Developmental light deprivation transiently reduces the expression of vGluT2 and GluN2B in the rat ventral suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the most important circadian clock in mammals. The SCN synchronizes to environmental light via the retinohypothalamic tract (RHT), which is an axon cluster derived from melanopsin-expressing intrinsic photosensitive retinal ganglion cells. Investigations on the development of the nonimage-forming pathway and the RHT are scarce. Previous studies imply that light stimulation during postnatal development is not needed to make the RHT functional at adult stages. Here, we examined the effects of light deprivation (i.e., constant darkness (DD) rearing) during postnatal development on the expression in the ventral SCN of two crucial proteins for the synchronization of circadian rhythms to light: the presynaptic vesicular glutamate transporter type 2 (vGluT2) and the GluN2B subunit of the postsynaptic NMDA receptor. We found that animals submitted to DD conditions exhibited a transitory reduction in the expression of vGluT2 (at P12-19) and of GluN2B (at P7-9) that was compensated at older stages. These findings support the hypothesis that visual stimulation during early ages is not decisive for normal development of the RHT-SCN pathway.

Light stimulation during postnatal development is not determinant for glutamatergic neurotransmission from the retinohypothalamic tract to the suprachiasmatic nucleus in rats.

The hypothalamic suprachiasmatic nucleus (SCN) is the leading circadian pacemaker in mammals, which synchronizes with environmental light through the retinohypothalamic tract (RHT). Although the SCN regulates circadian rhythms before birth, postnatal synaptic changes are needed for the RHT-SCN pathway to achieve total functional development. However, it is unknown whether visual experience affects developmental maturation. Here, we studied the effects of constant darkness (DD) rearing on the physiology (at pre- and postsynaptic levels) of glutamatergic neurotransmission between RHT and SCN during postnatal development in rats. Upon recording spontaneous and evoked excitatory postsynaptic currents (EPSCs) by electrical stimulation of RHT fibers, we found that DD animals at early postnatal ages (P3-19) exhibited different frequencies of spontaneous EPSCs and lower synaptic performance (short-term depression, release sites, and recruitment of RHT fibers) when compared with their normal light/dark (LD) counterparts. At the oldest age evaluated (P30-35), there was a synaptic response strengthening (probability of release, vesicular re-filling rate, and reduced synaptic depression) in DD rats, which functionally equaled (or surmounted) that of LD animals. Control experiments evaluating EPSCs in ventral SCN neurons of LD rats during day and night revealed no significant differences in spontaneous or evoked EPSCs by high-frequency trains in the RHT at any postnatal age. Our results suggest that DD conditions induce a compensatory mechanism in the glutamatergic signaling of the circadian system to increase the chances of synchronization to light at adult ages, and that the synaptic properties of RHT terminals during postnatal development are not critically influenced by environmental light.

An automated Y-maze based on a reduced instruction set computer (RISC) microcontroller for the assessment of continuous spontaneous alternation in rats.

Continuous spontaneous alternation behavior (SAB) in a Y-maze is used for evaluating working memory in rodents. Here, the design of an automated Y-maze equipped with three infrared optocouplers per arm, and commanded by a reduced instruction set computer (RISC) microcontroller is described. The software was devised for recording only true entries and exits to the arms. Experimental settings are programmed via a keyboard with three buttons and a display. The sequence of arm entries and the time spent in each arm and the neutral zone (NZ) are saved as a text file in a non-volatile memory for later transfer to a USB flash memory. Data files are analyzed with a program developed under LabVIEW® environment, and the results are exported to an Excel® spreadsheet file. Variables measured are: latency to exit the starting arm, sequence and number of arm entries, number of alternations, alternation percentage, and cumulative times spent in each arm and NZ. The automated Y-maze accurately detected the SAB decrease produced in rats by the muscarinic antagonist trihexyphenidyl, and its reversal by caffeine, having 100 % concordance with the alternation percentages calculated by two trained observers who independently watched videos of the same experiments. Although the values of time spent in the arms and NZ measured by the automated system had small discrepancies with those calculated by the observers, Bland-Altman analysis showed 95 % concordance in three pairs of comparisons, while in one it was 90 %, indicating that this system is a reliable and inexpensive alternative for the study of continuous SAB in rodents.

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.

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.

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.

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.

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.

Capsaicin produces antidepressant-like effects in the forced swimming test and enhances the response of a sub-effective dose of amitriptyline in rats.

