Mechanisms of stimulatory effects of mecamylamine on the dorsal raphe neurons.

Previous studies showed that mecamylamine a noncompetitive and nonspecific blocker of nicotinic acetylcholine receptors (nAChRs), stimulates the activity of the dorsal raphe nucleus (DRN) serotonergic neurons and DRN serotonin (5-HT) release. In the present study, the mechanisms involved in these mecamylamine-induced effects were examined using electrophysiology and calcium-imaging studies, both performed in Wistar rat midbrain slices. Mecamylamine (0.5-9 µM), bath administered, increased the firing frequency of identified 5-HT DRN neurons by a maximum of 5% at 3 µM. This effect was accompanied by a 112 % increase in the frequency of spontaneous excitatory postsynaptic currents of 5-HT DRN neurons. It was blocked by the AMPA/kainate receptor blocker CNQX (10 µM) and by the specific α4ß2 nAChRs blocker dihydro-ß-erythroidine (100 nM) but was not affected by tetrodotoxin (TTX, 500 nM). Simultaneously, mecamylamine produced a 58 % decrease in the frequency of GABAergic spontaneous inhibitory postsynaptic currents, an effect that was not influenced by TTX. Calcium-imaging studies support the results obtained with the electrophysiological studies by showing that mecamylamine (3 µM) increases the activity of a cell population located in the midline of the DRN, which was sensitive to the inhibitory effects of 8-OH-DPAT, an agonist at 5-HT1A receptors. It is assumed that mecamylamine, in low concentrations, acts as an agonist of α4ß2 nAChRs present on the glutamatergic DRN terminals, thus increasing intra-raphe glutamate release. This stimulatory effect is reinforced by the decrease in DRN GABA release, which is dependent on the mecamylamine-induced blockade of α7 nAChRs located on DRN GABAergic terminals. We hypothesize that at least a part of mecamylamine antidepressant effects described in animal models of depression are mediated by an increase in DRN 5-HT release.

Nicotine Increases Spontaneous Glutamate Release in the Rostromedial Tegmental Nucleus.

The rostromedial tegmental nucleus (RMTg) is a bilateral structure localized in the brainstem and comprise of mainly GABAergic neurons. One of the main functions of the RMTg is to regulate the activity of dopamine neurons of the mesoaccumbens pathway. Therefore, the RMTg has been proposed as a modulator of the reward system and adaptive behaviors associated to reward learning. The RMTg receives an important glutamatergic input from the lateral habenula. Also, it receives cholinergic inputs from the laterodorsal and pedunculopontine tegmental nuclei. Previously, it was reported that nicotine increases glutamate release, evoked by electric stimulation, in the RMTg nucleus. However, the mechanisms by which nicotine induces this effect were not explored. In the present work, we performed electrophysiological experiments in brainstem slices to study the effect of nicotine on spontaneous excitatory postsynaptic currents recorded from immunocytochemically identified RMTg neurons. Also, we used calcium imaging techniques to explore the effects of nicotine on multiple RMTg neurons simultaneously. We found that nicotine promotes the persistent release of glutamate through the activation of α7 nicotinic acetylcholine receptors present on glutamatergic afferents and by a mechanism involving calcium release from intracellular stores. Through these mechanisms, nicotine increases the excitability and synchronizes the activity of RMTg neurons. Our results suggest that the RMTg nucleus mediates the noxious effects of the nicotine, and it could be a potential therapeutic target against tobacco addiction.

Insulin Regulates GABAA Receptor-Mediated Tonic Currents in the Prefrontal Cortex.

Recent studies, have shown that insulin increases extrasynaptic GABAA receptor-mediated currents in the hippocampus, causing alterations of neuronal excitability. The prefrontal cortex (PFC) is another brain area which is involved in cognition functions and expresses insulin receptors. Here, we used electrophysiological, molecular, and immunocytochemical techniques to examine the effect of insulin on the extrasynaptic GABAA receptor-mediated tonic currents in brain slices. We found that insulin (20-500 nM) increases GABAA-mediated tonic currents. Our results suggest that insulin promotes the trafficking of extrasynaptic GABAA receptors from the cytoplasm to the cell membrane. Western blot analysis and immunocytochemistry showed that PFC extrasynaptic GABAA receptors contain α-5 and δ subunits. Insulin effect on tonic currents decreased the firing rate and neuronal excitability in layer 5-6 PFC cells. These effects of insulin were dependent on the activation of the PI3K enzyme, a key mediator of the insulin response within the brain. Taken together, these results suggest that insulin modulation of the GABAA-mediated tonic currents can modify the activity of neural circuits within the PFC. These actions could help to explain the alterations of cognitive processes associated with changes in insulin signaling.

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.

Nicotinic modulation of serotonergic activity in the dorsal raphe nucleus.

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.

Flattening plasma corticosterone levels increases the prevalence of serotonergic dorsal raphe neurons inhibitory responses to nicotine in adrenalectomised rats.

