A limited number of studies have used alternative approaches such as local infusions of small interfering RNAs or antisense morpholinos for the transient knockdown of HCRT [79C82], or local injection of shRNA-encoding viral vectors for the long-term knockdown of HCRT, HCRT-R1 or HCRT-R2 [83C87]. motivation for both cocaine and a Azasetron HCl highly palatable food reward (i.e., sweetened condensed milk; SCM) under a progressive ratio schedule of reinforcement, but did not alter responding for SCM under a fixed ratio schedule. Importantly, silencing did not affect food or water consumption, and had no consequence for general measures of arousal and stress reactivity. At the molecular level, chronic knockdown reduced the number of neurons expressing dynorphin (DYN), and to a smaller extent melanin-concentrating hormone (MCH), in the dorsal hypothalamus. These original findings support the hypothesis that HCRT neurotransmission promotes operant responding for both drug and nondrug rewards, preferentially under conditions requiring a high degree of motivation. Furthermore, the current study provides compelling evidence for the involvement of the HCRT system in cocaine self-administration also under low-effort conditions in rats allowed extended access, possibly via functional interactions with DYN and MCH signaling. Introduction Independently discovered in 1998 by de Lecea and Sakurai, hypocretin/orexin peptides hypocretin-1 (HCRT-1, also ORX-A) and hypocretin-2 (HCRT-2, also ORX-B), are derived from a common precursor, prepro-HCRT. These HCRT neuropeptides are synthesized in well-defined subregions of the dorsal hypothalamus: lateral hypothalamus proper, dorsomedial hypothalamus, and perifornical area [1C3]. HCRT projections are found throughout the brain and in regions known for their involvement in arousal, stress, and drug and nondrug reinforcement. These areas include, but are not limited to, the central amygdala, nucleus accumbens, ventral tegmental area, arcuate nucleus and paraventricular nucleus of the hypothalamus [2, 4C6]. HCRT neuropeptides bind two G-protein-coupled receptors, HCRT receptor 1 and receptor 2 (HCRT-R1 and HCRT-R2, respectively; [3]) that also are distributed widely throughout the brain [7, 8]. Accordingly, the HCRT system is involved in a multitude of physiological functions, such as the regulation of feeding, arousal, sleep/wake states, stress responses, energy homeostasis, and reward (for review, see refs. [9C12]). An abundant body of literature demonstrates the critical importance of HCRT transmission in the consumption and seeking of various reinforcers, including cocaine [13C24], nicotine [25C28], alcohol [29C35], heroin [36, 37], sucrose, and saccharin [35, 38, 39]. Importantly, HCRT receptor blockade generally does not influence drug self-administration under continuous, low-effort reinforcement, but rather blocks self-administration when the contingency of reinforcement requires higher levels of motivation to acquire the drug [40]. However, recent studies have indicated that acute blockade of HCRT signaling reduces not only the appetitive aspect but also the consummatory aspect of drug taking in dependent animals [20, 33, 36]. The aim of the present study was to investigate the role of HCRT neurotransmission in cocaine self-administration when rats are given extended access to the drug. The extended?access model produces a gradual escalation of cocaine self-administration and an increased motivation to obtain cocaine, along with increases in brain self-stimulation thresholds during withdrawal, stress reactivity, resistance to punishment and reinstatement susceptibility [41C50]. To examine the role of HCRT transmission in Azasetron HCl this model, expression was silenced long-term throughout the dorsal hypothalamus of adult rats using a short hairpin RNA (shRNA)-encoding adeno-associated viral (AAV) vector. To further investigate the function of HCRT in reward consumption and potentially dissociate its role in regulating motivation for drug versus food, the effects of silencing on self-administration of a highly palatable food reinforcer (sweetened condensed milk), as well as for regular food pellets and water, were assessed. In addition, given the modulatory part of HCRT in a multitude of behavioral and physiological functions, locomotor activity, anxiety-like behavior, and stress-induced analgesia and corticosterone response were measured to further evaluate the specificity of the behavioral effects of knockdown. Finally, molecular adaptations to Azasetron HCl long term reduction in HCRT signaling were investigated by analyzing the manifestation of prodynorphin (PDYN) and