ß4-Nicotinic Receptors Are Critically Involved in Reward-Related Behaviors and Self-Regulation of Nicotine Reinforcement.

Nicotine addiction, through smoking, is the principal cause of preventable mortality worldwide. Human genome-wide association studies have linked polymorphisms in the CHRNA5-CHRNA3-CHRNB4 gene cluster, coding for the α5, α3, and ß4 nicotinic acetylcholine receptor (nAChR) subunits, to nicotine addiction. ß4*nAChRs have been implicated in nicotine withdrawal, aversion, and reinforcement. Here we show that ß4*nAChRs also are involved in non-nicotine-mediated responses that may predispose to addiction-related behaviors. ß4 knock-out (KO) male mice show increased novelty-induced locomotor activity, lower baseline anxiety, and motivational deficits in operant conditioning for palatable food rewards and in reward-based Go/No-go tasks. To further explore reward deficits we used intracranial self-administration (ICSA) by directly injecting nicotine into the ventral tegmental area (VTA) in mice. We found that, at low nicotine doses, ß4KO self-administer less than wild-type (WT) mice. Conversely, at high nicotine doses, this was reversed and ß4KO self-administered more than WT mice, whereas ß4-overexpressing mice avoided nicotine injections. Viral expression of ß4 subunits in medial habenula (MHb), interpeduncular nucleus (IPN), and VTA of ß4KO mice revealed dose- and region-dependent differences: ß4*nAChRs in the VTA potentiated nicotine-mediated rewarding effects at all doses, whereas ß4*nAChRs in the MHb-IPN pathway, limited VTA-ICSA at high nicotine doses. Together, our findings indicate that the lack of functional ß4*nAChRs result in deficits in reward sensitivity including increased ICSA at high doses of nicotine that is restored by re-expression of ß4*nAChRs in the MHb-IPN. These data indicate that ß4 is a critical modulator of reward-related behaviors.SIGNIFICANCE STATEMENT Human genetic studies have provided strong evidence for a relationship between variants in the CHRNA5-CHRNA3-CHRNB4 gene cluster and nicotine addiction. Yet, little is known about the role of ß4 nicotinic acetylcholine receptor (nAChR) subunit encoded by this cluster. We investigated the implication of ß4*nAChRs in anxiety-, food reward- and nicotine reward-related behaviors. Deletion of the ß4 subunit gene resulted in an addiction-related phenotype characterized by low anxiety, high novelty-induced response, lack of sensitivity to palatable food rewards and increased intracranial nicotine self-administration at high doses. Lentiviral vector-induced re-expression of the ß4 subunit into either the MHb or IPN restored a "stop" signal on nicotine self-administration. These results suggest that ß4*nAChRs provide a promising novel drug target for smoking cessation.

Morphine Reward Promotes Cue-Sensitive Learning: Implication of Dorsal Striatal CREB Activity.

Different parallel neural circuits interact and may even compete to process and store information: whereas stimulus-response (S-R) learning critically depends on the dorsal striatum (DS), spatial memory relies on the hippocampus (HPC). Strikingly, despite its potential importance for our understanding of addictive behaviors, the impact of drug rewards on memory systems dynamics has not been extensively studied. Here, we assessed long-term effects of drug- vs food reinforcement on the subsequent use of S-R vs spatial learning strategies and their neural substrates. Mice were trained in a Y-maze cue-guided task, during which either food or morphine injections into the ventral tegmental area (VTA) were used as rewards. Although drug- and food-reinforced mice learned the Y-maze task equally well, drug-reinforced mice exhibited a preferential use of an S-R learning strategy when tested in a water-maze competition task designed to dissociate cue-based and spatial learning. This cognitive bias was associated with a persistent increase in the phosphorylated form of cAMP response element-binding protein phosphorylation (pCREB) within the DS, and a decrease of pCREB expression in the HPC. Pharmacological inhibition of striatal PKA pathway in drug-rewarded mice limited the morphine-induced increase in levels of pCREB in DS and restored a balanced use of spatial vs cue-based learning. Our findings suggest that drug (opiate) reward biases the engagement of separate memory systems toward a predominant use of the cue-dependent system via an increase in learning-related striatal pCREB activity. Persistent functional imbalance between striatal and hippocampal activity could contribute to the persistence of addictive behaviors, or counteract the efficiency of pharmacological or psychotherapeutic treatments.

