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.

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.

Treatment of Parkinson's disease: nanostructured sol-gel silica-dopamine reservoirs for controlled drug release in the central nervous system.

INTRODUCTION: We have evaluated the use of silica-dopamine reservoirs synthesized by the sol-gel approach with the aim of using them in the treatment of Parkinson's disease, specifically as a device for the controlled release of dopamine in the striatum. Theoretical calculations illustrate that dopamine is expected to assume a planar structure and exhibit weak interactions with the silica surface. METHODS: Several samples were prepared by varying the wt% of dopamine added during the hydrolysis of tetraethyl orthosilicate. The silica-dopamine reservoirs were characterized by N(2) adsorption, scanning and transmission electron microscopy, and Fourier transform infrared spectroscopy. The in vitro release profiles were determined using ultraviolet visible absorbance spectroscopy. The textural analyses showed a maximum value for the surface area of 620 m(2)/g nanostructured silica materials. The stability of dopamine in the silica network was confirmed by infrared and (13)C-nuclear magnetic resonance spectroscopy. The reservoirs were evaluated by means of apomorphine-induced rotation behavior in hemiparkisonian rats. RESULTS: The in vitro dopamine delivery profiles indicate two regimes of release, a fast and sustained dopamine delivery was observed up to 24 hours, and after this time the rate of delivery became constant. Histologic analysis of formalin-fixed brains performed 24-32 weeks after reservoir implantation revealed that silica-dopamine implants had a reddish-brown color, suggesting the presence of oxidized dopamine, likely caused by the fixation procedure, while implants without dopamine were always translucent. CONCLUSION: The major finding of the study was that intrastriatal silica-dopamine implants reversed the rotational asymmetry induced by apomorphine, a dopamine agonist, in hemiparkinsonian rats. No dyskinesias or other motor abnormalities were observed in animals implanted with silica or silica-dopamine.

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.

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.

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.