Postoperative delirium is common in older cardiac surgery patients and associated with negative short-term and long-term outcomes. The alpha-2-adrenergic receptor agonist dexmedetomidine shows promise as prophylaxis and treatment for delirium in intensive care units (ICU) and postoperative settings. Clonidine has similar pharmacological properties and can be administered both parenterally and orally. We aim to study whether repurposing of clonidine can represent a novel treatment option for delirium, and the possible effects of dexmedetomidine and clonidine on long-term cognitive trajectories, motor activity patterns and biomarkers of neuronal injury, and whether these effects are associated with frailty status.

Nuclei of the brainstem involved in behavioral state control are mutually interconnected. Histaminergic neurons of the posterior hypothalamus receive inputs from brainstem noradrenergic cell groups as well as from the locus coeruleus. The role of adrenergic inputs in histaminergic function is unclear. We examined the actions of adrenergic agonists on histaminergic neurons of the tuberomamillary nucleus (TM) using electrophysiological methods in a brain slice preparation. Evoked GABAergic inhibitory postsynaptic potentials (IPSPs) in histaminergic neurons were reduced in amplitude following the application of norepinephrine (NE) (2-20 microM) or clonidine (10 microM) but were not affected by isoproterenol (10 microM). Norepinephrine application caused no changes in membrane properties of TM neurons. Responses to exogenously applied GABA were unaffected by adrenergic agonists. Clonidine reduced the frequency of spontaneous IPSPs, an action that was blocked by yohimbine. Norepinephrine did not alter the amplitude distribution of bicuculline-sensitive miniature inhibitory postsynaptic currents (mIPSCs). Thus, GABA release onto TM neurons is modulated presynaptically by adrenergic alpha(2)-receptors. Inputs from noradrenergic neurons of the brainstem will reduce the inhibitory actions of GABAergic inputs resulting in disinhibition of histaminergic neurons.

The central component that establishes chronic pain from peripheral nerve injury is associated with increased tumor necrosis factor-alpha (TNFalpha) production in the brain. This study examined TNFalpha and its reciprocally permissive role with alpha(2)-adrenergic activation during peak and progressive decline of thermal hyperalgesia in sciatic nerve chronic constriction injury (CCI). Accumulation of TNFalpha mRNA (in situ hybridization) increases in the hippocampus and locus coeruleus during the onset of neuropathic pain and persists as hyperalgesia abates. Activation of alpha(2)-adrenergic receptors in control rats decreases TNFalpha mRNA accumulation in these brain regions. In contrast, during hyperalgesia, alpha(2)-adrenergic activation enhances TNFalpha mRNA accumulation. Whether this enhanced TNFalpha production is associated with changes in the regulation of norepinephrine (NE) release was tested. Hippocampal slices were electrically depolarized to evaluate alpha(2)-adrenergic and TNFalpha regulation of NE release. While inhibition of NE release by TNFalpha is maximal during peak hyperalgesia, it subsequently transforms to facilitate NE release. In addition, alpha(2)-adrenergic receptor activation with clonidine (0.2mg/kg, i.p.) in CCI rats experiencing hyperalgesia restores TNFalpha and alpha(2)-adrenergic inhibition of NE release. While TNFalpha directs the development of hyperalgesia, it also directs its resolution. Transformed sensitivity to alpha(2)-adrenergic agonists during hyperalgesia demonstrates a mechanism for therapy.

The alpha-2 adrenergic agonist dexmedetomidine (Dex), 3-300 microg/kg, i.p., decreased cerebellar cGMP in a dose-dependent manner. Fentanyl (F), an opioid agonist, increased cerebellar cGMP at 0.3 mg/kg, s.c., and decreased it at doses >/=1 mg/kg. The inhibitory effect was receptor specific, that of Dex being blocked by the alpha-2 adrenergic antagonist yohimbine, 5 mg/kg, i.p.; that of F by the opioid antagonist naloxone, 5 mg/kg, i.p. In contrast the stimulatory effect of F was blocked by both naloxone and yohimbine. Yohimbine also enhanced the inhibitory effect of F. In mice pretreated with pertussis toxin, 2 microgram/mouse, given i.c.v. 72 h before the agonists, the decrease in cGMP induced by Dex or F was not affected, while the stimulatory effect of F was reversed to an inhibitory effect. When inhibiting doses of F and Dex were administered together, the cGMP response was smaller than the sum of the individual responses. Dex attenuated in a dose-dependent manner the decrease in cGMP induced by F, and unmasked or enhanced the stimulatory effect of F. These results show that the alpha-2 adrenergic- and opioid-receptors are coupled to the cGMP effector system and suggest that the two pathways converge at a common post-receptor site in the cascade of events transducing the receptor signal to cGMP regulation.

