Stefan Mihailescu

Effects of nicotine and mecamylamine on rat dorsal raphe neurons.

This study investigates the hypothesis that serotonin mediates certain nicotine effects, such as mood improvement and the suppression of the ponto-geniculo-occipital spikes of rapid eye movement sleep. The influence of nicotine (10-300 microM) on the firing rate of dorsal raphe neurons and on serotonin release was therefore, studied in rat midbrain slices. Nicotine increased the firing rate, 10-90%, in 67.5% recorded neurons and decreased it, 8-100%, in the remaining 32.5%. Serotonin release increased 2-7 times after nicotine administration, regardless of firing frequency, but the absolute value of serotonin release was 3 times higher during the decreases than during the increases in firing rate. Mecamylamine (1-20 microM) transiently stimulated the dorsal raphe neurons and competitively antagonized the nicotine-induced serotonin release. The results support the working hypothesis and additionally show that mecamylamine also stimulates dorsal raphe neurons.

Mechanisms of nicotine actions on dorsal raphe serotoninergic neurons

Nicotine, locally administered into the dorsal raphe nucleus (DRN) of rat midbrain slices, increased the discharge rate of 70% of serotoninergic neurons, decreased it in 30% and induced reciprocal oscillatory increases in serotonin (5-hydroxytryptamine, 5-HT) and gamma-aminobutyric acid (GABA) release. All of nicotines stimulatory effects were maximal at 2.15 microM. Bicuculline, a GABA(A) receptor antagonist, increased the firing rate in 64% of serotoninergic neurons, decreased it in 36% and augmented serotonin and GABA release. Bicuculline increased nicotines stimulatory effects on firing rate but did not reverse the inhibitory ones. N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinil-cyclohexanecarboxamide (WAY-100635), a 5-HT(1A) receptor antagonist, increased the firing rate of 88% of serotoninergic neurons, as well as serotonin and GABA release and reversed nicotines inhibitory action on serotoninergic neurons. These data suggest that nicotine decreases the firing rate of one third of serotoninergic neurons through serotonin release and increases the firing rate of the remaining two thirds, due to stronger stimulatory than indirect inhibitory effects.

Presynaptic α4β2 nicotinic acetylcholine receptors increase glutamate release and serotonin neuron excitability in the dorsal raphe nucleus.

Several behavioral effects of nicotine are mediated by changes in serotonin (5-HT) release in brain areas that receive serotonergic afferents from the dorsal raphe nucleus (DRN). In vitro experiments have demonstrated that nicotine increases the firing activity in the majority of DRN 5-HT neurons and that DRN contains nicotinic acetylcholine receptors (nAChRs) located at both somata and presynaptic elements. One of the most common presynaptic effects of nicotine is to increase glutamate release. Although DRN receives profuse glutamatergic afferents, the effect of nicotine on glutamate release in the DRN has not been studied in detail. Using whole-cell recording techniques, we investigated the effects of nicotine on the glutamatergic input to 5-HT DRN neurons in rat midbrain slices. Low nicotine concentrations, in the presence of bicuculline and tetrodotoxin (TTX), increased the frequency but did not change the amplitude of glutamate-induced EPSCs, recorded from identified 5-HT neurons. Nicotine-induced increase of glutamatergic EPSC frequency persisted 10–20 min after drug withdrawal. This nicotinic effect was mimicked by exogenous administration of acetylcholine (ACh) or inhibition of ACh metabolism. In addition, the nicotine-induced increase in EPSC frequency was abolished by blockade of α4β2 nAChRs, voltage-gated calcium channels, or intracellular calcium signaling but not by α7 nAChR antagonists. These data suggest that both nicotine and endogenous ACh can increase glutamate release through activation of presynaptic α4β2 but not α7 nAChRs in the DRN. The effect involves long-term changes in synaptic function, and it is dependent on voltage-gated calcium channels and presynaptic calcium stores.

