Chronic Nicotine Treatment Impacts the Regulation of Opioid and Non-opioid Peptides in the Rat Dorsal Striatum

The chronic use of nicotine, the main psychoactive ingredient of tobacco smoking, alters diverse physiological processes and consequently generates physical dependence. To understand the impact of chronic nicotine on neuropeptides, which are potential molecules associated with dependence, we conducted qualitative and quantitative neuropeptidomics on the rat dorsal striatum, an important brain region implicated in the preoccupation/craving phase of drug dependence. We used extensive LC-FT-MS/MS analyses for neuropeptide identification and LC-FT-MS in conjunction with stable isotope addition for relative quantification. The treatment with chronic nicotine for 3 months led to moderate changes in the levels of endogenous dorsal striatum peptides. Five enkephalin opioid peptides were up-regulated, although no change was observed for dynorphin peptides. Specially, nicotine altered levels of nine non-opioid peptides derived from precursors, including somatostatin and cerebellin, which potentially modulate neurotransmitter release and energy metabolism. This broad but selective impact on the multiple peptidergic systems suggests that apart from the opioid peptides, several other peptidergic systems are involved in the preoccupation/craving phase of drug dependence. Our finding permits future evaluation of the neurochemical circuits modulated by chronic nicotine exposure and provides a number of novel molecules that could serve as potential therapeutic targets for treating drug dependence.Nicotine is the main psychoactive ingredient of tobacco (1). By acting on the nicotinic acetylcholine receptors located in diverse brain areas, nicotine generates psychoactive effects such as euphoria, reduced stress, increased energy, and enhanced cognitive functions (2). Chronic nicotine use alters various aspects of neurochemical transmission and has a strong impact on diverse physiological processes (2), resulting in drug-seeking and drug-taking behaviors for normal smokers and for a considerable number of patients suffering from schizophrenia and Alzheimer disease, who use nicotine for self-medication (3, 4). The dorsal striatum (DS)¹ is one of the key brain regions that has been associated with neural regulation during chronic nicotine exposure (5). In particular, the DS is involved in habit formation during the preoccupation/craving (later) phase of nicotine dependence characterized by compulsive drug-taking (6). Behavioral changes associated with nicotine dependence have been linked to small molecule neurotransmitter systems, including the glutamate and dopamine system in the DS (7). The DS is also known to contain diverse neuropeptides, many of which are probably critical mediators of physiological processes that are associated with nicotine, such as the regulation of reinforcement and energy metabolism. However, neuropeptides have not been extensively investigated in the DS during long periods of nicotine administration.Immunoassay studies have shown that neuropeptides, including substance P, neuropeptide Y, and opioid peptides, including the enkephalins, are expressed by inhibitory neurons (8), which make up a large majority of the neurons in the DS (9). Many of these inhibitory GABAergic neurons express nicotinic cholinergic receptors (10), suggesting that nicotine administration may regulate their activity, leading to variations in the release of neuropeptides, as well as the inhibitory neurotransmitter GABA. Previous investigations of peptide regulation during chronic nicotine administration in the striatum have exclusively focused on the class of opioid peptides, which are thought to play an important role in the control of diverse physiological processes, including reward processing, nociception, and regulation of emotions (11, 12). Available studies have focused on the analysis of three opioid peptides, their precursors, or receptors as follows: met-enkephalin, dynorphin, and β-endorphin, using conventional techniques like immunoassays (13, 14). There is considerable variability in reported changes of peptide levels in the striatum during chronic nicotine administration. For example, when animals are treated with 1 mg/kg free base nicotine (daily for 14 days), met-enkephalin increased in the striatum (15). By contrast, met-enkephalin is reduced in the striatum when rats are treated with 0.3 mg/kg nicotine (three times/day for 14 days) (16). A number of factors might contribute to this observed variability, including the exact dosing, daily frequency, time span of administration, and delivery method of nicotine. Furthermore, as individual studies have each so far generally examined a single opioid peptide, there is currently little reliable information about peptide co-regulation, even for these well studied opioid peptides. In addition to these opioid peptides, the DS expresses peptides from other peptide families, which are also potential targets under the regulation of chronic nicotine treatment. So far, however, there is no information available about changes of these non-opioid peptides during chronic nicotine administration.In this study, our aim was to use a neuropeptidomics approach (17) to provide a comprehensive characterization of dorsal striatal neuropeptides after long term nicotine chronic treatment in adult rats using oral administration. The main advantage of this approach is that it allows the simultaneous monitoring of many peptides from the same brain tissue derived from a single drug protocol. We used a combination of a robust sample preparation method (18), high accuracy LC-MS analysis (19, 20), and the use of multiple synthetic internal standards (21) to compare peptide levels in the DS between chronic nicotine and control animals. Our peptidome analysis determined 14 peptides exhibiting significant changes following chronic nicotine administration. Among these peptides were members of the opioid family that had previously been associated with nicotine dependence, as well as a number of newly identified peptides, including members of the secretogranin, cholecystokinin, and somatostatin families. This greatly expands the present scope of peptide involvement in drug dependence in the dorsal striatum.