Our previous work demonstrated dexmedetomidine-activated phosphorylation of extracellular regulated kinases 1 and 2 (ERK(1/2)) in primary cultures of mouse astrocytes and showed that it is evoked by alpha(2)-adrenoceptor-mediated transactivation of epidermal growth factor (EGF) receptors, a known response to activation of G(i/o)- or G(q)-coupled receptors [Li, B., Du, T., Li, H., Gu, L., Zhang, H., Huang, J., Hertz, L., Peng, L., 2008a. Signaling pathways for transactivation by dexmedetomidine of epidermal growth factor receptors in astrocytes and its paracrine effect on neurons. Br. J. Pharmacol. 154, 191-203]. Like most studies of transactivation, that study used cultured cells, raising the question whether a similar effect can be demonstrated in intact brain tissue and the brain in vivo. In the present study we have shown that (i) dexmedetomidine-mediated ERK(1/2) phosphorylation occurs in mouse brain slices with a similar concentration dependence as in cultured astrocytes (near-maximum effect at 50nM); (ii) intraperitoneal injection of dexmedetomidine (3microg/kg) in adult mice causes rapid phosphorylation of the EGF receptor (at Y845 and Y992) and of ERK(1/2) in the brain; (iii) both EGF receptor and ERK(1/2) phosphorylation are inhibited by intraventricular administration of (a) AG 1478, a specific inhibitor of the receptor-tyrosine kinase of the EGF receptor; (b) GM 6001, an inhibitor of metalloproteinase(s) required for release of EGF receptor agonists from membrane-bound precursors; or (c) heparin, neutralizing heparin-binding EGF (HB-EGF). Thus, in intact brain HB-EGF, known to be expressed in brain, may be the major EGF agonist released in response to stimulation of alpha(2)-adrenoceptors, the released agonist(s) activate(s) EGF receptors, and ERK(1/2) is phosphorylated as a conventional response to EGF receptor activation. Our previous paper (see above) showed that dexmedetomidine evokes no ERK(1/2) phosphorylation in cultured neurons, but neurons respond to astrocyte-conditioned medium (and to EGF) with ERK(1/2) phosphorylation. The present findings therefore suggest that EGF receptor transactivation in astrocytes in the mature brain in vivo is an important process in response to alpha(2)-adrenoceptor stimulation and may lead to phosphorylation of ERK(1/2) both in astrocytes themselves and in adjacent neurons.

Alpha-2 agonists have sedative, analgesic, and opioid-sparing effects. Moreover, intraoperative or postoperative systemic administration of alpha-2 adrenergic agonists is known to reduce postoperative pain and opioid consumption. This meta-analysis investigated whether preoperative administration of alpha-2 agonists can affect postoperative pain and opioid consumption.

Agonists acting at alpha(2)-adrenergic and opioid receptors (alpha(2)ARs and ORs, respectively) inhibit pain transmission in the spinal cord. When coadministered, agonists activating these receptors interact in a synergistic manner. Although the existence of alpha(2)AR/OR synergy has been well characterized, its mechanism remains poorly understood. The formation of heterooligomers has been proposed as a molecular basis for interactions between neuronal G-protein-coupled receptors. The relevance of heterooligomer formation to spinal analgesic synergy requires demonstration of the expression of both receptors within the same neuron as well as the localization of both receptors in the same neuronal compartment. We used immunohistochemistry to investigate the spatial relationship between alpha(2)ARs and ORs in the rat spinal cord to determine whether coexpression could be demonstrated between these receptors. We observed extensive colocalization between alpha(2A)-adrenergic and delta-opioid receptors (DOP) on substance P (SP)-immunoreactive (-ir) varicosities in the superficial dorsal horn of the spinal cord and in peripheral nerve terminals in the skin. alpha(2A)AR- and DOP-ir elements were colocalized in subcellular structures of 0.5 mum or less in diameter in isolated nerve terminals. Furthermore, coincubation of isolated synaptosomes with alpha(2)AR and DOP agonists resulted in a greater-than-additive increase in the inhibition of K(+)-stimulated neuropeptide release. These findings suggest that coexpression of the synergistic receptor pair alpha(2A)AR-DOP on primary afferent nociceptive fibers may represent an anatomical substrate for analgesic synergy, perhaps as a result of protein-protein interactions such as heterooligomerization.