Serotoninergic dorsal raphe neurons possess functional postsynaptic nicotinic acetylcholine receptors

Very few neurons in the telencephalon have been shown to express functional postsynaptic nicotinic acetylcholine receptors (nAChRs), among them, the noradrenergic and dopaminergic neurons. However, there is no evidence for postsynaptic nAChRs on serotonergic neurons. In this study, we asked if functional nAChRs are present in serotonergic (5‐HT) and nonserotonergic (non‐5‐HT) neurons of the dorsal raphe nucleus (DRN). In rat midbrain slices, field stimulation at the tegmental pedunculopontine (PPT) nucleus evoked postsynaptic currents (eEPSCs) with different components in DRN neurons. After blocking the glutamatergic and GABAergic components, the remaining eEPSCs were blocked by mecamylamine and reduced by either the selective α7 nAChR antagonist methyllycaconitine (MLA) or the selective α4β2 nAChR antagonist dihydro‐β‐eritroidine (DHβE). Simultaneous addition of MLA and DHβE blocked all eEPSCs. Integrity of the PPT‐DRN pathway was assessed by both anterograde biocytin tracing and antidromic stimulation from the DRN. Inward currents evoked by the direct application of acetylcholine (ACh), in the presence of atropine and tetrodotoxin, consisted of two kinetically different currents: one was blocked by MLA and the other by DHβE; in both 5‐HT and non‐5‐HT DR neurons. Analysis of spontaneous (sEPSCs) and evoked (eEPSCs) synaptic events led to the conclusion that nAChRs were located at the postsynaptic membrane. The possible implications of these newly described nAChRs in various physiological processes and behavioral events, such as the wake‐sleep cycle, are discussed. Synapse 62:601–615, 2008.

Nicotine stimulation of dorsal raphe neurons: effects on laterodorsal and pedunculopontine neurons

Previous studies showed that nicotine suppresses the ponto-geniculo-occipital (PGO) spikes of rapid eye movement (REM) sleep in cats. This effect may depend on stimulation of dorsal raphe nucleus (DRN) serotoninergic neurons that inhibit the pedunculopontine (PPT) and laterodorsal tegmental (LDT) cholinergic neurons, generators of PGO spikes. For testing this hypothesis 37 experiments were performed in rat midbrain slices. Nicotine (2 mM), administered locally into DRN, significantly increased the firing rate of 81.1% DRN neurons and serotonin release while simultaneously and significantly decreasing the firing rate of 80.8% LDT neurons and of 81.8% PPT neurons. The inhibition of LDT neurons by nicotine administered into DRN was blocked by the 5-HT1A receptor antagonist WAY-100635 (140 nM) administered into LDT. These results indicate that nicotine inhibits the activity of LDT and PPT neurons and consequently the generation of PGO spikes through stimulation of DRN serotoninergic neurons.

Subcutaneous administration of nicotine changes dorsal raphe serotonergic neurons discharge rate during REM sleep

In the present study nicotine (0.1 mg/kg, s.c.) increased discharge rate of putative dorsal raphe (DRN) serotonergic neurons of behaving rats during REM sleep (362.61%), without any significant change during waking and non-REM sleep. Since serotonergic DRN neurons gate PGO onset, these results suggest that nicotine-induced suppression of PGO spikes during REM sleep previously reported is achieved through stimulation of dorsal raphe serotonergic cells.

Nicotinic modulation of serotonergic activity in the dorsal raphe nucleus.

Abstract Cholinergic signaling mediated by nicotinic receptors has been associated to a large number of physiological and behavioral processes such as learning, memory, attention, food-intake and mood disorders. Although it is well established that many nicotinic actions are mediated through an increase in serotonin (5-HT) release, the physiological mechanisms by which nicotine produces these effects are still unclear. The dorsal raphe nucleus (DRN) contains the major amount of 5-HT neurons projecting to different parts of the brain. DRN also contains nicotinic acetylcholine receptors (nAChRs) located at somatic and presynaptic elements. Nicotine produces both inhibitory and excitatory effects on different subpopulations of 5-HT DRN neurons. In this review, we describe the presynaptic and postsynaptic mechanisms by which nicotine increases the excitability of DRN neurons as well as the subtypes of nAChRs involved. We also describe the inhibitory effects of nicotine and the role of 5-HT1A receptors in this effect. These nicotinic actions modulate the activity of different neuronal subpopulations in the DRN, changing the 5-HT tone in the brain areas where these groups of neurons project. Some of the physiological implications of nicotine-induced 5-HT release are discussed.

Effects of nicotine on alcohol intake in a rat model of depression.

