Lucas Salomon

Striatal Medium-Sized Spiny Neurons: Identification by Nuclear Staining and Study of Neuronal Subpopulations in BAC Transgenic Mice

Precise identification of neuronal populations is a major challenge in neuroscience. In the striatum, more than 95% of neurons are GABAergic medium-sized spiny neurons (MSNs), which form two intermingled populations distinguished by their projections and protein content. Those expressing dopamine D1-receptors (D1Rs) project preferentially to the substantia nigra pars reticulata (SNr), whereas those expressing dopamine D2- receptors (D2Rs) project preferentially to the lateral part of the globus pallidus (LGP). The degree of segregation of these populations has been a continuous subject of debate, and the recent introduction of bacterial artificial chromosome (BAC) transgenic mice expressing fluorescent proteins driven by specific promoters was a major progress to facilitate striatal neuron identification. However, the fraction of MSNs labeled in these mice has been recently called into question, casting doubt on the generality of results obtained with such approaches. Here, we performed an in-depth quantitative analysis of striatal neurons in drd1a-EGFP and drd2-EGFP mice. We first quantified neuronal and non-neuronal populations in the striatum, based on nuclear staining with TO-PRO-3, and immunolabeling for NeuN, DARPP-32 (dopamine- and cAMP-regulated phosphoprotein Mr∼32,000), and various markers for interneurons. TO-PRO-3 staining was sufficient to identify MSNs by their typical nuclear morphology and, with a good probability, interneuron populations. In drd1a-EGFP/drd2-EGFP double transgenic mice all MSNs expressed EGFP, which was driven in about half of them by drd1a promoter. Retrograde labeling showed that all MSNs projecting to the SNr expressed D1R and very few D2R (<1%). In contrast, our results were compatible with the existence of some D1R-EGFP-expressing fibers giving off terminals in the LGP. Thus, our study shows that nuclear staining is a simple method for identifying MSNs and other striatal neurons. It also unambiguously confirms the degree of segregation of MSNs in the mouse striatum and allows the full exploitation of results obtained with BAC-transgenic mice.

Monoamine Oxidase Inhibitors Allow Locomotor and Rewarding Responses to Nicotine

Although nicotine is generally considered to be the main compound responsible for the addictive properties of tobacco, experimental data indicate that nicotine does not exhibit all the characteristics of other abused substances, such as psychostimulants and opiates. For example, nicotine is only a weak locomotor enhancer in rats and generally fails to induce a locomotor response in mice. This observation contradicts the general consensus that all drugs of abuse release dopamine in the nucleus accumbens, a subcortical structure, and thus increase locomotor activity in rodents. Because tobacco smoke contains monoamine oxidase inhibitors (MAOIs) and decreases MAO activity in smokers, we have combined MAOIs with nicotine to determine whether it is possible to obtain a locomotor response to nicotine in C57Bl6 mice. Among 15 individual or combined MAOIs, including harmane, norharmane, moclobemide, selegiline, pargyline, clorgyline, tranylcypromine and phenelzine, only irreversible inhibitors of both MAO-A and -B (tranylcypromine, phenelzine, and clorgyline+selegiline) allowed a locomotor response to nicotine. The locomotor stimulant interaction of tranylcypromine and nicotine was absent in β2-nicotinic acetylcholine receptor subunit knockout mice. Finally, it was found that, whereas naïve rats did not readily self-administer nicotine (10 μg/kg/injection), a robust self-administration of nicotine occurred when animals were pretreated with tranylcypromine (3 mg/kg). Our data suggest that MAOIs contained in tobacco and tobacco smoke act in synergy with nicotine to enhance its rewarding effects.

Inhibition of Monoamine Oxidases Desensitizes 5-HT1A Autoreceptors and Allows Nicotine to Induce a Neurochemical and Behavioral Sensitization

Although nicotine is generally considered to be the main compound responsible for addictive properties of tobacco, experimental data indicate that nicotine does not exhibit all the characteristics of other substances of abuse. We recently showed that a pretreatment with mixed irreversible monoamine oxidases inhibitors (MAOIs), such as tranylcypromine, triggers a locomotor response to nicotine in mice and allows maintenance of behavioral sensitization to nicotine in rats. Moreover, we showed by microdialysis in mice that behavioral sensitization induced by compounds belonging to main groups of drugs of abuse, such as amphetamine, cocaine, morphine, or alcohol, was underlain by sensitization of noradrenergic and serotonergic neurons. Here, this neurochemical sensitization was tested after nicotine, tranylcypromine, or a mixture of both compounds. Data indicate that, whereas neither repeated nicotine nor repeated tranylcypromine alone has any effect by itself, a repeated treatment with a mixture of nicotine and tranylcypromine induces both behavioral sensitization and sensitization of noradrenergic and serotonergic neurons. The development of neurochemical and behavioral sensitizations is blocked by prazosin and SR46349B [(1Z,2E)-1-(2-fluoro-phenyl)-3-(4-hydroxyphenyl)-prop-2-en-one-O-(2-dimethylamino-ethyl)-oxime hemifumarate], two antagonists of α1b-adrenergic and 5-HT2A receptors, respectively, but not by SCH23390 [R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride], a D1 receptor antagonist. Finally, we found that pretreatments with WAY 100635 [N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclo-hexane carboxamide trihydrochloride], a 5-HT1A receptor antagonist, can also induce a behavioral and neurochemical sensitization to repeated nicotine. Complementary experiments with 8-OHDPAT (8-hydroxy-dipropylamino-tetralin), a 5-HT1A receptor agonist, and analysis of 5-HT1A receptors expression in the dorsal raphe nucleus after a tranylcypromine injection indicate that MAOIs contained in tobacco desensitize 5-HT1A autoreceptors to trigger the strong addictive properties of tobacco.

