Orexin B/hypocretin 2 increases glutamatergic transmission to ventral tegmental area neurons.

The orexins (hypocretins) play a crucial role in arousal, feeding and reward. Highly relevant to these functions, orexin-containing neurons from the lateral hypothalamus project densely to the ventral tegmental area (VTA), which is the origin of dopamine projections implicated in motivation and reward. Orexin A/hypocretin 1 (oxA/hcrt-1) can enable long-term changes associated with drugs of abuse; however, the effects of orexin B/hypocretin 2 (oxB/hcrt-2) on excitatory synaptic transmission in the VTA are unknown. We used whole-cell patch-clamp electrophysiology in rat horizontal midbrain slices to examine the effects of oxB/hcrt-2 on excitatory synaptic transmission. We observed that oxB/hcrt-2 has distinct effects from oxA/hcrt-1 in the VTA. oxB/Hcrt-2 (100 nM) increased presynaptic glutamate release in addition to a postsynaptic potentiation of NMDA receptors (NMDARs). The oxB/hcrt-2-mediated postsynaptic potentiation of NMDARs was mediated via activation of orexin/hypocretin 2 (OX2/Hcrt-2) receptors and protein kinase C (PKC). Furthermore, the increase in transmitter release probability was also PKC-dependent, but not through activation of orexin/hypocretin 1 (OX1/Hcrt-1) or OX2/Hcrt-2 receptors. Finally, oxB/hcrt-2 or the selective OX2/Hcrt-2 receptor agonist ala(11)-D-leu(15)-orexin B, significantly reduced spike-timing-induced long-term potentiation. Taken together, these results support a dual role for oxB/hcrt-2 in mediating enhanced glutamatergic transmission in the VTA, and suggest that oxA/hcrt-1 and oxB/hcrt-2 exert different functional roles in modulating the enhancement of the motivational components of arousal and feeding.

Regulation of the mesolimbic dopamine circuit by feeding peptides.

Polypeptides produced in the gastrointestinal tract, stomach, adipocytes, pancreas and brain that influence food intake are referred to as 'feeding-related' peptides. Most peptides that influence feeding exert an inhibitory effect (anorexigenic peptides). In contrast, only a few exert a stimulating effect (orexigenic peptides), such as ghrelin. Homeostatic feeding refers to when food consumed matches energy deficits. However, in western society where access to palatable energy-dense food is nearly unlimited, food is mostly consumed for non-homeostatic reasons. Emerging evidence implicates the mesocorticolimbic circuitry, including dopamine neurons of the ventral tegmental area (VTA), as a key substrate for non-homeostatic feeding. VTA dopamine neurons encode cues that predict rewards and phasic release of dopamine in the ventral striatum motivates animals to forage for food. To elucidate how feeding-related peptides regulate reward pathways is of importance to reveal the mechanisms underlying non-homeostatic or hedonic feeding. Here, we review the current knowledge of how anorexigenic peptides and orexigenic peptides act within the VTA.

Continued morphine modulation of calcium channel currents in acutely isolated locus coeruleus neurons from morphine-dependent rats.

1. The actions of the opioid agonists morphine and methionine-enkephalin (met-enkephalin) on the calcium channel currents (IBa) of acutely isolated locus coeruleus (LC) neurons from morphine-dependent and vehicle-treated rats were examined using whole cell patch clamp techniques. 2. In LC neurons maintained in 5 microM morphine, co-superfusion of naloxone (1 microM) or the mu-opioid receptor antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 1 microM) with morphine resulted in a significant increase in the amplitude of IBa. The increases in IBa were not different in neurons from morphine-dependent or vehicle rats. The increase in IBa was mimicked by washing off morphine, but not by co-superfusion of the kappa-receptor antagonist norbinaltorphimine (300 nM) or the delta-receptor antagonist ICI-174864 (1 microM). 3. In spontaneously withdrawn LC neurons from morphine-dependent rats, met-enkephalin (pD2 7.1, maximum inhibition 49%) and morphine (pD2 6.5, maximum inhibition 33%), inhibited IBa in all cells. In cells from vehicle rats the pD2 for met-enkephalin was 7.3, maximum inhibition 52%, while the pD2 for morphine was 6.6 and the maximum inhibition 43% (P<0.05 versus cells from morphine-dependent rats). 4. IBa in LC neurons was mostly comprised of omega-conotoxin GVIA- (N-type) and omega-agatoxin IVA- (P/Q-type) sensitive components, with lesser amounts of nimodipine-sensitive current and current resistant to all three blockers. Neither the density of IBa nor the proportion of any of the components of IBa differed between neurons from morphine-dependent or vehicle-treated rats. 5. This study demonstrates that in morphine-dependent rats, morphine and met-enkephalin modulation of somatic IBa in LC neurons displays modest tolerance compared with untreated rats. Further, chronic morphine treatment does not alter the type or density of IBa in LC neurons. These results provide more evidence that functional mu-opioid receptor coupling is not dramatically altered in the LC in morphine-dependent rats.