How external stimuli prevent the onset of sleep has been little studied. This is usually considered to be a non-specific type of phenomenon. However, the hypnotic drug dexmedetomidine, an agonist at α2 adrenergic receptors, has unusual properties that make it useful for investigating this question. Dexmedetomidine is considered to produce an 'arousable' sleep-like state, so that patients or animals given dexmedetomidine become alert following modest stimulation. We hypothesized that it might be more difficult to make mice unconscious with dexmedetomidine if there was a sufficient external stimulus. Employing a motorized rotating cylinder, which provided a continuous and controlled arousal stimulus, we quantitatively measured the ability of such a stimulus to prevent dexmedetomidine loss of righting reflex in two inbred strains of mice (C57BL/6 and 129X1). We found that whereas the C57BL/6 strain required a strong stimulus to prevent dexmedetomidine-induced hypnosis, the 129X1 strain stayed awake even with minimal stimuli. Remarkably, this could be calibrated as a simple threshold trait, i.e. a binary 'yes-no' response, which after crossing the two mouse strains behaved as a dominant-like trait. We carried out a genome-wide linkage analysis on the F2 progeny to determine if the ability of a stimulus to prevent dexmedetomidine hypnosis could be mapped to one or more chromosomal regions. We identified a locus on chromosome 4 with an associated Logarithm of Odds score exceeding the pre-established threshold level. These results show that complex traits, such as the ability of a stimulus to reverse drug-induced hypnosis, may have precise genetic determinants.

Mesenteric veins are more sensitive than arteries to the constrictor effects of sympathetic nerve stimulation and alpha-adrenoceptor agonists. We tested the hypothesis that alpha(1)- and alpha(2)-adrenoceptors interact to enhance adrenergic reactivity of mesenteric veins. We studied neurogenic and agonist-induced constrictions of mesenteric veins and arteries in vitro. Norepinephrine concentration-response curves were left-shifted in veins compared to arteries. UK 14,304 (0.01-1 microM, alpha(2)-adrenoceptor receptor agonist) did not constrict arteries or veins but enhanced constrictions and Ca(2+) signals mediated by alpha(1)-adrenoceptor stimulation in veins. Yohimbine (alpha(2)-adrenoceptor receptor antagonist) and MK912 (alpha(2C)-adrenoceptor receptor antagonist), but not alpha(2A)- or alpha(2B)-adrenoceptor antagonists, produced rightward shifts in norepinephrine concentration-response curves in veins. Pharmacological studies revealed that alpha(1D)-adrenoceptors mediate venous constrictions. Norepinephrine responses in veins from alpha(2C)-adrenoceptor knock-out (KO) mice were not different from wild type veins. Yohimbine inhibited norepinephrine constrictions in alpha(2C)-adrenoceptor KO veins suggesting that there is upregulation of other alpha(2)-adrenoceptors in alpha(2C)-KO mice. These data indicate that alpha(1D)- and alpha(2C)-adrenoceptors interact in veins but not in arteries. This interaction enhances venous adrenergic reactivity. Mesenteric vein-specific alpha(2)-adrenoceptor linked Ca(2+) and perhaps other signaling pathways account for enhanced venous adrenergic reactivity.

