Nicotinic effects on midbrain dopamine neurons : a dual mechanism of action

The neurons of the mesocorticolimbic dopamine system are located in the ventral tegmental area (VTA) in the mesencephalon and send their projections to forebrain structures, such as the prefrontal cortex, the amygdala and the nucleus accumbens. The dopamine system is important in regulating mood and motivation as well as cognitive function and appear to be primarily involved in drug dependence, since most drugs abused by man stimulate this system. Nicotine stimulates dopamine neurons both by increasing their single spike activity and burst activity. Burst activity is characterized by action potentials fired rapidly in short episodes with longer intervals in between and is particularly effective in releasing dopamine in the terminal areas and increasing the postsynatic activation of the protoonco gene c-fos. Nicotine-induced dopamine release in the nucleus accumbens seems to be more or less exclusively dependent on the neuronal activation elicited via stimulation of nicotinic acetylcholine receptors (nAChRs) in the VTA. Burst firing of dopamine neurons is controlled by excitatory amino acids (EAAs) acting at NMDA receptors in the VTA. In many parts of brain, nicotine, instead of exciting neurons directly, as first thought, has been shown to act preferentially indirectly, i.e. by facilitating the release of EAAs. Recent evidence also suggests that nAChRs of the [alpha]7* subtype may be involved in presynaptic facilitation of EAA release. Therefore, the specific aim of the present project has been to analyze the role of EAAs in the stimulatory effects of nicotine on the mesocorticolimbic dopamine system and, in particular, the role of [alpha]7* nAChRs in this regard. Using microdialysis we found that the effect of nicotine (0.5 mg/kg s.c. free base) on dopamine release in the nucleus accumbens was attenuated by concomitant infusion in the VTA of the NMDA receptor antagonist AP-5, but not the AMPA/kainate receptor antagonist CNQX Nicotine also increased the levels of EAAs in the VTA an effect that was prevented by concomitant infusion of the 0 selective nAChR antagonist methyllycaconitine (MLA). Local infusion of MLA in the VTA also blocked nicotine-induced dopamine release in the nucleus accumbens. These data indicate that nicotine may act presynaptically at [alpha]7* nAChRs to stimulate the release of EAAs. Presynaptic [alpha]7* nAChRs may reside on [alpha]7* originating in the prefrontal cortex, since they provide an important source of glutamatergic [alpha]7* for dopamine neurons in the VTA. Therefore, excitotoxic lesions were made in the prefrontal cortex and autoradiography binding with nAChR ligands was performed on sections of the VTA. [alpha]-bungarotoxin binding, which represents binding to 0 nAChRs, was reduced by 30% in lesioned animals compared to controls, providing anatomical support for our notion. The postsynaptic consequences in the nucleus accumbens and medial prefrontal cortex of the corresponding pharmacological manipulations within the VTA was measured with Fos-immunohistochemistry. Nicotine increased Fos-expression in the nucleus accumbens and the medial prefrontal cortex. nAChR and NMDA receptor blockade in the VTA by mecamylamine or AP-5 prevented the effect of systemic nicotine on Fosexpression in the nucleus accumbens but not in the medial prefrontal cortex. MLA (6.0 mg/kg i.p.) antagonized the effect of nicotine on Fos expression both in the nucleus accumbens and the prefrontal cortex. MLA also inhibited nicotine-induced burst firing in dopamine neurons without affecting nicotine's effect on firing rate. In contrast the [alpha]4[beta]2 selective antagonist dihydro-[beta]-erythroidine [DH[beta]E, 1.0 mg/kg s.c.) prevented nicotine's effect on firing rate but did nor antagonize the nicotine-induced increase in burst firing. Accordingly, DH[beta]E did not affect the nicotine-induced Fos-expression in the nucleus accumbens and the prefrontal cortex. Also, we found that an 0 agonist (AR-R-17779) increased burst firing in dopamine neurons without affecting single spike activity, whereas an [alpha]4[beta]4[beta2 agonist (A-85380) increased single spike firing without affecting burst activity. These data suggest that [alpha]7* nAChRs are primarily involved in the burst firing enhancing effects of nicotine, whereas [alpha]4[beta]2 nAChRs appear to be primarily responsible for the nicotine-induced increase in single spike firing. Consequently, nicotine may activate dopamine neurons by two separate but convergent mechanisms. These mechanisms are both likely to contribute to different aspects of nicotine dependence. Selective ligands for nAChRs subtypes may be less dependence producing and might, therefore be used therapeutically since nicotine exerts several potentially beneficial effects on a number of CNS functions

Antipsychotic-like effect by combined treatment with citalopram and WAY 100635: involvement ofthe 5-HT2C receptor.