Effect of adenosine receptor agonists on release of the nucleoside analogue [3H]formycin B from cultured smooth muscle DDT1 MF-2 cells.

Adenosine has receptor-mediated effects in a variety of cell types and is predominantly formed from ATP by a series of nucleotidase reactions. Adenosine formed intracellularly can be released by bidirectional nucleoside transport processes to activate cell surface receptors. We examined whether stimulation of adenosine receptors has a regulatory effect on transporter-mediated nucleoside release. DDT1 MF-2 smooth muscle cells, which possess nitrobenzylthioinosine-sensitive (ES) transporters as well as both adenosine A1 and A2 receptors, were loaded with the metabolically stable nucleoside analogue [3H]formycin B. N6-cyclohexyladenosine (CHA), a selective adenosine A1 receptor agonist, produced a concentration-dependent inhibition of [3H]formycin B release with an IC50 value of 2.7 microM. Further investigation revealed CHA interacts directly with nucleoside transporters with a Ki value of 3.3 microM. Neither 5'-N-ethylcarboxamidoadenosine (NECA), a mixed adenosine A1 and A2 receptor agonist, nor CGS 21680, a selective adenosine A2A receptor agonist, affected nucleoside release. We conclude that release of the nucleoside formycin B from DDT1 MF-2 cells is not regulated by adenosine A1 or A2 receptor activation.

Effect of insulin on excitatory synaptic transmission onto dopamine neurons of the ventral tegmental area in a mouse model of hyperinsulinemia.

Obesity has drastically increased over the last few decades. Obesity is associated with elevated insulin levels, which can gain access to the brain, including into dopamine neurons of the ventral tegmental area (VTA), a brain region critical for mediating reward-seeking behavior. Synaptic plasticity of VTA dopamine neurons is associated with altered motivation to obtain reinforcing substances such as food and drugs of abuse. Under physiological circumstances, insulin in the VTA can suppress excitatory synaptic transmission onto VTA dopamine neurons and reduce aspects of palatable feeding behavior. However, it is unknown how insulin modulates excitatory synaptic transmission in pathological circumstances such as hyperinsulinemia. Using patch-clamp electrophysiology, we demonstrate that, in a hyperinsulinemic mouse model, insulin has reduced capacity to cause a synaptic depression of VTA dopamine neurons, although both low-frequency stimulation-induced long-term depression and cannabinoid-induced depression were normal. These results suggest that insulin action in the VTA during pathological hyperinsulinemia is disrupted and may lead to increased feeding behavior.

Acute opioid receptor desensitization and tolerance: is there a link?