The sympathetic nervous system (SNS) controls various physiological functions via the neurotransmitter noradrenaline. Activation of the SNS in response to psychological or physical stress is frequently associated with weakened immunity. Here, we investigated how adrenoceptor signaling influences leukocyte behavior. Intravital two-photon imaging after injection of noradrenaline revealed transient inhibition of CD8+ and CD4+ T cell locomotion in tissues. Expression of β-adrenergic receptor in hematopoietic cells was not required for NA-mediated inhibition of motility. Rather, chemogenetic activation of the SNS or treatment with adrenergic receptor agonists induced vasoconstriction and decreased local blood flow, resulting in abrupt hypoxia that triggered rapid calcium signaling in leukocytes and halted cell motility. Oxygen supplementation reversed these effects. Treatment with adrenergic receptor agonists impaired T cell responses induced in response to viral and parasitic infections, as well as anti-tumor responses. Thus, stimulation of the SNS impairs leukocyte mobility, providing a mechanistic understanding of the link between adrenergic receptors and compromised immunity.

When agonists to alpha(2)adrenergic receptor (AR) and delta opioid receptor (DOR) are co-administered, they act synergistically to inhibit nociceptive elicited behavior. Some previous studies of synergism have used the DOR-selective agonist [D-Pen(2),D-Pen(5)]-enkehphalin (DPDPE), however, DPDPE has been shown to be less potent in mu opioid receptor-knockout (MOR-KO) mice. It is possible, therefore, that MOR contributes to the synergism of DPDPE with the alpha(2)AR agonists. We compared the interactions of spinally administered DPDPE with an alpha(2)AR-adrenergic agonist in MOR-KO and MOR-wildtype (WT) mice. In these mice, morphine is ineffective and the potency of spinally administered DOR agonists, deltorphin II (DELT II) and DPDPE decreased 16- and 250-fold, respectively. Antagonism studies using the MOR-selective antagonist, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH(2) (CTOP) and the DOR-selective antagonist, naltrindole HCl (naltrindole) demonstrated that while DOR mediates DPDPE-induced antinociception in MOR-KO, both MOR and DOR participate in DPDPE antinociception in WT mice, suggesting that DPDPE is less selective for DOR than previously observed in binding studies when given in vivo. The potency of the alpha(2)AR agonist UK14,304 was equivalent in WT and MOR-KO, demonstrating that the loss of opioid-mediated antinociception in the MOR-KO was not due to generalized impairment of antinociceptive processing. Interestingly, isobolographic analysis showed that, despite substantial loss of DPDPE potency in MOR-KO, DPDPE-UK14,304 synergism is fully retained. Collectively, these experiments demonstrate that although MOR participates in DELT II- and DPDPE-mediated spinal antinociception, DOR independently participates in synergistic antinociception with alpha(2)AR. Resolution of the roles of the opioid receptor subtypes in opioid agonist-induced effects may require comparison of the effects of multiple selective agonists in knockout animals.

We have previously demonstrated that the intrathecal administration of pertussis toxin produces a long-lasting thermal allodynia in mice. The purpose of the present studies was to compare the antinociceptive and the antiallodynic effects of drugs that are commonly used in treating neuropathic allodynia in untreated mice and in mice which had been administered vehicle or pertussis toxin intrathecally 7 days previously. In untreated mice, morphine, fentanyl, clonidine, oxymetazoline, desipramine and lidocaine, but not MK801, produced dose-related antinociception when tested using a 55 degrees C water tail-flick test. However, 7 days after the intrathecal injection of pertussis toxin, which induced a condition of thermal allodynia when tested using a 45 degrees C water bath, the full opioid and the full alpha(2)-adrenergic receptor agonists fentanyl and clonidine, but not the partial opioid nor the partial alpha(2)-adrenergic receptor agonists morphine and oxymetazoline, reversed the pertussis toxin-induced thermal allodynia. Moreover, lidocaine, desipramine, carbamazepine and MK801 failed to reverse the pertussis toxin-induced thermal allodynia. The present results suggest that decrements in G(i)/G(o)-protein function may be involved in initiating and/or maintaining some neuropathic pain states. Moreover, the results of the present study suggest that the use of full, but not partial, opioid or alpha(2)-agonists may be useful in the treatment of thermal allodynic pain states which may be due at least in part to inhibitory second messenger system dysfunction. Further, the underlying biochemistry of the apparent allodynic pain state induced by intrathecal administration of pertussis toxin warrants further investigation.