Catalepsy occurs following high dopamine (DA) D2 blockade by typical antipsychotic drugs (APDs). We showed that a combination of a high dose of citalopram, a selective serotonin reuptake inhibitor (SSRI) and the selective 5-HT1A receptor antagonist WAY 100635 produces significant catalepsy in rats, similar to APDs. Here, we investigated the potential antipsychotic activity of lower doses of citalopram+WAY 100635, using the conditioned avoidance response (CAR) test. Cataleptogenic liability of the combination was evaluated with the catalepsy test. Citalopram and WAY 100635 in combination, but not when givenalone, prod uced a significant antipsychotic action in CAR without significant catalepsy, similar to the effect selective 5-HT2C receptor antagonist, SB , completely prevent 242084ed the citalopram/WAY 100635-induced suppression of CAR indicating an involvement of the 5-HT2C receptor. In summary, treatment with an SSRI/5-HT1A antagonist combination might prove beneficial in psychiatric disorders withpsychotic/depressive symptoms.

Adjunctive galantamine, but not donepezil, enhances the antipsychotic-like effect of raclopride in rats.

Acetylcholine (ACh) esterase inhibitors like galantamine and donepezil have been tested as adjunct treatment in schizophrenia. Although ACh esterase inhibition might confer some antipsychotic activity, the role of allosteric potentiation of nicotinic ACh receptors (nAChRs), which is an additional mechanism of galantamine, remains elusive. Therefore, the potential antipsychotic-like effects of galantamine and donepezil, respectively, alone, and in combination with the dopamine D2/3 receptor antagonist, raclopride, were tested in the conditioned avoidance response (CAR) test and extrapyramidal side-effect liability was assessed with the catalepsy test. Neither galantamine nor donepezil alone suppressed CAR selectively. Galantamine, but not donepezil, enhanced the raclopride-induced suppression of CAR, predicting augmentation of antipsychotic activity. In contrast to donepezil, galantamine did not increase catalepsy, alone or combined with raclopride. These data suggest that allosteric potentiation of nAChRs may mediate the antipsychotic-like effect of adjunctive galantamine and provide support for the development of α7 nAChR-selective allosteric potentiators for schizophrenia

The Importance of Ventral Hippocampal Dopamine and Norepinephrine in Recognition Memory

Dopaminergic neurons originating from the ventral tegmental area (VTA) and the locus coeruleus are innervating the ventral hippocampus and are thought to play an essential role for efficient cognitive function. Moreover, these VTA projections are hypothesized to be part of a functional loop, in which dopamine regulates memory storage. It is hypothesized that when a novel stimulus is encountered and recognized as novel, increased dopamine activity in the hippocampus induces long-term potentiation and long-term storage of memories. We here demonstrate the importance of increased release of dopamine and norepinephrinein the rat ventral hippocampus on recognition memory, using microdialysis combined to a modified novel object recognition test. We found that presenting rats to a novel object significantly increased dopamine and norepinephrine output in the ventral hippocampus. Two hours after introducing the first object, a second object (either novel or familiar) was placed in the same position as the first object. Presenting the animals to a second novel object significantly increased dopamine and norepinephrine release in the ventral hippocampus, compared to a familiar object. In conclusion, this study suggests that dopamine and norepinephrine output in the ventral hippocampus has a crucial role in recognition memory and signals novelty.De två sista författarna delar sistaförfattarskapet.</p

The enzyme scheme used in the detection of glutamate.

