Results indicated that superfusion of
Results indicated that superfusion of orexin alone, glutamate alone or orexin-glutamate co-administration significantly increases the spontaneous discharge rate of LC neurons in both morphine dependent and non-dependent rats (Fig. 2, Fig. 3). However, co-application of orexin and glutamate resulted in a synergistic increase in firing rate only in morphine dependent animals (Fig. 4). This finding was revealed by comparing the values of net increase in firing rate induced by orexin-glutamate co-application with additive effect of orexin and glutamate. As shown, no synergistic effect was observed in control group. In a previous study by this group, the effect of orexin on firing rate (at different concentrations) was tested in vitro and finally orexin 100 nM was found to be more effective (18,40). The same dose-response finding has also been reported by other researchers in this field (30). In addition, glutamate dose was chosen based on the results of another study in which co-application of orexin and glutamate resulted in synergism (36). The results of present study revealed the occurrence of a new interaction between the two endogenous (orexinergic and glutamatergic) systems triggered by long-term morphine exposure. This hypothesis is in line with the previous reports demonstrating an interaction between orexin and glutamate in rat prefrontal PR957 sale (36) and ventral tegmental area (VTA) (41). Orexin-A has been shown to enhance NMDA receptor-mediated Ca2+ release in LC neurons which in turn results in the subsequent increase in cellular excitability (30,42). Thus, orexin-A, in addition to the direct excitatory effect on LC (43), may also potentiate the effect of glutamate on these neurons. On the other hand, long-term use of morphine has been shown to affect the intracellular events within the LC (44). For example, chronic morphine exposure increases the activity of adenylyl cyclase. This leads to an increase in cyclic adenosine monophosphate (cAMP) which in turn activates protein kinase A (PKA) and enhances cAMP-dependent Na+ current (45,46). Furthermore, it has been shown that chronic activation of opioid receptors increases the activity of NMDA receptors (47). This also enhances intracellular Ca2+ concentration and the activity of CaM-kinase and phosphorylated cAMP-response element-binding protein (CREB) (48). These events finally result in further increase in cAMP concentration which is followed by enhanced neuronal excitability. In addition, activation of orexin type 1 receptors (OX1Rs) has been reported to stimulate cAMP synthesis (49). Moreover, chronic use of morphine has been shown to enhance both the activity of orexinergic neurons and the expression of orexin receptors (23). In 2010, Henny et al., showed that orexinergic terminals release glutamate in addition to orexin within the LC (50).Thus the increased tone of the orexin system during opiate dependence could promote LC neuronal excitation by increasing glutamate release in this brain region. It is currently well established that the hyperactivity of LC neurons temporally coincides with the expression of behavioral manifestations induced by opiate-withdrawal (51) Previous studies have shown that intra-LC injection of glutamate elicits opiate withdrawal-like behavioral manifestations in morphine dependent rats (52). Similarly, a recent study by our group indicated that intra-LC microinjection of orexin-A induces the same somatic signs in both morphine-dependent and naïve animals (53). Consistently, there is evidence indicating that withdrawal-induced over-excitation of LC neurons is mediated by enhanced release of glutamate (3). In addition, the neuronal activity of orexin releasing fibers has been suggested to be enhanced during opiate withdrawal (51). Thus, the mentioned cellular events may somehow explain the more potent (mentioned as synergistic) increase in spontaneous discharge rate of LC neurons following orexin-glutamate co-application in morphine dependent rats compared to their naïve counterparts. In other words, neuroadaptations occurred at the results of prolonged opioid exposure seem to be potentiated within the LC neurons receiving both glutamatergic and orexinergic inputs. Another possibility could be attributed to withdrawal-induced enhancement of orexin-glutamate co-release from the same synaptic terminals (50). These mechanisms might involve either increased intracellular concentration of cAMP or up-regulation of NMDA/orexin receptors. However, further studies are required to elucidate the molecular pathways underlying the suggested interaction during the development of morphine dependence within the coerulear neurons.