Luis F. Alguacil

Effects of histamine H3 receptor ligands in experimental models of anxiety and depression

Abstract Histamine H3 receptor ligands have been proposed to be of potential therapeutic interest for the treatment of different central nervous system disorders; however, the psychopharmacological properties of these drugs have not been studied extensively. In this work, we investigated the possible involvement of histamine H3 receptor function in experimental models of anxiety (elevated plus-maze) and depression (forced swimming test). Male Sprague-Dawley rats were treated IP with the histamine H3 receptor agonist R-α-methylhistamine (10 mg/kg) or the histamine H3 receptor antagonist thioperamide (0.2, 2 and 10 mg/kg) and 30 min afterwards the time spent in the open arms of an elevated plus-maze was registered for 5 min. The immobility time of male OF1 mice in the forced swimming test was recorded for 6 min, 1 h after the IP administration of R-α-methylhistamine (10 and 20 mg/kg), thioperamide (0.2, 2, 10 and 20 mg/kg) or another histamine H3 receptor antagonist, clobenpropit (5 mg/kg). The locomotor activity of mice was checked in parallel by means of an activity meter. Both saline controls and active drug controls were used in all the paradigms. Neither thioperamide nor R-α-methylhistamine significantly changed animal behaviour in the elevated plus-maze. R-α-methylhistamine and the higher dose of thioperamide assayed (20 mg/kg) were also inactive in the forced swimming test. By contrast, thioperamide (0.2–10 mg/kg) dose-dependently decreased immobility, the effect being significant at 10 mg/kg (33% reduction of immobility); clobenpropit produced an effect qualitatively similar (24% reduction of immobility). None of these histamine H3 receptor antagonists affected locomotor activity. These preliminary results suggest that the histamine H3 receptor blockade could be devoid of anxiolytic potential but have antidepressant effects. Besides, the stimulation of these receptors does not seem to be followed by changes in the behavioural parameters studied.

Effects of yohimbine on the antinociceptive and place conditioning effects of opioid agonists in rodents

The pharmacological modulation of opioid actions by drugs acting on heterologous mechanisms could be useful to overcome some of the main problems associated with the use of opiate agonists. Based on previous findings on the interactions between yohimbine and opioid drugs, we have further studied the effects of yohimbine on the antinociceptive and positive‐negative reinforcing effects of morphine (μ opioid receptor‐preferring agonist), U‐50,488 (κ agonist) and SNC80 (δ agonist). Pretreatment with yohimbine completely blocked the antinociception provided by the three opioid agonists in the mouse tail‐immersion test. A similar blockade of SNC80 and U‐50,488‐induced antinociception was observed with yohimbine in the mouse hot plate test at the same doses. In this paradigm, the effect of the κ agonist was very slight and the actions of yohimbine rather variable. In place conditioning experiments with SD (Sprague – Dawley) male rats, yohimbine alone was inactive but it limited the preference induced by morphine and SNC80 and the aversive effect of U‐50,488. Impaired novelty preference was also observed with the combination of yohimbine and U‐50,488. It is concluded that yohimbine tends to limit opioid antinociception and the addictive potential of μ and δ opioid agonists. More selective drugs could help to understand the mechanisms involved in these actions.

Effects of yohimbine on morphine analgesia and physical dependence in the rat.

The effects of yohimbine on morphine analgesia and on the development of opiate physical dependence were studied to find out more about the involvement of alpha 2-adrenergic mechanisms in opioid actions. Male Sprague-Dawley rats (250-300 g) were used. The acute effect of morphine (5 mg/kg i.p.) in the tail-flick test was reduced significantly by pretreatment with a single dose of yohimbine (2 mg/kg i.p.). Alone yohimbine, produced a slight hyperalgesia. Animals treated with a sustained-release preparation of morphine (300 mg/kg s.c.) showed the same sensitivity to opiate analgesia 72 h later whether they were treated concomitantly with yohimbine or not, but they exhibited fewer withdrawal symptoms upon naloxone injection after yohimbine (2 or 4 mg/kg i.p. 24, 28, 48 and 52 h after the start of systemic morphine treatment). The results obtained confirm previous data on the effects of yohimbine on morphine analgesia and reveal the importance of alpha 2-adrenergic activation in the development of opioid physical dependence.

