Elena Krstew

The GABAB receptor allosteric modulator CGP7930, like baclofen, reduces operant self-administration of ethanol in alcohol-preferring rats

Abstract GABA systems have been implicated as targets for ethanol at the cellular, molecular and behavioural level. The present study was designed to further examine the potential of the GABAB receptor as a target for regulating operant alcohol responding. Given that the prototypic agonist, baclofen, reduces the self-administration of alcohol, we hypothesized that the GABAB receptor allosteric modulator, CGP7930, might have similar actions but a reduced side-effect profile. In this context, inbred alcohol-preferring (iP) rats were trained to respond for 10% v/v ethanol in a fixed ratio paradigm; all drug testing was performed under an FR3 schedule. Both baclofen and CGP7930 independently reduced voluntary responding for 10% ethanol in a dose-related manner. Neither drug impacted upon responding for water. A combination of subthreshold doses of baclofen and CGP7930 was also able to reduce operant responding for ethanol, suggesting that CGP7930 is indeed acting to facilitate GABAB receptor-mediated signalling in this paradigm. These data demonstrate the potential of positive allosteric modulators of metabotropic GABAB receptors to regulate alcohol responding.

The orexin1 receptor antagonist SB-334867 dissociates the motivational properties of alcohol and sucrose in rats

A role for orexin A in mediating the primary and conditioned reinforcing effects of alcohol has been established. It is unclear however whether the contribution of orexins to alcohol reward occurs independently of effects on appetite and feeding, and whether orexins regulate the motivation to consume alcohol compared to other rewards. To examine this further here we investigate the effect of the orexin(1) receptor antagonist, SB-334867, on self-administration of alcohol (10% v/v) under both fixed (FR) and progressive ratio (PR) schedules of reinforcement, and whether this differs from the motivation to administer a natural food reward, sucrose (0.2-0.7% w/v) in alcohol preferring (iP) rats. SB-334867 treatment significantly reduced responding for both alcohol and sucrose under a FR3 schedule; however, at the same dose, reduced responding and break point for ethanol, but not sucrose, under a PR schedule. These findings for the first time implicate a role for orexins in the motivation to self-administer alcohol and suggest that this may occur independent of any generalized effect on appetitive drive.

Acamprosate Produces Its Anti-Relapse Effects Via Calcium

Alcoholism is one of the most prevalent neuropsychiatric diseases, having an enormous health and socioeconomic impact. Along with a few other medications, acamprosate (Campral—calcium-bis (N-acetylhomotaurinate)) is clinically used in many countries for relapse prevention. Although there is accumulated evidence suggesting that acamprosate interferes with the glutamate system, the molecular mode of action still remains undefined. Here we show that acamprosate does not interact with proposed glutamate receptor mechanisms. In particular, acamprosate does not interact with NMDA receptors or metabotropic glutamate receptor group I. In three different preclinical animal models of either excessive alcohol drinking, alcohol-seeking, or relapse-like drinking behavior, we demonstrate that N-acetylhomotaurinate by itself is not an active psychotropic molecule. Hence, the sodium salt of N-acetylhomotaurinate (i) is ineffective in alcohol-preferring rats to reduce operant responding for ethanol, (ii) is ineffective in alcohol-seeking rats in a cue-induced reinstatement paradigm, (iii) and is ineffective in rats with an alcohol deprivation effect. Surprisingly, calcium salts produce acamprosate-like effects in all three animal models. We conclude that calcium is the active moiety of acamprosate. Indeed, when translating these findings to the human situation, we found that patients with high plasma calcium levels due to acamprosate treatment showed better primary efficacy parameters such as time to relapse and cumulative abstinence. We conclude that N-acetylhomotaurinate is a biologically inactive molecule and that the effects of acamprosate described in more than 450 published original investigations and clinical trials and 1.5 million treated patients can possibly be attributed to calcium.

Presynaptic adenosine A2a receptors on soma and central terminals of rat vagal afferent neurons.

