Jörgen A. Engel

Centrally administered neuropeptide Y (NPY) produces anxiolytic-like effects in animal anxiety models

Effects of intracerebroventircular (ICV), neuropeptide Y (NPY) (0.2–5.0 nmol) and its C-terminal 13–36 amino acid (AA) fragment (0.4–2.0 nmol) have been examined with respect to anxiolytic properties in two rat anxiety models, Montgomerys conflict test (MT), and Vogels drinking conflict test (VT). In the MT, 1.0 and 5.0 nmol NPY abolished the normal preference for the closed arms of the maze. At 5.0 nmol, the total number of entries made into both closed and open arms was decreased by 50%. In the VT, both 0.2 and 1.0 nmol NPY markedly increased the number of shocks accepted. The effect of 5.0 nmol NPY was less pronounced. In control experiments, NPY (0.2 nmol) did not affect pain sensitivity or thirst. Pretreatment with the selective alpha2-adrenergic receptor antagonist idazoxan, at a dose which by itself did not affect behaviour (2.0 mg/kg), antagonized the effect of 1.0 nmol NPY in the VT. NPY 13-36 was without significant effect in both models. The results suggest that NPY exerts anxiolytic-like effects, and that these effects are mediated through an interaction with noradrenergic systems. Higher doses of NPY produce sedation and ataxia, which decrease overall activity in the MT, and interfere with the ability fully to express behaviourally the anxiolytic-like effect in the VT. The findings are discussed in relation to the noradrenaline hypothesis of anxiety, and to observations indicating involvement of NPY in the pathophysiology of major depression.

Ghrelin administration into tegmental areas stimulates locomotor activity and increases extracellular concentration of dopamine in the nucleus accumbens.

Ghrelin stimulates appetite, increases food intake and causes adiposity by mechanisms that include direct actions on the brain. Previously, we showed that intracerebroventricular administration of ghrelin has stimulatory and dopamine‐enhancing properties. These effects of ghrelin are mediated via central nicotine receptors, suggesting that ghrelin can activate the acetylcholine–dopamine reward link. This reward link consists of cholinergic input from the laterodorsal tegmental area (LDTg) to the mesolimbic dopamine system that originates in the ventral tegmental area (VTA) and projects to the nucleus accumbens. Given that growth hormone secretagogue receptors (GHSR‐1A) are expressed in the VTA and LDTg, brain areas involved in reward, the present series of experiments were undertaken to examine the hypothesis that these regions may mediate the stimulatory and dopamine‐enhancing effects of ghrelin, by means of locomotor activity and in vivo microdialysis in freely moving mice. We found that local administration of ghrelin into the VTA (1 µg in 1 µl) induced an increase in locomotor activity and in the extracellular concentration of accumbal dopamine. In addition, local administration of ghrelin into the LDTg (1 µg in 1 µl) caused a locomotor stimulation and an increase in the extracellular levels of accumbal dopamine. Taken together, this indicates that ghrelin might, via activation of GHSR‐1A in the VTA and LDTg, stimulate the acetylcholine–dopamine reward link, implicating that ghrelin is a part of the neurochemical overlap between the reward systems and those that regulate energy balance.

Ghrelin stimulates locomotor activity and accumbal dopamine-overflow via central cholinergic systems in mice: implications for its involvement in brain reward.

It is becoming increasingly apparent that there is a degree of neurochemical overlap between the reward systems and those regulating energy balance. We therefore investigated whether ghrelin, a stomach‐derived and centrally derived orexigenic peptide, might act on the reward systems. Central ghrelin administration (1 µg/µL, to the third ventricle) induced an acute increase in locomotor activity as well as dopamine‐overflow in the nucleus accumbens, suggesting that ghrelin can activate the mesoaccumbal dopamine system originating in the ventral tegmental area, a system associated with reward and motivated behaviour. The cholinergic afferents to the ventral tegmental area have been implicated in natural reward and in regulating mesoaccumbal dopamine neurons. The possibility that nicotinic receptors are involved in mediating the stimulatory and dopamine‐enhancing effects of ghrelin is supported by the findings that peripheral injection of the unselective nicotinic antagonist mecamylamine (2.0 mg/kg) blocked these ghrelin‐induced effects. Tentatively, ghrelin may, via activation of the acetylcholine–dopamine reward link, increase the incentive values of signals associated with motivated behaviours of importance for survival such as feeding behaviour. It will be important to discover whether this has therapeutic implications for compulsive addictive behaviours, such as eating behaviour disorders and drug dependence.