Generation of specific antisera directed against D-amino acids: focus on the neuroanatomical distribution of D-glutamate and other D-amino acids.

This review updates the findings about the anatomical distribution (using immunohistochemical techniques) and possible functions of D-glutamate in the central nervous system of mammals, as well as compares the distribution of D-glutamate with the distribution of the most studied D-amino acids: D-serine and D-aspartate. The protocol used to obtain highly specific antisera directed against D-amino acids is also reported. Immunoreactivity for D-glutamate was found in dendrites and cell bodies, but not in nerve fibers. Perikarya containing D-glutamate were found in the mesencephalon and thalamus. The highest density of cell bodies was found in the dorsal raphe nucleus, the mesencephalic central grey matter, the superior colliculus, and in the subparafascicular thalamic nucleus. In comparison with the distribution of immunoreactive cell bodies containing D-serine or D-aspartate, the distribution of D-glutamate-immunoreactive perikarya is less widespread. Currently, the physiological actions mediated by D-glutamate in the brain are unknown but the restricted neuroanatomical distribution of this D-amino acid suggests that D-glutamate could be involved in very specific physiological mechanisms. In this sense, the possible functional roles of D-glutamate are discussed.

Detection of pantothenic acid-immunoreactive neurons in the rat lateral septal nucleus by a newly developed antibody.

INTRODUCTION: The available immunohistochemical techniques have documented restricted distribution of vitamins in the mammalian brain. The aim of the study was to develop a highly specific antiserum directed against pantothenic acid to explore the presence of this vitamin in the mammalian brain. MATERIAL AND METHODS: According to ELISA tests, the anti-pantothenic acid antiserum used showed a good affinity (10-8 M) and specificity. The antiserum was raised in rabbits. Using an indirect immunoperoxidase technique, the mapping of pantothenic acid-immunoreactive structures was carried out in the rat brain. RESULTS: Pantothenic acid-immunoreactive perikarya were exclusively found in the intermediate part of the lateral septal nucleus. These cells were generally small, round, fusiform or pyramidal and showed 2-3 long (50-100 µm) immunoreactive dendrites. Any immunoreactive axons containing pantothenic acid were detected. CONCLUSIONS: The very restricted anatomical distribution of the pantothenic acid suggests that this vitamin could be involved in some specific neurophysiological mechanisms.

Disrupting effect of drug-induced reward on spatial but not cue-guided learning: implication of the striatal protein kinase A/cAMP response element-binding protein pathway.

The multiple memory systems hypothesis posits that different neural circuits function in parallel and may compete for information processing and storage. For example, instrumental conditioning would depend on the striatum, whereas spatial memory may be mediated by a circuit centered on the hippocampus. However, the nature of the task itself is not sufficient to select durably one system over the other. In this study, we investigated the effects of natural and pharmacological rewards on the selection of a particular memory system during learning. We compared the effects of food- or drug-induced activation of the reward system on cue-guided versus spatial learning using a Y-maze discrimination task. Drug-induced reward severely impaired the acquisition of a spatial discrimination task but spared the cued version of the task. Immunohistochemical analysis of the phosphorylated form of the cAMP response element binding (CREB) protein and c-Fos expression induced by behavioral testing revealed that the spatial deficit was associated with a decrease of both markers within the hippocampus and the prefrontal cortex. In contrast, drug reward potentiated the cued learning-induced CREB phosphorylation within the dorsal striatum. Administration of the protein kinase A inhibitor 8-Bromo-adenosine-3',5'-cyclic monophosphorothioate Rp isomer (Rp-cAMPS) into the dorsal striatum before training completely reversed the drug-induced spatial deficit and restored CREB phosphorylation levels within the hippocampus and the prefrontal cortex. Therefore, drug-induced striatal hyperactivity may underlie the declarative memory deficit reported here. This mechanism could represent an important early step toward the development of addictive behaviors by promoting conditioning to the detriment of more flexible forms of memory.

