Figure 1.
Systemic injections of almorexant reduced ethanol and sucrose self-administration.
A. After 8 weeks of 20% ethanol self-administration, almorexant significantly attenuated active lever responding for ethanol in Long-Evans rats. B. After 8 weeks of 5% sucrose self-administration, almorexant significantly attenuated active lever responding for sucrose in Long-Evans rats. The values are expressed as mean number of active lever presses ± SEM, (repeated measures ANOVA followed by Newman-Keuls post hoc test) ***p<0.001, **p<0.01, *p<0.05 compared to vehicle, n = 14 for ethanol, n = 12 for sucrose.
Table 1.
Number of inactive lever presses, amount of ethanol consumed (g/kg), and reinforcers earned following systemic almorexant treatment in animals trained to self-administer either ethanol or sucrose.
Table 2.
Locomotor responses following systemic almorexant treatment in naïve animals.
Figure 2.
Intra-VTA, but not intra-SNr, infusions reduce ethanol self-administration, but have no effect on sucrose self-administration.
A. Infusions of almorexant into the VTA significantly reduced active lever responding for 20% ethanol but (B.) had no effect on 5% sucrose responding. C. Schematic representation of the VTA cannula placements. D. Infusions of almorexant into the SNr had no effect on active lever responding for 20% ethanol. E. Schematic representation of the SNr cannula placements. Values are expressed as mean active lever presses ± SEM (repeated measures ANOVA followed by Newman Keuls post hoc testing,* p<0.05 compared to vehicle for VTA groups and paired t-test for SNr data), n = 12 for VTA ethanol, n = 8 for VTA sucrose and n = 9 for the SNr ethanol.
Table 3.
Number of inactive lever presses, amount of intake (g/kg), and reinforcers earned following intra-VTA almorexant treatment.
Table 4.
Number of inactive lever presses, amount of intake (g/kg), and reinforcers earned following intra-SNr almorexant treatment.
Figure 3.
Almorexant inhibits intracellular calcium release in HEK-293 cells expressing either Ox-R1 or Ox-R2.
A. Activation of intracellular calcium release in HEK-293 cells expressing human Ox-R1 by orexin-A (1 pM-10 µM) (closed squares). Almorexant alone did not stimulate intracellular calcium release in Ox-R1 expressing cells (open triangles). B. Activation of intracellular calcium release in HEK-293 cells expressing human Ox-R2 by orexin-B (1 fM- 10 µM, closed squares). Almorexant alone did not stimulate intracellular calcium release in Ox-R2 expressing cells (open triangles). C. Inhibition of orexin-A (100 nM, closed squares) induced stimulation calcium release in Ox-R1 by SB-334867 (closed triangles) or almorexant (open triangles) (10 pM-100 µM). D. Inhibition of orexin-B (10 nM; closed square) induced stimulation of calcium release in Ox-R2 by SB-334867 (closed triangles) or almorexant (10 pM-100 µM, open triangles). Results are expressed as mean ± SEM relative fluorescence units (RFU) calculated as agonist-induced maximum calcium peak/cell number ×1000.
Figure 4.
Orexins enhance firing rate in VTA neurons.
A–E. Graphs of firing rate responses of individual cells to application of orexin peptides. One minute of baseline and the last minute of orexin application are plotted. Sample cell-attached recordings next to the plots demonstrate effect of orexin-A or -B on firing rate. Baseline before addition of orexin peptide (top trace) and last minute after addition of orexin (bottom trace) are shown, scale bar denotes 10 s. A. Responses of Long-Evans putative DA neurons to orexin-A, where 6 out of 7 neurons increased in firing with orexin-A. B. Responses of Long-Evans putative non DA neurons, where 2 out of 4 neurons increased in firing with orexin-A. C. Responses of Long-Evans putative DA neurons to orexin-B, where 2 out of 9 neurons responded to orexin-B. D. Responses of Long-Evans putative non-DA neurons, where all cells increased in firing rate upon orexin-B application. E. Responses of Sprague-Dawley putative DA neurons, where 2 out of 4 significantly increased in firing with orexin-B. Paired t-test was used to assume significance.
Figure 5.
Almorexant blocks orexin-A induced increase in firing rate.
A. Almorexant (1 µM) blocked orexin-A (100 nM) induced enhancement of firing rate in Long-Evans VTA neurons, n = 4. B. Percent change in firing rate from baseline, denoted as 100%, in the presence (grey bar, ore-A+Almx.) or absence of almorexant (black bar, ore-A) upon application of orexin-A. One-way ANOVA followed by Newman-Keuls post hoc test comparing the baseline (1 min) and last one min of orexin-A application (** p<0.01, plotted from fig. 5A, control). *p<0.05 denotes comparison between last min of orexin-A only (control, Fig. 5A) and orexin-A+Almorexant (+Almorexant, Fig. 5A). C. No change in firing rate upon orexin-B (100 nM) application was observed in presence of almorexant, n = 8. A & C. Each point represents mean ± SEM over a period of 10 seconds. D. Percent change in firing rate from baseline, denoted as 100%, in the presence (grey bar, ore-B+Almx.) or absence of almorexant (black bar, ore-B) upon application of orexin-B.