Requirement of central ghrelin signaling for alcohol reward.

The stomach-derived hormone ghrelin interacts with key CNS circuits regulating energy balance and body weight. Here we provide evidence that the central ghrelin signaling system is required for alcohol reward. Central ghrelin administration (to brain ventricles or to tegmental areas involved in reward) increased alcohol intake in a 2-bottle (alcohol/water) free choice limited access paradigm in mice. By contrast, central or peripheral administration of ghrelin receptor (GHS-R1A) antagonists suppressed alcohol intake in this model. Alcohol-induced locomotor stimulation, accumbal dopamine release and conditioned place preference were abolished in models of suppressed central ghrelin signaling: GHS-R1A knockout mice and mice treated with 2 different GHS-R1A antagonists. Thus, central ghrelin signaling, via GHS-R1A, not only stimulates the reward system, but is also required for stimulation of that system by alcohol. Our data suggest that central ghrelin signaling constitutes a potential target for treatment of alcohol-related disorders.

Effects of GABAergic agonists and antagonists on various ethanol-induced behavioral changes

The interaction between ethanol and various GABAergic drugs (muscimol, bicuculline, picrotoxin) with regard to their effects on locomotor activity, drug-induced sleep, body temperature, and convulsions was studied. It was demonstrated that the GABA receptor agonist muscimol potentiated the sedative properties of ethanol, while the opposite effect, a reduction of ethanol-produced sedation, was seen upon administration of the GABA receptor blocking agent picrotoxin. Consequently, the results from the present series of experiments indicate that ethanol enhances central GABAergic activity.

Anticonflict effect of the putative serotonin receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT)

The putative 5-HT agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT) produced in rats an increase in the number of shocks accepted in a modified Vogels conflict test design. Subchronic pretreatment with p-chlorophenylalanine (PCPA) similarly caused release of the punished behavior. This anticonflict effect of PCPA was antagonized by both 5-hydrotryptophan and 8-OH-DPAT. Thus in naive animals 8-OH-DPAT exerting anticonflict effects acted like a 5-HT antagonist, whereas in subchronically PCPA-pretreated animals with presumably supersensitive 5-HT receptors, 8-OH-DPAT decreasing the number of accepted shocks acted like a 5-HT agonist.

Voluntary ethanol intake in the rat: effects of nicotinic acetylcholine receptor blockade or subchronic nicotine treatment.

It has been suggested that the mesolimbic dopamine activating and the reinforcing properties of ethanol involve activation of central nicotinic acetylcholine receptors. To test this hypothesis, the effects of two nicotinic receptor antagonists and of subchronic nicotine treatment on voluntary ethanol consumption (ethanol 6% v/v or water) were studied in ethanol low-, medium- or high-preferring Wistar rats. After systemic mecamylamine (2 mg/kg) but not hexamethonium (0 mg/kg) high- but not low-preferring rats decreased their ethanol intake but, however, not their ethanol preference. When subchronically exposed to nicotine (0.35 mg/kg, s.c. daily) medium-preferring rats markedly increased their ethanol intake and preference. This effect lasted for more than 1 week after interrupting nicotine administration. Ethanol intake levels did not correlate with locomotor activity scores after nicotine challenge (0.35 mg/kg, s.c.) or with exploratory locomotor activity. However, exploratory locomotor activity correlated with locomotor activity scores both after nicotine (0.35 mg/kg, s.c.) and ethanol (0.125 g/kg i.p.) challenge. Dopamine release, as indicated by accumulation of 3-methoxytyramine after monoamine oxidase inhibition, was increased in the limbic forebrain (including the nucleus accumbens, the olfactory tubercles, the amygdala and the septum) after acute nicotine (0.35 mg/kg s.c.) or ethanol (2.5 g/kg i.p.) in animals subchronically exposed to nicotine compared to subchronically vehicle-treated controls. The present results further implicate central nicotinic receptors in the molecular events mediating the reinforcing properties of ethanol, and suggest that subchronic nicotine enhances the responsiveness of mesolimbic dopamine neurons both to nicotine and to ethanol. Clinical implications are discussed.

Ghrelin increases intake of rewarding food in rodents.

