Francisco J. Heredia-López

Chronic caffeine consumption prevents cognitive decline from young to middle age in rats, and is associated with increased length, branching, and spine density of basal dendrites in CA1 hippocampal neurons.

Chronic caffeine consumption has been inversely associated with the risk of developing dementia and Alzheimers disease. Here we assessed whether chronic caffeine treatment prevents the behavioral and cognitive decline that male Wistar rats experience from young (≈3 months) to middle age (≈10 months). When animals were young they were evaluated at weekly intervals in three tests: motor activity habituation in the open field (30-min sessions at the same time on consecutive days), continuous spontaneous alternation in the Y-maze (8 min), and elevated plus-maze (5 min). Afterward, rats from the same litter were randomly assigned either to a caffeine-treated group (n=13) or a control group (n=11), which received only tap water. Caffeine treatment (5 mg/kg/day) began when animals were ≈4 months old, and lasted for 6 months. Behavioral tests were repeated from day 14 to day 28 after caffeine withdrawal, a time period that is far in excess for the full excretion of a caffeine dose in this species. Thirty days after caffeine discontinuation brains were processed for Golgi-Cox staining. Compared with controls, we found that middle-aged rats that had chronically consumed low doses of caffeine (1) maintained their locomotor habituation during the second consecutive day exposure to the open field (an index of non-associative learning), (2) maintained their exploratory drive to complete the conventional minimum of nine arm visits required to calculate the alternation performance in the Y-maze in a greater proportion, (3) maintained their alternation percentage above chance level (an index of working memory), and (4) did not increase the anxiety indexes assessed by measuring the time spent in the open arms of the elevated plus maze. In addition, morphometric analysis of hippocampal neurons revealed that dendritic branching (90-140 μm from the soma), length of 4th and 5th order branches, total dendritic length, and spine density in distal dendritic branches were greater in the basal but not the apical dendrites of CA1 pyramidal neurons from rats chronically treated with caffeine, in comparison with their age- and littermate-matched controls. Altogether, the present findings strengthen the epidemiological observations suggesting that prolonged caffeine intake prevents the cognitive decline associated with aging, and open the possibility that this process could be mediated by promoting the growth of dendrites and spines in neurons of the adult mammalian brain.

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 Parkinsons disease.

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 N2 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 m2/g nanostructured silica materials. The stability of dopamine in the silica network was confirmed by infrared and 13C-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.

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.

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.

Clinical doses of citalopram or reboxetine differentially modulate passive and active behaviors of female Wistar rats with high or low immobility time in the forced swimming test.

The sensitivity of immobility time (IT) to antidepressant-drugs differs in rats expressing high or low motor activity during the forced swimming test (FST). However, whether this heterogeneity is expressed after the administration of the most selective serotonin and norepinephrine reuptake inhibitors (SSRIs and SNRIs, respectively) is unknown. We compared the influence of either the SSRI citalopram or the SNRI reboxetine with the tricyclic antidepressant amitriptyline on two subgroups of female Wistar rats expressing high IT (HI; at or above the mean value) or low IT (LI; below the mean) during the initial 5 min of the first session of the FST. None of the tested drugs increased motor activity in the open field test. When vehicle was applied to either HI or LI rats, IT increased in the second session of the FST. This increment concurred with a simultaneous climbing time (CT) decrement. When amitriptyline (15 mg/kg) was tested the CT increased for both HI and LI rats. This increment was accompanied by an IT decrement in HI and LI rats. Reboxetine (0.16 or 1 mg/kg) precluded IT and CT changes in both HI and LI rats and produced a swimming time reduction. Citalopram (0.4, 1, and 3 mg/kg) essentially mimicked the influence of reboxetine on the IT and CT in LI rats, as well as in HI rats, but in the latter case only at 3 mg/kg. Yet, at the dose of 10 mg/kg citalopram lacked this effect in both subgroups. No differences were detected when the IT of LI rats was evaluated with citalopram (3 mg/kg) during estrus or diestrus stage. These results show that clinical doses of citalopram produced an antidepressant-like effect selectively in LI rats, while amitriptyline or reboxetine produced this effect in both LI and HI animals.

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.

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 Parkinsons 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 Parkinsons disease.

A novel rotometer based on a RISC microcontroller

A new, low-cost rotometer, based on a reduced instruction set computer (RISC) microcontroller, is presented. Like earlier devices, it counts the number and direction of full turns for predetermined time periods during the evaluation of turning behavior induced by drug administration in rats. The present stand-alone system includes a nonvolatile memory for long-term data storage and a serial port for data transmission. It also contains a display for monitoring the experiments and has battery backup to avoid interruptions owing to power failures. A high correlation was found (r > .988,p < 2 × 10−14) between the counts of the rotometer and those of two trained observers. The system reflects quantitative differences in turning behavior owing to pharmacological manipulations. It provides the most common counting parameters and is inexpensive, flexible, highly reliable, and completely portable (weight including batteries, 159 g).

Improvement of postural adjustment steps in hemiparkinsonian rats chronically treated with caffeine is mediated by concurrent blockade of A1 and A2A adenosine receptors.

Chronic treatment with the non-selective adenosine receptor antagonist caffeine produces full recovery of the contralateral adjusting steps in hemiparkinsonian rats. In order to disclose which adenosine receptor subtype mediates this effect, a group of hemiparkinsonian rats (n=9) was treated with caffeine (5.15 mumol/kg/day), or equimolar doses of selective A1 (DPCPX) or A2A (ZM 241385) adenosine receptor antagonists, administered in a counterbalanced order over periods of 3 weeks, interspersed with equivalent washout intervals. Treatment with ZM 241385 caused full recovery (102+/-6%) of the contralateral forepaw stepping, while the maximal effect of DPCPX was only 73+/-7% of that produced by caffeine. The maximal effect of caffeine and ZM 241385 remained stable throughout the treatment period. The response to DPCPX showed more fluctuations, but tolerance did not develop. Stepping improvement was significantly faster with DPCPX than with ZM 241385, while caffeine had intermediate values. Stepping decrease after treatment interruption was faster with ZM 241385 than with caffeine, while DPCPX had intermediate values. In other experiments with the same rats, addition of the A2AR agonist CGS 21680 (5.15 mumol/kg) or the A1R agonist CCPA (2.71 mumol/kg) during the second week of caffeine treatment reversed the improvement of contralateral stepping by 59+/-4% and 30+/-3%, respectively. The combined treatment with CGS 21680 and CCPA caused complete reversal of the contralateral stepping recovery afforded by caffeine, which was more than additive (114+/-5%) compared with the sum of the maximal inhibition produced by either agonist administered alone (89+/-4%). In all cases, after interrupting the adenosine agonists, the effect of caffeine was fully restored. None of the aforementioned treatments induced significant changes in the stepping of the ipsilateral forepaw. Collectively, these results suggest that the improvement of postural adjustments induced by chronic treatment with low doses of caffeine in hemiparkinsonian rats is mediated by concurrent blockade of A1 and A2A adenosine receptors, with a larger involvement of the latter.