[35S]GTPgammaS autoradiography of slide-mounted tissue sections was used to examine G-protein coupling in the rat spinal cord, as stimulated by dopamine, the D1 receptor agonist SKF 38393, noradrenaline, and noradrenaline in the presence of the alpha adrenoceptor antagonist, phentolamine. Measurements were obtained from the different laminae of spinal grey and from the dorsal, lateral, and ventral columns of white matter, at cervical, thoracic, and lumbar levels. At every level, there was a relatively strong basal incorporation of GTPgammaS in laminae II-III>lamina IV-X of spinal grey, even in presence of DPCPX to block endogenous activation by adenosine A1 receptors. Dopamine, and to a lesser degree SKF 38393, but not the D2 receptor agonist quinpirole, stimulated G-protein coupling in laminae IV-X. Both dopamine and SKF 38393 also induced a weak but significant activation throughout the white matter. In both grey and white matter, the activation by dopamine was markedly reduced in presence of a selective D1 receptor antagonist. Noradrenaline strongly stimulated coupling throughout the spinal grey at all levels, an effect that was uniformly reduced in the presence of phentolamine. With or without phentolamine, there was also significant stimulation by noradrenaline in the white matter. Under the same experimental conditions, alpha 1, alpha 2, and beta adrenergic receptor agonists failed to activate GTPgammaS incorporation in either grey or white matter. However, in the presence of selective alpha 1 or alpha 2 receptor antagonist, significant reductions of noradrenaline-stimulated GTPgammaS incorporation were observed in both grey and white matter. The beta antagonist propanolol reduced GTPgammaS incorporation in grey matter only. Thus, the results confirmed the existence of D1 dopamine receptors and of alpha 1, alpha 2, and beta adrenergic receptors in the grey matter of rat spinal cord. In white matter, they strongly suggested the presence of dopamine D1, and of alpha 1 and alpha 2 adrenergic receptors on glia and/or microvessels, that might be activated by diffuse transmission in vivo.

The purpose of the present study was to clarify whether acute injection of stress-related hormones, corticosterone (CORT), norepinephrine (NE) and epinephrine (E) affect food passage in the crop of chicks (Gallus gallus). Subcutaneous (SQ) injection of CORT significantly retarded the food passage in the crop of chicks. Intraperitoneal (IP) injection of NE and E also significantly decreased the crop emptying rate. Additional experiments by using agonists of adrenergic receptors found that IP injection of phenylephrine and clonidine but not isoproterenol retarded the food passage in the crop of chicks. These results demonstrated that the effect of NE and E would be mediated by alpha-1-, alpha-2- rather than beta-adrenergic receptor. Finally, we found that injection of CORT, NE and E had no effect on the number of defecations while intracerebroventricular injection of corticotropin-releasing hormone and urocortin-3 significantly increased it. These results suggest that CORT, NE and E might affect the food passage in the upper digestive tract in chicks.

Although it is known that noradrenaline (NA) powerfully controls spinal motor networks, few data are available regarding the noradrenergic (NAergic) modulation of intrinsic and synaptic properties of neurons in motor networks. Our work explores the cellular basis of NAergic modulation in the rat motor spinal cord. We first show that lumbar motoneurons express the three classes of adrenergic receptors at birth. Using patch-clamp recordings in the newborn rat spinal cord preparation, we characterized the effects of NA and of specific agonists of the three classes of adrenoreceptors on motoneuron membrane properties. NA increases the motoneuron excitability partly via the inhibition of a K(IR) like current. Methoxamine (alpha(1)), clonidine (alpha(2)) and isoproterenol (beta) differentially modulate the motoneuron membrane potential but also increase motoneuron excitability, these effects being respectively inhibited by the antagonists prazosin (alpha(1)), yohimbine (alpha(2)) and propranolol (beta). We show that the glutamatergic synaptic drive arising from the T13-L2 network is enhanced in motoneurons by NA, methoxamine and isoproterenol. On the other hand, NA, isoproterenol and clonidine inhibit both the frequency and amplitude of miniature glutamatergic EPSCs while methoxamine increases their frequency. The T13-L2 synaptic drive is thereby differentially modulated from the other glutamatergic synapses converging onto motoneurons and enhanced by presynaptic alpha(1) and beta receptor activation. Our data thus show that the NAergic system exerts a powerful and complex neuromodulation of lumbar motor networks in the neonatal rat spinal cord.