1. Morphine, used long-term for the treatment of pain, results in drug tolerance. The therapeutic benefits, as well as side effects, of morphine are mediated predominantly via activation of mu-opioid receptors. Although the underlying mechanisms for opioid tolerance remains unclear, early adaptive processes, such as acute receptor desensitization and receptor downregulation, have been suggested to be crucial to the development of opioid tolerance. 2. Other neuroadaptations resulting from chronic opioid use include upregulation of the cAMP pathway, an increase in the cAMP response element-binding protein and Fos-related antigens. However, the connection between upregulation of these cellular elements and the mechanism behind the behavioural phenomenon remains unclear. 3. Acute receptor desensitization is thought to occur via uncoupling of the receptor and G-protein, which is followed by internalization of the receptor from the cell membrane. This process occurs after a few minutes of agonist exposure. Receptor-G-protein uncoupling is mediated via phosphorylation of putative sites on the intracellular loops of activated receptors. 4. Acute desensitization and downregulation of receptors both result in a reduction of agonist efficacy. These events occur early in the cascade of cellular adaptation; however, it is uncertain whether these processes contribute to the long-term changes in receptor sensitivity that occur after repeated exposure to opioids. 5. Acute desensitization may, in fact, be a protective mechanism whereby cells adapt to avoid the development of physiological drug tolerance by rapidly attenuating receptor-mediated signalling. Those drugs that do not cause receptor internalization, such as morphine, may have higher propensities to develop tolerance.

Uptake and release of [3H]formycin B via sodium-dependent nucleoside transporters in mouse leukemic L1210/MA27.1 cells.

At least seven functionally distinct nucleoside transport processes exist; however, mouse leukemic L1210/MA27.1 cells possess only one subtype, a Na+-dependent transporter termed N1/cif. The capacity of this transporter subtype to release nucleosides from L1210/MA27.1 cells was investigated with the poorly metabolized inosine analog [3H]formycin B. Uptake of [3H]formycin B into these cells was inhibited by replacement of Na+ in the buffer with choline, or by blocking Na+/K+ ATPase with 2 mM ouabain, inhibiting glycolysis with 5 mM iodoacetic acid or inhibiting nucleoside transport with 1 mM phloridzin. Sodium stimulated uptake with an EC50 value of 12 mM. To measure release of [3H]formycin B, cells were loaded with [3H]formycin B (10 microM) then washed and resuspended in buffer. Replacement of Na+ in the buffer with choline enhanced [3H]formycin B release by 20 to 47%, and significant stimulation of release was observed with Na+ concentrations of 30 mM or less. Resuspending loaded cells into Na+ buffer containing 2 mM ouabain or 10 microM monensin, a Na+ ionophore, significantly enhanced [3H]formycin B release during 20 min by 39% or 29%, respectively. Release of [3H]formycin B into choline buffer was inhibited 26.5% by 10 mM phloridzin and 39.6% by 10 mM propentofylline, compounds known to inhibit various transporters including Na+-dependent nucleoside transporters. Release was also inhibited significantly by 100 microM concentrations of dilazep, dipyridamole and nitrobenzylthioinosine, inhibitors with selectivity for Na+-independent nucleoside transporters. In the absence of Na+, the permeants adenosine and uridine enhanced [3H]formycin B release by up to 40.9% and 21.4%, respectively. These data indicate that in the absence of an inwardly directed Na+ gradient, Na+-dependent nucleoside transporters can function in the release of nucleosides.

Nociceptin inhibits calcium channel currents in a subpopulation of small nociceptive trigeminal ganglion neurons in mouse.

1. The effects of nociceptin/orphanin FQ (N/OFQ) and opioid receptor agonists on voltage-activated calcium channel currents (I(Ca)) were examined in acutely isolated mouse trigeminal ganglion neurons using whole-cell patch-clamp recordings. These effects were correlated with responses of the neurons to capsaicin and binding of Bandeiraea simplicifolia isolectin B4 (IB4). 2. Trigeminal neurons were divided into two populations based on the presence (type 2) or absence (type 1) of a prominent T-type I(Ca). N/OFQ potently (EC(50) of 19 nM) inhibited high-voltage-activated (HVA) I(Ca) in most (82 %) small (capacitance < 12 pF) type 1 neurons, but few (9 %) larger (> 12 pF) type 1 neurons. N/OFQ inhibited I(Ca) in few (23 %) type 2 cells, and did not affect the T-type I(Ca) in any cell. 3. The mu-opioid agonists DAMGO and morphine inhibited I(Ca) in most type 1 neurons, more often (95 % versus 77 %) in the small cells. The inhibition of I(Ca) by DAMGO and morphine was more efficacious in small versus large type 1 neurons. mu-Opioids did not inhibit I(Ca) in type 2 neurons. 4. Most small type 1 neurons were sensitive to capsaicin (93 %) and bound IB4 (86 %). Fewer larger type 1 neurons responded to capsaicin (30 %) or bound IB4 (58 %). Type 2 neurons did not respond to capsaicin, although some bound IB4 (35 %). 5. Thus, N/OFQ preferentially inhibits HVA I(Ca) in a subpopulation of small nociceptive trigeminal ganglion neurons that is also highly sensitive to mu-opioid agonists.