The dorsal vagal complex of the medulla oblongata is a key centre involved in the regulation of numerous autonomic functions, including cardiovascular control. Adenosine has been implicated as a potential neuromodulator of the baroreceptor reflex, and therefore the current study has investigated the presence and characteristics of adenosine receptors on rat vagal afferent neurons. In the nodose-vagal grease gap preparation, the adenosine A2a agonist CGS-21680 evoked a depolarisation only in the presence of the selective adenosine A1 antagonist PACPX. Autoradiography using [3H]NECA (4 nM) with suppression of A1 binding enabled the first visualisation of high affinity adenosine A2 receptors in the nucleus tractus solitarius (NTS). Unilateral nodose ganglionectomy resulted in over 90% reduction in binding in the lesioned (ipsilateral) NTS compared to a sham control. Furthermore, local administration of CGS-21680 increased evoked glutamate release in the NTS, as measured by in vivo microdialysis. These data suggest the presence of presynaptic adenosine A2a receptors on both the soma and central terminals of rat vagal afferent neurons, and thereby support the hypothesis that adenosine may have a modulatory role in the baroreceptor reflex.

Discrete cue‐conditioned alcohol‐seeking after protracted abstinence: pattern of neural activation and involvement of orexin1 receptors

BACKGROUND AND PURPOSE The enduring propensity for alcoholics to relapse even following years of abstinence presents a major hurdle for treatment. Here we report a model of relapse following protracted abstinence and investigate the pattern of neuronal activation following cue‐induced reinstatement and administration of the orexin1 receptor antagonist SB‐334867 in inbred alcohol‐preferring rats.

Relaxin-3/RXFP3 system regulates alcohol-seeking

Significance Relapse and hazardous drinking represent the most difficult clinical problems in treating patients with alcohol use disorders. Increasing our understanding of the brain circuits and chemicals that regulate alcohol intake and relapse offers the potential for more targeted therapeutic approaches to assist in relapse prevention. Using a rat model of alcohol use and alcohol-seeking, we provide the first evidence that a neuropeptide, namely relaxin-3, acts upon specific receptors (relaxin family peptide 3) within the brain to regulate alcohol self-administration and relapse-like behavior. In the case of relapse-like alcohol-seeking, this system appears particularly involved in stress-mediated relapse via actions within a brain region called the bed nucleus of the stria terminalis. Relapse and hazardous drinking represent the most difficult clinical problems in treating patients with alcohol use disorders. Using a rat model of alcohol use and alcohol-seeking, we demonstrated that central administration of peptide antagonists for relaxin family peptide 3 receptor (RXFP3), the cognate receptor for the highly conserved neuropeptide, relaxin-3, decreased self-administration of alcohol in a dose-related manner and attenuated cue- and stress-induced reinstatement following extinction. By comparison, RXFP3 antagonist treatment did not significantly attenuate self-administration or reinstatement of sucrose-seeking, suggesting a selective effect for alcohol. RXFP3 is densely expressed in the stress-responsive bed nucleus of the stria terminalis, and bilateral injections of RXFP3 antagonist into the bed nucleus of the stria terminalis significantly decreased self-administration and stress-induced reinstatement of alcohol, suggesting that this brain region may, at least in part, mediate the effects of RXFP3 antagonism. RXFP3 antagonist treatment had no effect on general ingestive behavior, activity, or procedural memory for lever pressing in the paradigms assessed. These data suggest that relaxin-3/RXFP3 signaling regulates alcohol intake and relapse-like behavior, adding to current knowledge of the brain chemistry of reward-seeking.

Assessing appetitive and consummatory phases of ethanol self-administration in C57BL/6J mice under operant conditions: regulation by mGlu5 receptor antagonism

RationaleThe development of mouse models of ethanol consumption and ethanol-seeking behavior is of particular importance in understanding the underlying mechanisms of drug abuse because these models can enable an analysis of an effect of specific genotype on drug-seeking behavior and the interaction of potential therapeutics with genotype. However, there are some limitations with present models, notably the inability to examine appetitive and consummatory behavior separately.Materials and methodsIn the present study, C57BL/6 mice were trained to self-administer 10% ethanol in a modified operant protocol that allowed a clear delineation of consummatory and appetitive phases. The utility of this procedure was confirmed with the use of the metabotropic glutamate 5 (mGlu5) receptor antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP).ResultsLimited-access consumption during the dark phase of the light–dark cycle with intermittent access (every second or third day) led to a high level of consumption by the mice. MTEP caused a dose-dependent decrease in both the consumption of ethanol and the appetitive response for ethanol. Furthermore, this effect was unrelated to any effect of MTEP on locomotor activity.ConclusionsThe model provides a useful paradigm for examining both the appetitive and consummatory phases of ethanol consumption in mice; furthermore, the data indicate mGlu5 receptors are involved in both phases.