We investigated whether ghrelin action at the level of the ventral tegmental area (VTA), a key node in the mesolimbic reward system, is important for the rewarding and motivational aspects of the consumption of rewarding/palatable food. Mice with a disrupted gene encoding the ghrelin receptor (GHS‐R1A) and rats treated peripherally with a GHS‐R1A antagonist both show suppressed intake of rewarding food in a free choice (chow/rewarding food) paradigm. Moreover, accumbal dopamine release induced by rewarding food was absent in GHS‐R1A knockout mice. Acute bilateral intra‐VTA administration of ghrelin increased 1‐hour consumption of rewarding food but not standard chow. In comparison with sham rats, VTA‐lesioned rats had normal intracerebroventricular ghrelin‐induced chow intake, although both intake of and time spent exploring rewarding food was decreased. Finally, the ability of rewarding food to condition a place preference was suppressed by the GHS‐R1A antagonist in rats. Our data support the hypothesis that central ghrelin signaling at the level of the VTA is important for the incentive value of rewarding food.

Nicotinic mechanisms involved in the dopamine activating and reinforcing properties of ethanol

Ethanol shares with all major dependence producing drugs the ability to activate brain mesocorticolimbic dopamine neurons, an important part of the brain reward systems. This dopamine activation may be involved in mediating the positive reinforcing effects of ethanol. The mechanisms of action of ethanol in its activation of this dopamine system remain, however, to be elucidated. A selective pharmacological interference with these mechanisms may offer a possibility to reduce the reinforcing properties of ethanol without simultaneously interfering with the reinforcing properties of natural rewards. Ethanol has been shown to directly influence the function of various ligand-gated ion-channels. Several of these are located on or nearby mesocorticolimbic dopamine neurons. One such receptor is the nicotinic acetylcholine receptor (nAChR). The present article reviews a series of investigations aimed at investigating whether nAChRs are involved in the dopamine activating and reinforcing properties of ethanol. To this end acute and chronic behavioral and neurochemical experiments were performed in mice and rats. The results obtained indicate that central nAChRs in the ventral tegmental area are involved in mediating the mesolimbic dopamine activating and reinforcing effects of ethanol. Furthermore, the ethanol-induced activation of these receptors is probably indirect, subsequent to a primary interference of ethanol in the nucleus accumbens. Moreover, subchronic nicotine treatment enhances the reinforcing and dopamine activating properties of ethanol. This long-term effect may, however, derive from autonomic adaptations in response to intermittent blockade of peripheral nAChRs (rather than from intermittent stimulation of central receptors), and appears to be associated with development of a disinhibitory behavior that could involve also other neurotransmitters, e.g. serotonin. Taken together, these findings could provide a neurobiological explanation to the often observed co-abuse of nicotine and ethanol in man. Furthermore, since the behavioral models applied previously have predicted therapeutic drug effects in the clinic, the results suggest that selective blockade of the ventral tegmental nAChRs that are involved in the above effects may provide a new pharmacological alternative in the treatment of alcoholism.

Neurochemical and behavioral studies on ethanol and nicotine interactions

The most commonly abused drugs, alcohol and nicotine, are likely also the most costly drugs in terms of health and societal costs. A large body of evidence from epidemiological studies indicate that smoking and alcohol-intake are positively correlated. The mesocorticolimbic dopamine system has been implicated in mediating some of the reinforcing effects of ethanol, however, the mechanism(s) of action remains to be elucidated; consideration as to ethanols ability to interact with ligand-gated ion channels should be considered. Accumulating evidence from electrophysiological, pharmacological and neurochemical studies suggest that ethanol may interact with the nicotinic acetylcholine receptor (nAChR). Thus, it has been shown that the ethanol-induced stimulation of the mesolimbic dopamine system and of locomotor activity as well as ethanol intake and preference in rodents may involve central nicotinic acetylcholine receptors. Additionally, data has been presented that nAChRs located in the ventral tegmental area may be of particular importance for these effects of ethanol. Studies aimed at defining the nAChR subpopulation(s) involved in mediating ethanol-induced locomotor stimulation and accumbal dopamine overflow as well as ethanol-intake have revealed that alpha(3)beta(2) or alpha(6) (using alpha-Conotoxin MII) but not alpha(4)beta(2) (using dihydro-beta-erythroidine) or alpha(7) (using methyllycaconitine), could represent targets for developing new drugs in the treatment of alcoholism. These results do not allow any conclusion as to whether the involvement nAChRs in mediating the effects of ethanol is direct and/or indirect. With regard to an indirect effect, evidence has accumulated indicating that the cholinergic excitatory input to the dopaminergic neurons in the ventral tegmental area may be an important part of the neuronal circuits mediating natural as well as drug-rewarded behavior. The possibility may thus be considered that ethanol activates the cholinergic afferents causing a release of acetylcholine in the ventral tegemental area leading to a stimulation of nAChRs and thereby excite the mesocorticolimbic dopamine system.