Clinical studies suggest that depression facilitates alcohol abuse. Depressed individuals also have increased rates of smoking, and it has been suggested that nicotine may improve depression. It is therefore possible that nicotine may reduce alcohol use in depression. To investigate this potential relationship, we evaluated alcohol intake in an animal model of depression, which consists of administering clomipramine (CLI), a preferential serotonin reuptake inhibitor, to neonatal rats. This pharmacological manipulation produces adult depression-like behaviors, such as reduced aggressiveness, decreased pleasure seeking, diminished sexual activity, increased locomotor activity and increased REM sleep. In this study, we found that CLI rats exhibited significantly higher locomotor activity, lower aggressiveness and higher alcohol intake than control rats. Chronic administration of a low dose of nicotine (0.25 mg/kg/day) or a sham operation did not modify these behaviors. However, chronic administration of nicotine at a higher dose (1.5 mg/kg/day) significantly increased aggressive behavior and reduced alcohol intake in CLI rats. The effect of nicotine on alcohol intake lasted at least 1 month after cessation of nicotine administration. These results indicate that nicotine reverted some depression signs and reduced alcohol self-administration in the CLI model of depression.

Bupropion-induced inhibition of α7 nicotinic acetylcholine receptors expressed in heterologous cells and neurons from dorsal raphe nucleus and hippocampus.

The pharmacological activity of bupropion was compared between α7 nicotinic acetylcholine receptors expressed in heterologous cells and hippocampal and dorsal raphe nucleus neurons. The inhibitory activity of bupropion was studied on GH3-α7 cells by Ca2+ influx, as well as on neurons from the dorsal raphe nucleus and interneurons from the stratum radiatum of the hippocampal CA1 region by using a whole-cell voltage-clamp technique. In addition, the interaction of bupropion with the α7 nicotinic acetylcholine receptor was determined by [3H]imipramine competition binding assays and molecular docking. The fast component of acetylcholine- and choline-induced currents from both brain regions was inhibited by methyllycaconitine, indicating the participation of α7-containing nicotinic acetylcholine receptors. Choline-induced currents in hippocampal interneurons were partially inhibited by 10 µM bupropion, a concentration that could be reached in the brain during clinical administration. Additionally, both agonist-induced currents were reversibly inhibited by bupropion at concentrations that coincide with its inhibitory potency (IC50=54 µM) and binding affinity (Ki=63 µM) for α7 nicotinic acetylcholine receptors from heterologous cells. The [3H]imipramine competition binding and molecular docking results support a luminal location for the bupropion binding site(s). This study may help to understand the mechanisms of actions of bupropion at neuronal and molecular levels related with its therapeutic actions on depression and for smoking cessation.

Nicotine increases GABAergic input on rat dorsal raphe serotonergic neurons through alpha7 nicotinic acetylcholine receptor.

The dorsal raphe nucleus (DRN) contains large populations of serotonergic (5-HT) neurons. This nucleus receives GABAergic inhibitory afferents from many brain areas and from DRN interneurons. Both GABAergic and 5-HT DRN neurons express functional nicotinic acetylcholine receptors (nAChRs). Previous studies have demonstrated that nicotine increases 5-HT release and 5-HT DRN neuron discharge rate by stimulating postsynaptic nAChRs and by increasing glutamate and norepinephrine release inside DRN. However, the influence of nicotine on the GABAergic input to 5-HT DRN neurons was poorly investigated. Therefore, the aim of this work was to determine the effect of nicotine on GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) of 5-HT DRN neurons and the subtype of nAChR(s) involved in this response. Experiments were performed in coronal slices obtained from young Wistar rats. GABAergic sIPSCs were recorded from post hoc-identified 5-HT DRN neurons with the whole cell voltage patch-clamp technique. Administration of nicotine (1 μM) increased sIPSC frequency in 72% of identified 5-HT DRN neurons. This effect was not reproduced by the α4β2 nAChR agonist RJR-2403 and was not influenced by TTX (1 μM). It was mimicked by the selective agonist for α7 nAChR, PNU-282987, and exacerbated by the positive allosteric modulator of the same receptor, PNU-120596. The nicotine-induced increase in sIPSC frequency was independent on voltage-gated calcium channels and dependent on Ca(2+)-induced Ca(2+) release (CICR). These results demonstrate that nicotine increases the GABAergic input to most 5-HT DRN neurons, by activating α7 nAChRs and producing CICR in DRN GABAergic terminals.