP, both drugs were tested in reserpinized rats, another preclinical model of Parkinson's disease. Four groups of rats (n = 7) were treated with reserpine (5 mg/kg, i.p.). A control group (n = 7) was treated only with the vehicle for reserpine (dimethylsulphoxide). The spontaneous locomotor behavior was tested 24 h later in a box with infrared sensors, 30 min after receiving one of the following treatments: distilled water (1 ml/kg), caffeine (1 mg/kg), THP (0.1 mg/kg) or caffeine plus THP. The levels of horizontal locomotion (14 +/- 5%) and vertical exploration (15 +/- 10%) were significantly lower in reserpinized rats treated with distilled water, compared with the mean activity values (100%) recorded in animals pretreated only with the vehicle for reserpine. The reserpine-induced hypokinesia was neither reversed by caffeine alone nor by THP alone. However, the combination of caffeine plus THP restored locomotion (141 +/- 19%) and vertical exploration (82 +/- 17%) to levels not significantly different to those of non-reserpinized rats. Moreover, the time-course of locomotion and exploration displayed the characteristic habituation over time, in which short-term memory processes are involved. Also, the thigmotaxis index indicated that the combined treatment did not induce anxiety-like behavior. Hence, these results support the proposal that low, subthreshold doses of caffeine plus THP have the potential to alleviate the motor disabilities in parkinsonian patients, with a low risk of causing anxiety or memory impairment.

Caffeine and muscarinic antagonists act in synergy to inhibit haloperidol-induced catalepsy.

The possible synergism between caffeine and muscarinic antagonists to inhibit haloperidol-induced catalepsy was investigated with the bar test in rats. Pretreatment with low doses of caffeine (1-3 mg/kg), a non-selective adenosine antagonist, dose dependently reduced the intensity and increased the onset latency of catalepsy induced by haloperidol (0.5-2 mg/kg). Similar effects were produced by the muscarinic antagonists atropine (4.1 mg/kg), and trihexyphenidyl (THP, 0.01-3 mg/kg). THP inhibited catalepsy intensity with an ED(50) of 0.38 mg/kg, and increased its onset latency with an ED(50) of 0.52 mg/kg. The anticataleptic effect of anticholinergics was potentiated when a low dose of caffeine (1 mg/kg) was applied simultaneously. In the presence of caffeine, THP inhibited catalepsy intensity with an ED(50) of 0.19 mg/kg, and prolonged the latency with an ED(50) of 0.30 mg/kg. The synergism was more evident when THP was administered at subthreshold doses that were unable to modify haloperidol-induced catalepsy when applied alone, but produced a clear inhibition of catalepsy when injected with caffeine. To assess whether repeated administration of caffeine could induce tolerance to the synergism with THP, a group of rats was pretreated with three daily doses of caffeine (1 mg/kg) for seven days, and the catalepsy test was performed on the eighth day. In these animals, caffeine was still able to enhance the anticataleptic actions of THP, suggesting that repeated administration of 1 mg/kg caffeine does not induce tolerance to the synergism with anticholinergics. These results indicate that low doses of caffeine enhance the anticataleptic actions of muscarinic antagonists, and leave open the possibility of using caffeine as adjunctive therapy to reduce the doses and the adverse effects of anticholinergics in Parkinson's disease.

Selective A2A, but not A1 adenosine antagonists enhance the anticataleptic action of trihexyphenidyl in rats.

In rats made cataleptic with haloperidol (5.32 micromol/kg), the bar test was used to assess the possible synergism between the muscarinic antagonist trihexyphenidyl (THP) and selective adenosine A(1) and A(2A) receptor antagonists. Neither catalepsy intensity nor latency were affected by a subthreshold dose of THP (0.33 micromol/kg). The selective adenosine A(1) antagonist 8-cyclopentyl-1,3-dipropyl-xanthine (DPCPX) (5.15 micromol/kg) caused a small, but significant reduction of catalepsy intensity that remained unchanged when combined with THP. DPCPX had no effect on catalepsy latency, either alone or combined with THP. In contrast, an equimolar dose of the selective adenosine A(2A) antagonist 4-(2-[7-amino-2-(2-furyl)1,2,4-triazolo[2,3-a]-[1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) (5.15 micromol/kg) produced a significant reduction of catalepsy intensity and increased catalepsy latency. Both effects were potentiated when ZM 241385 was combined with THP. The synergism was more evident when rats were pretreated with a subthreshold dose of ZM 241385 (1.55 micromol/kg) that was unable to modify catalepsy parameters when applied alone, but produced a significant reduction in catalepsy intensity and an increase in catalepsy latency when administered with THP. Catalepsy was unaffected by a combination of equimolar, subthreshold doses of DPCPX (1.55 micromol/kg) and ZM 241385 (1.55 micromol/kg). These findings indicate that the anticataleptic effect of anticholinergics is enhanced only by the selective blockade of adenosine A(2A) receptors.