Retinoic acid normalizes nuclear receptor mediated hypo-expression of proteins involved in beta-amyloid deposits in the cerebral cortex of vitamin A deprived rats.

Recent data have revealed that disruption of vitamin A signaling observed in Alzheimer's disease (AD) leads to a deposition of beta-amyloid (Abeta). The aim of this study was to precise the role of vitamin A and its nuclear receptors (RAR) in the processes leading to the Abeta deposits. Thus, the effect of vitamin A depletion and subsequent administration of retinoic acid (RA, the active metabolite of vitamin A) on the expression of RARbeta, and of proteins involved in amyloidogenic pathway, e.g., amyloid precursor protein (APP), beta-secretase enzyme (BACE), and APP carboxy-terminal fragment (APP-CTF) was examined in the whole brain, hippocampus, striatum, and cerebral cortex of rats. Rats fed a vitamin A-deprived diet for 13 weeks exhibited decreased amount of RARbeta, APP695, BACE, and of APP-CTF in the whole brain and in the cerebral cortex. Administration of RA is able to restore all expression. The results suggest that fine regulation of vitamin A mediated gene expression seems fundamental for the regulation of APP processing.

Age-related effects of ethanol consumption on triiodothyronine and retinoic acid nuclear receptors, neurogranin and neuromodulin expression levels in mouse brain.

The effects of ethanol consumption and ageing were investigated on the expression levels of retinoic acid (RA) and triiodothyronine (T3) nuclear receptors (RAR, RXR and TR) and of associated target genes involved in synaptic plasticity, neurogranin (RC3) and neuromodulin (GAP-43) in mice brain. For this purpose, C57BL/6 adult and aged mice were subjected to 5-month ethanol consumption and the mRNA expression of RAR, RXR, TR, RC3 and GAP-43 was measured using a real-time RT-PCR method. GAP-43 and RC3 protein levels also were measured by Western blot. Results showed that 12% ethanol consumption in adult mice (11 months) induced an increase in RARbeta, RXRbetagamma and TRalphabeta mRNA level in the brain with only an increase in RC3 expression. The same ethanol consumption in aged mice (22 months) reversed the age-related hypo-expression in brain RARbeta, TRalphabeta and target genes RC3 and GAP-43. Compared with our previous behavioral data showing that ethanol is able to partially suppress a selective age-related cognitive deficit, these results suggest that the ethanol-induced increase in RA and T3 nuclear receptors expression could be one of the mechanisms involved in the normalization of synaptic plasticity-associated gene expression altered in aging brain.

Triiodothyronine administration reverses vitamin A deficiency-related hypo-expression of retinoic acid and triiodothyronine nuclear receptors and of neurogranin in rat brain.

Recent studies have revealed that retinoids play an important role in the adult central nervous system and cognitive functions. Previous investigations in mice have shown that vitamin A deficiency (VAD) generates a hypo-expression of retinoic acid (RA, the active metabolite of vitamin A) receptors and of neurogranin (RC3, a neuronal protein involved in synaptic plasticity) and a concomitant selective behavioural impairment. Knowing that RC3 is both a triiodothyronine (T3) and a RA target gene, and in consideration of the relationships between the signalling pathways of retinoids and thyroid hormones, the involvement of T3 on RA signalling functionality in VAD was investigated. Thus, the effects of vitamin A depletion and subsequent administration with RA and/or T3 on the expression of RA nuclear receptors (RAR, RXR), T3 nuclear receptor (TR) and on RC3 in the brain were examined. Rats fed a vitamin A-deficient diet for 10 weeks exhibited a decreased expression of RAR, RXR and TR mRNA and of RC3 mRNA and proteins. RA administration to these vitamin A-deficient rats reversed only the RA hypo-signalling in the brain. Interestingly, T3 is able to restore its own brain signalling simultaneously with that of vitamin A and the hypo-expression of RC3. These results obtained in vivo revealed that one of the consequences of VAD is a dysfunction in the thyroid signalling pathway in the brain. This seems of crucial importance since the down regulation of RC3 observed in the depleted rats was corrected only by T3.