<p>The tip of the microelectrode consists of two pairs of platinum recording sites. One pair, the glutamate sensitive sites, were coated with a mixture of glutamate oxidase (GluOx), bovine serum albumin (BSA) and glutaraldehyde (0.125%). The remaining pair was coated only with BSA and glutaraldehyde and they served as control (background/sentinel) channels sensitive to the oxidation of endogenous molecules other than glutamate. <i>m</i>-phenylenediamine dihydrochloride (<i>m</i>-PD) was electropolymerized onto all sites of the microelectrode in order to reduce access of potential electroactive interferents, like ascorbic acid (AA) and catecholamines, to the platinum recording sites (Mitchell, 2004). Released glutamate is oxidized by GluOx at the glutamate-sensitive sites, generating α-ketoglutarate and H₂O₂. Since the microelectrode is maintained at a constant potential (+0.7 V versus an Ag/AgCl reference), the H₂O₂ reporting molecule is further oxidized, yielding two electrons. The resulting current is then amplified and recorded by a FAST-16 recording system (Quanteon, LLC, Nicholasville, KY, USA). Extracellular glutamate reaches the platinum surface of control sentinels (without GluOx) but no oxidation current is generated. Therefore, any current detected at these sites is due to electrochemically active interferents other than glutamate.</p

Basal glutamate on experimental day 1

<p>a) Basal glutamate levels expressed as a function of age range and their corresponding weights on the day of recording. Each circle represents an individual animal. b) The cortical levels (μM) of glutamate in animals in postnatal day (PND) 28–38 were more than three times higher compared to that of adults (3–5 months old). Glutamate levels decreased with increasing age. Significance was tested using two-way ANOVA followed by Bonferroni´s post-hoc comparison test. *p<0.05, ***p<0.001.</p

Effects of saline and ethanol injections on spontaneous glutamate transients in adolescent and adult animals.

<p>Experimental recordings were performed over two days: Experimental day 1 (saline injection, 6 ml/kg, unfilled bars) and experimental day 2 (ethanol injection, 1 g/kg, filled bars). Baseline recordings are represented as 0–1 hour on the <i>x</i> axis. Following baseline recordings, the animals received an intraperitoneal (i.p.) injection and recordings were continued for three hours. Every hour post injection is represented as 1–2 (first hour), 2–3 (second hour) and 3–4 (third hour) hour on the <i>x</i> axis. a) Experimental day 1 baseline recordings show that the transient frequency was higher in adolescent when compared with transients of adult animals (*p<0.05). The transient frequency was unaffected in any hour in both age groups post-saline injection compared to their respective baseline values. On experimental day 2, the transient frequency was higher in adolescent compared to adult animals (*p<0.05) during baseline recordings. An ethanol injection inhibited the transient frequency (*p<0.05) in the first hour and potentiated it in the third hour compared to the baseline values (*p<0.05) in adolescent animals. b) The averaged transient amplitude was higher in adolescent when compared to adult animals on both experimental day 1 and 2 during baseline recordings (p = 0.055 and p = 0.06, respectively). Averaged transient amplitudes were unaffected following saline injection in both age groups (p>0.05). Post-ethanol injection in adolescent animals, the average amplitudes decreased within the first hour and increased in the third hour. However, these changes in amplitude were not significant when compared to baseline values (p>0.05). In the adult animals, the average amplitude was unaffected following ethanol injection (p>0.05). c) We did not observe any significant difference between the averaged T80 value during baseline recordings of adolescent and adult animals (p>0.05). In adolescent animals, post-ethanol injection, in the third hour, the averaged T80 value decreased significantly (*p<0.05) when compared to the baseline values. Due to a low number of transients in adult animals, we could not perform any statistical analysis using T80 values. Also, note that the values missing or bars without SEM are due to the fact that the transient were absent in certain hours or, very low in frequency, respectively. All statistical comparisons were made using two-way ANOVA with Bonferroni´s comparison test and paired student´s t-test was used for analysis of T80. *p<0.05, **p<0.01.</p

Experimental design.

<p>The number of animals in each age group, the experimental days, data collection and how age groups were pooled for statistical analysis are provided above. PND: postnatal day, i.p: intraperitoneal injection.</p

Recordings from a freely moving animal.

<p>a) A representative trace showing spontaneous glutamate transients in an animal postnatal day 34, within the third hour post-ethanol injection. b) Representative picture of a single glutamate transient from the subtracted channel. Amplitude (μM) is represented by the vertical axis and time in seconds on horizontal axis. T80 represents the time in seconds from maximum peak rise to 80% decay of signal (a measure of glutamate clearance).</p

Values from <i>in vitro</i> calibrations of microelectrodes prior to implantation.

<p>Values from <i>in vitro</i> calibrations of microelectrodes prior to implantation.</p