Shift of Circadian Feeding Pattern by High-Fat Diets Is Coincident with Reward Deficits in Obese Mice

Recent studies provide evidence that high-fat diets (HF) trigger both i) a deficit of reward responses linked to a decrease of mesolimbic dopaminergic activity, and ii) a disorganization of circadian feeding behavior that switch from a structured meal-based schedule to a continuous snacking, even during periods normally devoted to rest. This feeding pattern has been shown to be a cause of HF-induced overweight and obesity. Our hypothesis deals with the eventual link between the rewarding properties of food and the circadian distribution of meals. We have investigated the effect of circadian feeding pattern on reward circuits by means of the conditioned-place preference (CPP) paradigm and we have characterized the rewarding properties of natural (food) and artificial (cocaine) reinforcers both in free-feeding ad libitum HF mice and in HF animals submitted to a re-organized feeding schedule based on the standard feeding behavior displayed by mice feeding normal chow (“forced synchronization”). We demonstrate that i) ad libitum HF diet attenuates cocaine and food reward in the CPP protocol, and ii) forced synchronization of feeding prevents this reward deficit. Our study provides further evidence that the rewarding impact of food with low palatability is diminished in mice exposed to a high-fat diet and strongly suggest that the decreased sensitivity to chow as a positive reinforcer triggers a disorganized feeding pattern which might account for metabolic disorders leading to obesity.

Proteomic analysis of the nucleus accumbens of rats with different vulnerability to cocaine addiction

Vulnerability to the addictive effects of drugs of abuse varies among individuals, but the biological basis of these differences are poorly known. This work tries to increase this knowledge by comparing the brain proteome of animals with different rate of extinction of cocaine-seeking behaviour. To achieve this goal, we used a place-preference paradigm to separate Sprague Dawley rats in two groups: rats that extinguished (E) and rats that did not extinguish (NE) cocaine-seeking behaviour after a five-day period of drug abstinence. Once the phenotype was established, we compared the protein expression in the nucleus accumbens (NAC) of these animals after a single injection of either saline (SAL) or cocaine (COC, 15 mg/kg). The analysis of protein expression was performed by 2-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization time of flight mass spectrometry. When comparing E SAL and NE SAL animals we found significant differences in the expression level of 5 proteins: ATP synthase subunit alpha, fumarate hydratase, transketolase, NADH dehydrogenase [ubiquinone] flavoprotein 2 and glutathione transferase omega-1. A single injection of COC differently alters the NAC proteome of E and NE rats; thus in E COC animals there was an alteration in the expression of 6 proteins, including dihydropyrimidinase-related protein 2 and NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 10; whereas in NE COC rats 9 proteins were altered (including alpha-synuclein, peroxiredoxin-2 and peroxiredoxin-5). These proteins could be potential biomarkers of individual vulnerability to cocaine abuse and may be helpful in designing new treatments for cocaine addiction.

Yohimbine prevents morphine-induced changes of glial fibrillary acidic protein in brainstem and α2-adrenoceptor gene expression in hippocampus

The alpha(2)-adrenoceptor antagonist yohimbine is known to oppose to several pharmacological effects of opioid drugs, but the consequences and the mechanisms involved remain to be clearly established. In the present study we have checked the effects of yohimbine on morphine-induced alterations of the expression of key proteins (glial fibrillary acidic protein, GFAP) and genes (alpha(2)-adrenoceptors) in rat brain areas known to be relevant in opioid dependence, addiction and individual vulnerability to drug abuse. Rats were treated with morphine in the presence or absence of yohimbine. The effects of the treatments on GFAP expression were studied by immunohistochemical staining in Locus Coeruleus (LC) and Nucleus of the Solitary Tract (NST), two important noradrenergic nuclei. In addition, drug effects on alpha(2)-adrenoceptor gene expression were determined by real time RT-PCR in the hippocampus, a brain area that receives noradrenergic input from the brainstem. Morphine administration increased GFAP expression both in LC and NST as it was previously reported in other brain areas. Yohimbine was found to efficiently prevent morphine-induced GFAP upregulation. Chronic (but not acute) morphine downregulated mRNA levels of alpha(2A)- and alpha(2C)-adrenoceptors in the hippocampus, while simultaneously increased the expression of the alpha(2B)-adrenoceptor gene. Again, yohimbine was able to prevent morphine-induced changes in the levels of expression of the three alpha(2)-adrenoceptor genes. These results correlate the well-established reduction of opioid dependence and addiction by yohimbine and suggest that this drug could interfere with the neural plasticity induced by chronic morphine in central noradrenergic pathways.

The neurotrophic factor pleiotrophin modulates amphetamine-seeking behaviour and amphetamine-induced neurotoxic effects: evidence from pleiotrophin knockout mice