Irreversible blockade of monoamine oxidases reveals the critical role of 5-HT transmission in locomotor response induced by nicotine in mice

Although nicotine is generally considered as the main compound responsible for addictive properties of tobacco, some experimental data indicate that nicotine does not exhibit all the characteristics of other substances of misuse such as psychostimulants and opiates. For example, nicotine generally fails to induce locomotor response in mice and self‐administration of nicotine is difficult to obtain in rats. We have shown recently that a pretreatment with mixed irreversible monoamine oxidase inhibitors (MAOIs), such as tranylcypromine, triggers a locomotor response to nicotine in mice and induces a robust self‐administration of nicotine in rats. We show here that when mice were pretreated with enhancers of extracellular levels of noradrenaline, dopamine or serotonin (d‐amphetamine, GBR12783 or para‐chloro‐amphetamine, respectively) and injected with nicotine (1 mg/kg), only those animals pretreated with para‐chloro‐amphetamine exhibited a specific locomotor response to nicotine. These data indicate a critical role of serotonin in nicotine‐induced locomotor activity in mice. This was further confirmed in microdialysis experiments showing that nicotine induces an increase in extracellular serotonin levels in the ventral striatum in mice pretreated with tranylcypromine. This effect of nicotine on extracellular serotonin levels was absent in mice lacking the β2‐subunit of the nicotinic acetylcholine receptor. Our data suggest that mixed irreversible MAOIs contained in tobacco facilitate the effects of nicotine on serotonin release, thus allowing the locomotor and rewarding effects of nicotine.

Repeated exposure to MDMA triggers long-term plasticity of noradrenergic and serotonergic neurons.

3,4-Methylenedioxymethamphetamine (MDMA or ‘ecstasy’) is a psychostimulant drug, widely used recreationally among young people in Europe and North America. Although its neurotoxicity has been extensively described, little is known about its ability to strengthen neural circuits when administered in a manner that reproduces human abuse (i.e. repeated exposure to a low dose). C57BL/6J mice were repeatedly injected with MDMA (10 mg kg−1, intraperitoneally) and studied after a 4-day or a 1-month withdrawal. We show, using in vivo microdialysis and locomotor activity monitoring, that repeated injections of MDMA induce a long-term sensitization of noradrenergic and serotonergic neurons, which correlates with behavioral sensitization. The development of this phenomenon, which lasts for at least 1 month after withdrawal, requires repeated stimulation of α1B-adrenergic and 5-hydroxytryptamine (5-HT)2A receptors. Moreover, behavioral and neuroendocrine assays indicate that hyper-reactivity of noradrenergic and serotonergic networks is associated with a persistent desensitization of somatodendritic α2A-adrenergic and 5-HT1A autoreceptor function. Finally, molecular analysis including radiolabeling, western blot and quantitative reverse transcription-polymerase chain reaction reveals that mice repeatedly treated with MDMA exhibit normal α2A-adrenergic and 5-HT1A receptor binding, but a long-lasting downregulation of Gαi proteins expression in both locus coeruleus and dorsal raphe nucleus. Altogether, our results show that repeated MDMA exposure causes strong neural and behavioral adaptations and that inhibitory feedback mediated by α2A-adrenergic and 5-HT1A autoreceptors has an important role in the physiopathology of addictive behaviors.

α7 and β2* nicotinic receptors control monoamine-mediated locomotor response

Earlier studies reported exacerbated locomotor response to stress and tranylcypromine in &bgr;2 nicotinic acetylcholine receptor (nAChR) knockout (KO) mice. This study aimed to further assess the role of &bgr;2 and coexpressed nAChR subunits in the brain (&agr;4, &agr;6 and &agr;7) to control monoamine-mediated locomotor response, that is, response to novelty, saline, nicotine with tranylcypromine pretreatment, cocaine, d-amphetamine and morphine treatments. Results show that &bgr;2 KO mice were hyperreactive to novelty, cocaine and morphine. In contrast, &agr;7 KO mice were hyporeactive to tranylcypromine and cocaine. These results suggest that endogenous nAChR stimulation may exert a tonic control on monoamine-mediated locomotor responses. &bgr;2 and &agr;7-containing nAChR may contribute, respectively, to the inhibitory and permissive pathways of this tonic control.