Changes in function and ultrastructure of striatal dopaminergic terminals that regenerate following partial lesions of the SNpc

Following partial substantia nigra lesions, remaining dopaminergic neurones sprout, returning terminal density in the dorsal striatum to normal by 16 weeks. This suggests regeneration and maintenance of terminal density is regulated to release appropriate levels of dopamine. This study examined the structure and function of these reinnervated terminals, defining characteristics of dopamine uptake and release, density and affinity of the dopamine transporter (DAT) and ultrastructural morphology of dopamine terminals in the reinnervated dorsal striatum. Finally, rotational behaviour of animals in response to amphetamine was examined 4 and 16 weeks after substantia nigra pars compacta (SNpc) lesions. Dopamine transport was markedly reduced 16 weeks after lesioning along with reduced density and affinity of DAT. Rate of dopamine release and peak concentration, measured electrochemically, was similar in lesioned and control animals, while clearance was prolonged after lesioning. Ultrastructurally, terminals after lesioning were morphologically distinct, having increased bouton size, vesicle number and mitochondria, and more proximal contacts on post-synaptic cells. After 4 weeks, tendency to rotate in response to amphetamine was proportional to lesion size. By 16 weeks, rotational behaviour returned to near normal in animals where lesions were less than 70%, although some animals demonstrated unusual rotational patterns at the beginning and end of the amphetamine effect. Together, these changes indicate that sprouted terminals are well compensated for dopamine release but that transport mechanisms are functionally impaired. We discuss these results in terms of implications for dyskinesia and other behavioural states.

Functional GABAA receptors on rat vagal afferent neurones

1 In the present study, in vitro electrophysiology and receptor autoradiography were used to determine whether rat vagal afferent neurones possess γ‐aminobutyric acid (GABA)A receptors. 2 GABA (1‐100 μM) and isoguvacine (3–100 μm) caused a concentration‐dependent depolarization of the rat isolated nodose ganglion preparation at room temperature. When applied to the tissue 20 min before the agonist, SR95531 (3 μm) and bicuculline (3μm) caused a parallel shift to the right of the GABA and isoguvacine concentration‐response curves, yielding shifts of 81 fold and 117 fold for SR95531 and 4 fold and 12 fold for bicuculline, respectively. 3 Baclofen (10 nM‐100 μm) was unable to elicit a depolarization of the rat isolated nodose ganglion preparation at either room temperature or at 36°C, whilst 5‐aminovaleric acid (10 μm), a GABAB receptor antagonist, was unable to antagonize significantly the GABA‐induced depolarization at either room temperature or at 36°C. 4 [3H]‐SR95531 (7.2 nM), a GABAA receptor‐selective antagonist, bound topographically to sections of rat brainstem. Specific binding was highest in the medial nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus nerve (DMVN). Binding was also observed in certain medullary reticular nuclei, in particular the parvocellular reticular nucleus. 5 Unilateral nodose ganglionectomy caused a reduction in GABAA binding site density in the medial NTS from 93 ± 7 to 68 ± 6 d.p.m./mm2. This procedure also caused a reduction in GABAA binding site density in the side of the NTS contralateral to the lesion, from 151 ± 12 to 93 ± 7 d.p.m./mm2. Sham surgery had no effect on the binding of [3H]‐SR95531 in rat brainstem. 6 The present data provide evidence for the presence of GABAA receptors located on the soma and central terminals of rat vagal afferent neurones. Additionally, a population of GABAA receptors is evidenced postsynaptically in the rat NTS with respect to vagal afferent terminals. These data are discussed in relation to the functional pharmacology of GABA in this region of the NTS.

Actions of nitric oxide and expression of the mRNA encoding nitric oxide synthase in rat vagal afferent neurons

The present study has investigated whether nitric oxide (NO) is involved in neurotransmission of rat vagal afferent neurons. The diethylamine-NO complex (diethylamine-NO, 10-100 microM) and S-nitroso-N-acetylpenicillamine (3-100 microM) both elicited a concentration-dependent depolarisation of the isolated rat nodose ganglion preparation. Pre-treatment with 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 300 nM), 6-(phenylamino)-5,8-quinolinedione (LY83,583, 30 microM) and Methylene blue (100 microM) all caused a significant shift to the right in the concentration-response curve to diethylamine-NO. Incubation of rat nodose ganglion sections with a 35S-labeled antisense oligonucleotide to neuronal NO synthase resulted in visualisation of the mRNA encoding NO synthase over vagal afferent perikarya. The anatomical findings, therefore, suggest that a number of rat vagal afferent perikarya possess the ability to produce the enzyme required for the biosynthesis of NO. Collectively, these data suggest that NO may be functionally important as a neuromodulator of rat vagal afferent neurons.