Pleiotrophin (PTN), a neurotrophic factor with important roles in survival and differentiation of dopaminergic neurons, is up‐regulated in the nucleus accumbens after amphetamine administration suggesting that PTN could modulate amphetamine‐induced pharmacological or neuroadaptative effects. To test this hypothesis, we have studied the effects of amphetamine administration in PTN genetically deficient (PTN −/−) and wild type (WT, +/+) mice. In conditioning studies, we found that amphetamine induces conditioned place preference in both PTN −/− and WT (+/+) mice. When these mice were re‐evaluated after a 5‐day period without amphetamine administration, we found that WT (+/+) mice did not exhibit amphetamine‐seeking behaviour, whereas, PTN −/− mice still showed a robust drug‐seeking behaviour. In immunohystochemistry studies, we found that amphetamine (10 mg/kg, four times, every 2 hours) causes a significant increase of glial fibrillary acidic protein positive cells in the striatum of amphetamine‐treated PTN −/− mice compared with WT mice 4 days after last administration of the drug, suggesting an enhanced amphetamine‐induced astrocytosis in the absence of endogenous PTN. Interestingly, we found in concomitant in vitro studies that PTN (3 µM) limits amphetamine (1 mM)‐induced loss of viability of PC12 cell cultures, effect that could be related to the ability of PTN to induce the phosphorylation of Akt and ERK1/2. To test this possibility, we used specific Akt and ERK1/2 inhibitors uncovering for the first time that PTN‐induced protective effects against amphetamine‐induced toxicity in PC12 cells are mediated by the ERK1/2 signalling pathway. The data suggest an important role of PTN to limit amphetamine‐induced neurotoxic and rewarding effects.

Noradrenergic and opioidergic alterations in neuropathy in different rat strains

The Fischer 344 (F344) rat strain differs from the Lewis strain in the response to neuropathic pain. Recently, we found that F344 rats totally recover from mechanical allodynia induced by chronic constriction injury (CCI) of the sciatic nerve 28 days after surgery whereas Lewis rats are initiating their recovery at this time point. Thus, the use of this neuropathic pain model in these different rat strains constitutes a good strategy to identify possible target genes involved in the development of neuropathic pain. Since differences between Lewis and F344 rats in their response to pain stimuli in acute pain models have been related to differences in the endogenous opioid and noradrenergic systems, we aimed to determine the levels of expression of key genes of both systems in the spinal cord and dorsal root ganglia (DRG) of both strains 28 days after CCI surgery. Real time RT-PCR revealed minimal changes in gene expression in the spinal cord after CCI despite the strain considered, but marked changes in DRG were observed. A significant upregulation of prodynorphin gene expression occurred only in injured DRG of F344 rats, the most resistant strain to neuropathic pain. In addition, we found a significant downregulation of tyrosine hydroxylase and proenkephalin gene expression levels in both strains whereas delta-opioid receptor was found to be significantly downregulated only in injured DRG of Lewis rats although the same trend was observed in F344 rats. The data strongly suggest that dynorphins could be involved in strain differences concerning CCI resistance.

Effect of Yohimbine on the development of morphine dependence in the rat : Lack of involvement of cortical β-adrenoceptor modifications

The alpha-2 receptor antagonist yohimbine has been previously shown to prevent the development of morphine dependence in a rat behavioral model. This study was directed to clarify the mechanism of this interaction, which is presently unknown. Since upregulation of cortical beta-adrenoceptors has been suggested to be involved in morphine withdrawal, we have tested the possible correlation between receptor density and withdrawal behaviors in the presence of yohimbine. Sprague-Dawley male rats received a s.c. suspension of morphine (300 mg/kg) or the vehicle. Animals received saline or yohimbine (4 mg/kg, IP) 24, 28, 48 and 52 h after morphine and finally naloxone (1 mg/kg i.p) at 72 h; the subsequent signs of withdrawal (mainly wet-dog shakes and escape attempts) were recorded and the cerebral cortex dissected to study [3H]-CGP 12177 binding. Morphine-treated animals displayed a marked withdrawal behavior together with beta-adrenoceptor upregulation; nevertheless, these effects were not correlated. As expected, yohimbine prevented morphine withdrawal behavior but did not reverse the beta-adrenoceptor upregulation induced by the opiate. These results confirm previous evidence against the involvement of beta-adrenoceptor upregulation on morphine withdrawal behaviors and also permit to discard beta-adrenoceptor regulation as the neurochemical basis of the antiwithdrawal effect of yohimbine. The possible contribution of some other neurochemical effects of yohimbine are discussed to explain the inhibition of morphine dependence by that drug.

The α2-adrenoceptor antagonist yohimbine reduces glial fibrillary acidic protein upregulation induced by chronic morphine administration

Previous literature data show prominent interactions between alpha(2)-adrenoceptor ligands and opioid drugs, however, the nature of such interactions is still largely unknown. In the present study, we aimed to examine the potential protective effect of yohimbine, a alpha(2)-adrenoceptor antagonist, against glial fibrillary acidic protein (GFAP) alterations elicited by chronic morphine treatment. Increased astrogliosis, as indicated by increased GFAP immunohistochemical staining, was observed in the ventral tegmental area, nucleus accumbens shell, and frontal cortex of chronic morphine-treated (10 mg kg(-1), i.p., every 12 h for 13 days) rats compared with those treated with saline. Pretreatment with yohimbine (2 mg kg(-1), i.p., 30 min before each morphine injection) provided protection against morphine-induced GFAP upregulation. The present study demonstrates that yohimbine pretreatment reduces long-term morphine exposure-induced alterations in the astroglial reaction, suggesting that alpha(2)-adrenergic mechanisms may play an important role in mediating morphine-induced pathological effects in the brain.