Effects of propentofylline on adenosine receptor activity in Chinese hamster ovary cell lines transfected with human A1, A2A, or A2B receptors and a luciferase reporter gene.

Propentofylline is neuroprotective in vivo, but its mechanism of action is not completely understood. Previously, propentofylline was shown to block adenosine transport processes, to inhibit three adenosine receptor subtypes, and to inhibit cAMP phosphodiesterase. We tested the effect of propentofylline on adenosine receptor function in Chinese hamster ovary (CHO) cells transfected with human adenosine A1, A2A, or A2B receptors and a luciferase reporter gene under control of a promoter sequence containing several copies of the cAMP response element. We investigated the concentration-dependent inhibitory effects of propentofylline on cAMP phosphodiesterase, adenosine transport processes, and adenosine A1, A2A, and A2B receptors. At concentrations > or = 1 mM, propentofylline increased luciferase activity probably as a result of inhibition of cAMP phosphodiesterase. Inhibition of [3H]adenosine uptake by propentofylline was concentration dependent, with IC50 values of 37-39 microM for the three cell types. Agonist-activated adenosine A1 receptors were antagonized by 100 microM propentofylline, but inhibition of agonist-stimulated A2A or A2B receptors was not observed. In contrast, A1 and A2A receptor mediated effects of adenosine were enhanced by propentofylline at concentrations of 1 and 100 microM, respectively. These data indicate that the net effects of propentofylline in vivo will be dependent on the concentrations of propentofylline and adenosine available and on the subtypes of adenosine receptors, phosphodiesterases, and nucleoside transporters present.

Adenovirus vector-induced expression of the C-X-C chemokine IP-10 is mediated through capsid-dependent activation of NF-kappaB.

The use of adenovirus vectors for gene therapy has been limited by well-defined cellular and humoral immune responses. We have previously shown that adenovirus vectors rapidly induce the expression of the C-X-C chemokine, interferon-inducible protein 10 (IP-10), in vivo. Various first-generation, type 5 adenovirus vectors, including adCMVbetagal and UV-psoralen-inactivated adenovirus, equally induced the expression of IP-10 mRNA as early as 3 h following infection in mouse renal epithelial cells (REC). Luciferase reporter experiments using deletional mutants of the murine IP-10 5'-flanking region revealed that transcriptional activation of the IP-10 promoter by adCMVbetagal was dependent on the -161- to -96-bp region upstream of the transcription start site. In electrophoretic mobility shift assays, adCMVbetagal, adCMV-GFP, FG140, and transcription-defective adenovirus induced protein binding to oligonucleotides containing a consensus sequence for NF-kappaB at position -113 of the IP-10 promoter. Supershift assays confirmed an increase in binding activity of NF-kappaB p65 but not p50 or cRel in REC cells infected with various replication-deficient adenoviruses. Coinfection of REC cells with adCMVbetagal and an adenoviral vector expressing IkappaBalpha resulted in suppression of adCMVbetagal-induced expression of IP-10 at 6 and 16 h, further strengthening the conclusion that adenovirus-induced activation of IP-10 is dependent on NF-kappaB. The induction of IP-10 appeared to be direct because infection with adenovirus vectors failed to induce the expression of the potent IP-10 stimulators, interferon gamma and tumor necrosis factor alpha. Together, these findings demonstrate that adenovirus vectors directly induce the expression of IP-10 through capsid dependent activation of NF-kappaB.