Shridhar Narayanan

Endogenous enkephalins, not endorphins, modulate basal hedonic state in mice

The aversive response to naloxone administration observed in human and animal studies suggests the presence of an endogenous opioid tone regulating hedonic state but the class(es) of opioid peptides mediating such opioid hedonic tone is uncertain. We sought to address this question using mice deficient in either beta‐endorphin or pro‐enkephalin in a naloxone‐conditioned place aversion paradigm. Mice received saline in the morning in one chamber and either saline or naloxone (0.1, 1 or 10 mg/kg, s.c.) in the afternoon in another chamber, each day for 3 days. On the test day they were given free access to the testing chambers in the afternoon and the time spent in each chamber was recorded. Whereas wild‐type and beta‐endorphin‐deficient mice exhibited a robust conditioned place aversion to naloxone, pro‐enkephalin knockout mice failed to show aversion to naloxone at any dose tested. In contrast, these mice showed a normal conditioned aversion to the kappa opioid receptor agonist, U50,488 (5 mg/kg), and to LiCl (100 mg/kg) indicating that these mice are capable of associative learning. In a separate experiment, pro‐enkephalin knockout mice, similar to wild‐type and beta‐endorphin‐deficient mice, demonstrated a significant conditioned place preference to morphine (2.5, 5 and 10 mg/kg s.c.). These data suggest that enkephalins, but not endorphins, may mediate an endogenous opioid component of basal affective state and also indicate that release of neither endogenous enkephalins nor endorphins is critical for the acquisition or expression of the association between contextual cues and the rewarding effect of exogenously administered opiates.

Orphanin FQ and behavioral sensitization to cocaine.

The recently identified endogenous ligand for the ORL-1 (opioid receptor-like) receptor, orphanin FQ, has been shown to induce hypolocomotion and to decrease extracellular dopamine levels in the nucleus accumbens (N.Acc) after intraventricular (ICV) administration. This study investigated the effect of intraventral tegmental area (VTA) administration of orphanin FQ on the hyperlocomotor effects of peripheral cocaine administration and on the development of behavioral sensitization to cocaine. The administration of cocaine (40 mg/kg IP) once daily for 3 days to male Sprague-Dawley rats resulted in an enhanced locomotor response to a subsequent challenge of cocaine (10 mg/kg IP) 5 days later. The bilateral administration of orphanin FQ (10 microg/side or 30 microg/side) into the VTA 5-10 min prior to the administration of cocaine (40 mg/kg IP) produced a transient (15-30 min) decrease in the hyperlocomotor response to cocaine on day 1 but not on days 2 and 3 of the sensitization paradigm. Such orphanin FQ pretreatment on days 1-3 had no effect on the development of a sensitized response to cocaine (10 mg/kg IP) 5-7 days after the last orphanin FQ injection. However, repeated intra-VTA administration of orphanin FQ (30 microg/side) alone for 3 days resulted in a sensitized response to a single dose of cocaine (10 mg/kg IP) given 5-7 days later. These results indicate that orphanin FQ decreases the activity of mesolimbic dopamine neurons via an action in the VTA, an effect that is both transient and demonstrates rapid tolerance, and consequently, is insufficient to prevent the development of cocaine sensitization. The ability of the peptide to induce cocaine sensitization when administered alone despite its acute inhibitory effects is unique and requires further study to elucidate the mechanisms responsible.

Endogenous opioids mediate basal hedonic tone independent of dopamine d-1 or d-2 receptor activation

Exogenously administered opiates are recognized as rewarding and the involvement of dopamine systems in mediating their apparent pleasurable effects is contentious. The aversive response to naloxone administration observed in animal studies suggests the presence of an endogenous opioid tone regulating hedonic state. We sought evidence for the requirement for dopamine systems in mediating this action of endogenous opioids by determining whether mice deficient in dopamine D-1 or D-2 receptors were able to display conditioned place aversion to naloxone. Mice received saline in the morning in one chamber and either saline or naloxone (10 mg/kg, s.c.) in the afternoon in another chamber, each day for 3 days. On the test day they were given free access to the testing chambers in the afternoon. Similar to their wild-type littermates, D-1 and D-2 receptor knockout mice receiving naloxone in the afternoon spent significantly less time on the test day in the compartment in which they previously received naloxone, compared with animals receiving saline in the afternoon. The persistence of naloxone-conditioned place aversion in D-1 and D-2 knockout mice suggests that endogenous opioid peptides maintain a basal level of positive affect that is not dependent on downstream activation of dopamine systems involving D-1 or D-2 receptors.