melanin-concentrating hormone (MCH), two neuropeptides also synthesized in the dorsal hypothalamus. Material and Methods Animals Forty adult male Wistar rats (Charles River,.HCRT-R1 antagonists display no or minimal effects about low-effort, FR1 responding for cocaine self-administration in animals allowed limited access to cocaine [14, 20, 21]. the molecular level, chronic knockdown reduced the number of neurons expressing dynorphin (DYN), and to a smaller degree melanin-concentrating hormone (MCH), in the dorsal hypothalamus. These unique findings support the hypothesis that HCRT neurotransmission promotes operant responding for both drug and nondrug rewards, preferentially under conditions requiring a high degree of motivation. Furthermore, the current study provides persuasive evidence for the involvement of the HCRT system in cocaine self-administration also under low-effort conditions in rats allowed prolonged access, probably via functional relationships with DYN and MCH signaling. Intro Independently found out in 1998 by de Lecea and Sakurai, hypocretin/orexin peptides hypocretin-1 (HCRT-1, also ORX-A) and hypocretin-2 (HCRT-2, also ORX-B), are derived from a common precursor, prepro-HCRT. These HCRT neuropeptides are synthesized in well-defined subregions of the dorsal hypothalamus: lateral hypothalamus appropriate, dorsomedial hypothalamus, and perifornical area [1C3]. HCRT projections are found throughout the mind and in areas known for his or her involvement in arousal, stress, and drug and nondrug encouragement. These areas include, but are not limited to, Mouse monoclonal to E7 the central amygdala, nucleus accumbens, ventral tegmental area, arcuate nucleus and paraventricular nucleus of the hypothalamus [2, 4C6]. HCRT neuropeptides bind two G-protein-coupled receptors, HCRT receptor 1 and receptor 2 (HCRT-R1 and HCRT-R2, respectively; [3]) that also are distributed widely throughout the mind [7, Azasetron HCl 8]. Accordingly, the HCRT system is involved in a multitude of physiological functions, such as the rules of feeding, arousal, sleep/wake states, stress reactions, energy homeostasis, and incentive (for review, observe refs. [9C12]). An abundant body of literature demonstrates the essential importance of HCRT transmission in the usage and seeking of various reinforcers, including cocaine [13C24], nicotine [25C28], alcohol [29C35], heroin [36, 37], sucrose, and saccharin [35, 38, 39]. Importantly, HCRT receptor blockade generally does not influence drug self-administration under continuous, low-effort reinforcement, but rather blocks self-administration when the contingency of encouragement requires higher levels of motivation to acquire the drug [40]. However, recent studies possess indicated that acute blockade of HCRT signaling reduces not only the appetitive element but also the consummatory aspect of drug taking in dependent animals [20, 33, 36]. The aim Azasetron HCl of the present study was to investigate the part of HCRT neurotransmission in cocaine self-administration when rats are given extended access to the drug. The extended?access model produces a progressive escalation of cocaine self-administration and an increased motivation to obtain cocaine, along with raises in mind self-stimulation thresholds during withdrawal, stress reactivity, resistance to consequence and reinstatement susceptibility [41C50]. To examine the part of HCRT transmission with this model, manifestation was silenced long-term throughout the dorsal hypothalamus of adult rats using a short hairpin RNA (shRNA)-encoding adeno-associated viral (AAV) vector. To further investigate the function of HCRT in praise consumption and potentially dissociate its part in regulating motivation for drug versus food, the effects of silencing on self-administration of a highly palatable food reinforcer (sweetened condensed milk), as well as for regular food pellets and water, were assessed. In addition, given the modulatory part of HCRT in a multitude of behavioral and physiological functions, locomotor activity, anxiety-like behavior, and stress-induced analgesia and corticosterone response were measured to further evaluate the specificity of the behavioral effects of knockdown. Finally, molecular adaptations to long term reduction in HCRT signaling were investigated by analyzing the manifestation of prodynorphin (PDYN) and melanin-concentrating hormone (MCH), two neuropeptides also synthesized in the dorsal hypothalamus. Material and Methods Animals Forty adult male Wistar rats (Charles River, Raleigh, NC), weighing between 225C275?g at the beginning of the experiments, were housed in groups of 2C3 per cage inside a.