Transient receptor potential vanilloid 1 (TRPV1) channels have been implicated in depression and anxiety. The aim of this study was to evaluate the antidepressant-like properties of the TRPV1 agonist capsaicin using the forced swimming test (FST) in rats. Capsaicin (0.001-0.25 mg/kg, i.p.) produced a reduction of immobility in the FST. A maximally effective dose of the tricyclic antidepressant amitriptyline (12 mg/kg) reduced immobility as well. Notably, doses of capsaicin (1 pg/kg, 1 ng/kg, and 0.001 mg/kg) that were ineffective when applied alone produced a significant decrease in immobility when combined with a subthreshold dose of amitriptyline (5 mg/kg). Rats treated with capsaicin (0.01 mg/kg) + amitriptyline (5 mg/kg) displayed less immobility than those treated with a maximally effective dose of amitriptyline. The non-pungent TRPV1 channel agonist palvanil (0.05-0.1 mg/kg, i.p.) also decreased immobility in the FST. Capsaicin (0.05 mg/kg) did not affect general locomotion in the open field test, nor performance in the elevated plus maze, or skeletal muscle contraction strength measured in vitro after the FST (at 0.25 mg/kg). Altogether, our results imply that low doses of capsaicin produce antidepressant-like effects, and enhance the effect of a subthreshold dose of amitriptyline in the FST.

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.

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.

A novel automated rat catalepsy bar test system based on a RISC microcontroller.

Catalepsy tests performed in rodents treated with drugs that interfere with dopaminergic transmission have been widely used for the screening of drugs with therapeutic potential in the treatment of Parkinson's disease. The basic method for measuring catalepsy intensity is the "standard" bar test. We present here an easy to use microcontroller-based automatic system for recording bar test experiments. The design is simple, compact, and has a low cost. Recording intervals and total experimental time can be programmed within a wide range of values. The resulting catalepsy times are stored, and up to five simultaneous experiments can be recorded. A standard personal computer interface is included. The automated system also permits the elimination of human error associated with factors such as fatigue, distraction, and data transcription, occurring during manual recording. Furthermore, a uniform criterion for timing the cataleptic condition can be achieved. Correlation values between the results obtained with the automated system and those reported by two independent observers ranged between 0.88 and 0.99 (P<0.0001; three treatments, nine animals, 144 catalepsy time measurements).

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.

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.

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.

Circling behavior induced by microinjection of serotonin reuptake inhibitors in the substantia nigra.

The nigrostriatal dopaminergic neurons of the substantia nigra pars compacta (SNc) and the nondopaminergic neurons of the substantia nigra pars reticulata (SNr) receive a dense synaptic input from the serotonergic neurons of the raphe nuclei. To assess whether serotonin [5-hydroxytryptamine (5-HT)] spontaneously released at the substantia nigra could modulate motor activity, the 5-HT reuptake inhibitors (SRIs), duloxetine (6-12 nmol) and clomipramine (12 nmol), were unilaterally microinjected either into the SNc or the SNr of freely moving rats, and the circling behavior was counted with an automated rotometer. In the SNc, the main effect of the SRIs was a contraversive circling behavior that was not observed when applied at distances > or = 0.2 mm above the SNc. The circling induced by clomipramine was blocked by microinjection of haloperidol (53 nmol) into the ipsilateral neostriatum, suggesting that the circling elicited by microinjection of the SRIs into the SNc depends on an intact striatal dopaminergic transmission. Microinjection of 5-HT (21 nmol) only produced a significant contraversive circling response when it was coinjected with the SRIs. Pretreatment with methysergide (1 mg/kg ip), a nonselective 5-HT(2) antagonist, did not block the circling elicited by microinjection of clomipramine into the SNc, either alone or in combination with 5-HT. However, microinjection of the 5-HT(2) antagonist mianserin (2 nmol) into the SNc partially inhibited the circling induced by duloxetine (6 nmol), alone or coinjected with 5-HT. Since current theories of circling behavior hypothesize that the animal turns away from the cerebral hemisphere where dopamine neurotransmission predominates, these results suggest that the contraversive circling induced by the unilateral microinjection of SRIs into the SNc could be mediated by a 5-HT-induced increase of firing frequency of nigrostriatal dopaminergic neurons. When applied into the SNr, clomipramine and duloxetine also elicited a contraversive circling behavior and enhanced the circling induced by 5-HT. Systemic methysergide (1 mg/kg i.p.), but not intranigral mianserin (2 nmol), blocked the circling elicited by microinjection of clomipramine into the SNr, either alone or in combination with 5-HT. These results suggest that 5-HT(2)-like receptors are involved in the contraversive circling induced by enhancement of serotonergic transmission in the SNr.

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.