Mechanisms of stimulatory effects of mecamylamine on the dorsal raphe neurons
Introduction
Mecamylamine is an autonomic ganglion blocker, developed originally as an antihypertensive agent (Stone et al., 1956). However, at doses used to treat arterial hypertension, mecamylamine produces severe parasympatolithic effects (constipation, dry mouth, miction disturbances) (Young et al., 2001). In the period of the eighties, mecamylamine was shown to be a nonspecific and noncompetitive blocker of neural nicotinic acetylcholine receptors (nAChRs), at doses much lower than the ones required for the treatment of hypertension (Papke et al., 2011). During this same period, new therapeutic uses of mecamylamine were discovered, most of them derived from its nAChRs blocking properties: add for smoking cessation (Rose et al., 1998) treatment of alcoholism (Blomqvist et al., 1993) and of cocaine addiction (Reid et al., 1999). Surprisingly, mecamylamine also exhibited antidepressant effects in animal models of depression and clinical studies (Philip et al., 2010, for review). This effect was explained using the cholinergic hypothesis of the pathogeny of depression (Janowsky et al., 1972) according to which the prevalence of the cholinergic over the sympathetic tone in the central nervous system favors depressive states. Consequently, the blockers of acetylcholine (ACh) receptors, including mecamylamine, should produce antidepressant effects. Experimental data from different sources, showing that nAChRs blockers produce indeed antidepressant effects, support this hypothesis (Philip et al., 2010, for review).
Other laboratory studies also suggested that low concentrations of mecamylamine increase the firing rate of 5-HT DRN neurons and DRN 5-HT release (Mihailescu et al., 1998). The mechanism of these mecamylamine effects which may explain its antidepressant actions was not dilucidated. A more recent study demonstrated that nicotine and other nicotinic agonists are also able to increase the excitability of DRN serotonergic (5-HT) neurons (Garduño et al., 2012). These results are incongruent with the effect of mecamylamine which, being a nAChRs blocker, should not have similar effects to nicotine in this nucleus. In the present study, we analyzed the mechanisms involved in the mecamylamine-induced increase in the firing rate of 5-HT DRN neurons. The results show that low concentrations mecamylamine (0.5−9 μM) induce both an increase in the glutamatergic and a decrease in the GABAergic input of 5-HT DRN neurons. These effects involved α4β2 and α7 nAChRs, respectively. Calcium imaging experiments revealed that mecamylamine increases global activity in the DRN and this effect is exerted mainly on 5-HT neurons.
Section snippets
Brain slice preparation
All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care Committee of the Universidad Nacional Autónoma de México. Experiments were performed in young (postnatal day 18–21) Wistar rats that were deeply anesthetized with isoflurane and then decapitated. Their brains were quickly removed and placed into ice-cold (4 °C) artificial cerebrospinal fluid (ACSF)
Results
For electrophysiological experiments, we recorded 91 neurons located at the midline of the DRN. Recorded neurons fulfilled the criteria for a 5-HT nature: low firing frequency (<10 Hz) after applying depolarizing electrical stimuli, long-lasting action potential (>5 ms, measured at the base), high electrical membrane resistance and firing adaptation (Allers and Sharp, 2003; Garduño et al., 2012) the cells were also identified by using an anti-5-HT antibody (see Material and methods).
Discussion
The electrophysiological and calcium imaging data presented in this study argue that low concentrations of mecamylamine (<20 μM) favors the firing activity of identified 5-HT DRN neurons. This effect is mediated by an increase of the glutamatergic input and, at the same time, a decrease of the GABAergic input to the 5-HT DRN cells. The action mechanisms are presynaptic and involve the stimulation of α4β2 and the inhibition of α7 nAChRs. These conclusions are based on various observations.
Conclusions
In the present work we found that low concentrations of mecamylamine promotes the firing frequency of 5-HT DRN neurons by two mechanisms. One of them is mediated by the stimulation of presynaptic α4β2 nAChRs to increase glutamate release. The other one involves a decrease in GABA release by blockage of presynaptic α7 nAChRs. Combined, these mechanisms produce a global increase of the activity of 5-HT DRN neurons as shown in calcium imaging experiments. The increased activity of serotonergic
Author statements
OHG Performed experiments and analyzed data.
AMG Performed experiments and analyzed data.
SHL Performed experiments, analyzed data, and wrote the manuscript.
DECR Performed experiments.
GAL Immunocytochemistry and analysis.
DT Immunocytochemistry and analysis.
SM Conceived and planned the experiments and analyzed data.
All the author discussed the results and contributed to the final manuscript.
We manifest that there are no conflict of interest for any of the authors.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
This work was supported by the DGAPA –PAPIIT, IN216416 grant to Stefan Mihailescu and DGAPA –PAPIIT, IN216319 grant to Salvador Hernández-López.
Andrea Mondragón-García is a student of Maestría from the Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México (UNAM) and received the fellowship 856364 from CONACyT.
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These authors contributed equally to this work.