Sensitization to cocaine after a single intra-cerebral injection of orphanin FQ/nociceptin

Orphanin FQ/nociceptin (OFQ/N) has been shown to modulate mesolimbic dopaminergic neurotransmission. Repeated administration of OFQ/N into the ventral tegmental area results in a sensitized locomotor response to subsequent peripheral cocaine administration. The aim of the present study was to examine the potential for OFQ/N to produce a sensitized locomotor response to cocaine after a single intra-VTA administration and to determine if this effect of OFQ/N extrapolates to other points along the mesolimbic or nigrostriatal dopaminergic axes. Bilateral administration of OFQ/N (30 microg/side) into the VTA on day 1 to male Sprague--Dawley rats resulted in an enhanced locomotor response to cocaine (10 mg/kg i.p) administered on day 2. However, OFQ/N (3, 10 and 30 microg per side) administered on day 2, 5 mins prior to the administration of cocaine (10 mg/kg i.p), in animals treated with aCSF or OFQ/N on day 1, similarly blocked the action of cocaine, suggesting that the sensitized response was not due to tolerance to the effect of endogenously released OFQ/N. The administration of OFQ/N into the substantia nigra or nucleus accumbens failed to produce a significant sensitized response to a cocaine challenge 24 h later. A significant increase in cocaine stimulated locomotor response on day 2 was observed after injection of OFQ/N into the striatum on day 1. These results demonstrate the ability of a single intra-VTA or intra-striatal administration of OFQ/N to produce increases in the sensitivity to cocaine and may indicate a role for endogenous OFQ/N systems in regulating responses to psychostimulant drugs.

Alterations in the level of OFQ/N-IR in rat brain regions by cocaine.

We have previously shown that administration of orphanin FQ/nociceptin (OFQ/N), the endogenous ligand of the opioid receptor-like (ORL-1) receptor, into the lateral ventricles or VTA blocked cocaine sensitization. In the present study, we determined the effect of acute and chronic cocaine treatment on the level of endogenous OFQ/N in rat brain regions. Male Sprague Dawley rats were tested for motor activity in response to saline or cocaine (20 mg/kg) injection once daily for three consecutive days. To determine the effect of single or repeated cocaine administration on the level of OFQ/N, rats were sacrificed 1 h following saline or cocaine injection either on day 1 or 3, respectively. Additional groups of rats were treated similarly with saline or cocaine on days 1-3 and sacrificed or tested for locomotor sensitization on day 8. Consistent with previous studies, repeated cocaine administration induced locomotor sensitization to a challenge dose of cocaine (7.5 mg/kg) given on day 8. Measurements of tissue content of OFQ/N-IR using radioimmunoassay indicated that the rat hypothalamus and striatum, respectively, contained the highest and lowest levels of the peptide among the brain regions tested. Acute cocaine decreased the level of OFQ-IR in the rat midbrain and to a lesser extent in the striatum. On the other hand, the level of OFQ/N was higher in rats treated with cocaine on days 1-3 and sacrificed on day 8. These findings suggest that endogenous OFQ/N may be involved in the actions of cocaine and possibly in cocaine-induced motor stimulation and locomotor sensitization.

Characterization of active and inactive states of CB1 receptor and the differential binding state modulation by cannabinoid agonists, antagonists and inverse agonists

Cannabinoid 1 (CB1) receptors have the ability to change conformation between active (R*) and inactive (R) receptor states. Herein, we further characterize these receptor states using series of saturation radioligand binding studies and their differential displacement binding by various CB1 receptor ligands. Binding experiments were carried out in naïve rat/dog whole brain membranes using radioligands [(3)H]CP55,940 (for R* state) & [(3)H]SR141716A (both R* and R states) and various agonist, antagonist & inverse agonist ligands at CB1 receptors. In the saturation binding experiments, of the total number of CB1 receptor binding sites (R* + R) in the rat and dog whole brain membranes, only about 18.3 and 11.6% were in the active (R*) state recognized by [(3)H]CP55,940, respectively. In the competitive binding studies, all the CB1 receptor agonists investigated had significantly very high affinity for the active R* state recognized by [(3)H]CP55,940 and lower affinity for the inactive R state mainly recognized by [(3)H]SR141716A in the presence of a non-hydrolyzable analogue of GTP [Gpp(NH)p]. In contrast, various CB1 receptor antagonists/inverse agonists had similar nanomolar affinities at both [(3)H]CP55,940 and [(3)H]SR141716A recognized binding states. These results clearly characterize the significant differences between the active R* and inactive R binding states of CB1 receptors in naive rat and dog brain. In addition, these results also demonstrates that the CB1 agonists and antagonists/inverse agonists can be differentiated by their relative affinities at active (R*) and inactive (R) binding states of the CB1 receptor.