Major depression is characterized by a diminished activity of the brain serotonergic system as well as by the flattening of plasma cortisol levels. Nicotine improves mood in patients with major depression and in experimentally depressed animals by increasing brain serotonin (5-HT), noradrenaline and dopamine levels. The present study was directed to determine if flattening plasma glucocorticoid levels changes nicotine's stimulatory effects upon 5-HT DRN neurons. The experiments were performed in brain slices obtained from rats previously (14 days) adrenalectomised and implanted subcutaneously with one pellet containing 75mg of corticosterone (Adx+CSR rats). Whole cell voltage and current clamp techniques were used to study the activity of immunocitochemically identified 5-HT DRN neurons. Administration of nicotine (1µM) in sham-operated animals produced stimulatory effects in all 5-HT DRN neurons studied. In Adx+CSR rats however, nicotine inhibited 75% of 5-HT DRN neurons and increased the potassium-dependent inward rectifying current. The inhibitory effect of nicotine upon 5-HT DRN neurons was dependent on serotonin release inside the DRN, since it was converted into a stimulatory response by the selective antagonist of 5-HT1A receptors N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridyl)cyclohexanecarboxamide (WAY100635, 25nM). Adx+CSR rats also presented an increased function of 5-HT1A autoreceptors, since, in these rats, serotonin (1-10µM) produced a higher increase in the potassium dependent inward rectifying current in comparison with sham-operated animals. Serotonin release inside DRN was mediated by α4ß2 nicotinic acetylcholine receptors since the selective antagonist of these receptors dihydro-ß-erytroidine hydrobromide (DHßE, 100nM) blocked the inhibitory effects of nicotine 5-HT DRN neurons. These data indicate that, in the experimental model of adrenalectomised rats implanted with corticosterone pellets, nicotine increases the function of 5-HT1A receptors of 5-HT DRN neurons.

Presynaptic α4ß2 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 α4ß2 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 α4ß2 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 alpha7 nAChR antagonist methyllycaconitine (MLA) or the selective alpha4beta2 nAChR antagonist dihydro-beta-eritroidine (DHbetaE). Simultaneous addition of MLA and DHbetaE 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 DHbetaE; 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.

Design and synthesis of novel biologically active thrombin receptor non-peptide mimetics based on the pharmacophoric cluster Phe/Arg/NH2 of the Ser42-Phe-Leu-Leu-Arg46 motif sequence: platelet aggregation and relaxant activities.

The identification of the thrombin receptor has promoted the interest for the development of new therapeutic agents capable of selectively inhibiting unwanted biological effects of thrombin on various cell types. In this study we have designed and synthesized two series of new thrombin receptor antagonists based on the thrombin receptor motif sequence S42FLLR46, one possessing two (Phe/Arg) pharmacophoric groups and the other possessing three (Phe/Arg/NH2). N-(6-Guanidohexanoyl)-N'-(phenylacetyl)piperazine (1), N-(phenylacetyl)-4-(6-guanidohexanoylamidomethyl)piperidine (2), and N-(phenylacetyl)-3-(6-guanidohexanoylamido)pyrrolidine (3) (group A) carry the two pharmacophoric side chains of Phe and Arg residues incorporated on three different templates (piperazine, 4-aminomethylpiperidine, and 3-aminopyrrolidine). Compounds with three pharmacophoric groups (group B) were built similarly to group A using the same templates with the addition of an extra methylamino group leading to (S)-N-(6-guanidohexanoyl)-N'-(2-amino-3-phenylpropionyl)piperazine (4), (S)-N-(2-amino-3-phenylpropionyl)-4-(6-guanidohexanoylamidomethyl)piperidine (5), and (S)-N-(2-amino-3-phenylpropionyl)-3-(6-guanidohexanoylamido)pyrrolidine (6). Compounds were able to inhibit thrombin-induced human platelet activation even at low concentrations. In particular, among compounds in group A, compound 3 was found to be the most powerful thrombin receptor activation inhibitor, showing an IC50 of approximately 0.11 mM on platelet aggregation assay. Among compounds in group B, compound 4 was the most powerful to inhibit thrombin-induced platelet aggregation, showing an IC50 of approximately 0.09 mM. All compounds were also found to act as agonists in the rat aorta relaxation assay. Interestingly, the order of potency of these compounds as agonists of the endothelial thrombin receptor was the inverse of the order of potency of the same compounds as antagonists of the platelet thrombin receptor. Such compounds that are causing vasodilation while simultaneously inhibiting platelet aggregation would be very useful in preventing the installation of atherosclerotic lesions and deserve further investigation as potential drugs for treating cardiovascular diseases. The above findings coupled with computational analysis molecular dynamics experiments support also our hypothesis that a cluster of phenyl, guanidino, and amino groups is responsible for thrombin receptor triggering and activation.

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 nicotine's 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 nicotine's 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 nicotine's 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.

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.