Recordings in an integrating central neuron provide a quick way for identifying appropriate anaesthetic use in fish.

In animal husbandry, livestock industry and research facilities, anaesthetic agents are frequently used to moderate stressful intervention. For mammals and birds, procedures have been established to fine-tune anaesthesia according to the intervention. In ectothermic vertebrates, however, and despite changes in legislation and growing evidence on their cognitive abilities, the presently available information is insufficient to make similarly informed decisions. Here we suggest a straightforward way for rapidly filling this gap. By recording from a command neuron in the brain of fish whose crucial role requires it to integrate and process information from all sensory systems and to relay it to motor output pathways, the specific effects of candidate anaesthesia on central processing of sensory information can directly and efficiently be probed. Our approach allows a rapid and reliable way of deciding if and at which concentration a given anaesthetic affects the central nervous system and sensory processing. We employ our method to four anaesthetics commonly used in fish and demonstrate that our method quickly and with small numbers of animals provides the critical data for informed decisions on anaesthetic use.

Critical role of somatostatin receptor 2 in the vulnerability of the central noradrenergic system: new aspects on Alzheimer's disease.

Alzheimer's disease and other age-related neurodegenerative disorders are associated with deterioration of the noradrenergic locus coeruleus (LC), a probable trigger for mood and memory dysfunction. LC noradrenergic neurons exhibit particularly high levels of somatostatin binding sites. This is noteworthy since cortical and hypothalamic somatostatin content is reduced in neurodegenerative pathologies. Yet a possible role of a somatostatin signal deficit in the maintenance of noradrenergic projections remains unknown. Here, we deployed tissue microarrays, immunohistochemistry, quantitative morphometry and mRNA profiling in a cohort of Alzheimer's and age-matched control brains in combination with genetic models of somatostatin receptor deficiency to establish causality between defunct somatostatin signalling and noradrenergic neurodegeneration. In Alzheimer's disease, we found significantly reduced somatostatin protein expression in the temporal cortex, with aberrant clustering and bulging of tyrosine hydroxylase-immunoreactive afferents. As such, somatostatin receptor 2 (SSTR2) mRNA was highly expressed in the human LC, with its levels significantly decreasing from Braak stages III/IV and onwards, i.e., a process preceding advanced Alzheimer's pathology. The loss of SSTR2 transcripts in the LC neurons appeared selective, since tyrosine hydroxylase, dopamine ß-hydroxylase, galanin or galanin receptor 3 mRNAs remained unchanged. We modeled these pathogenic changes in Sstr2(-/-) mice and, unlike in Sstr1(-/-) or Sstr4(-/-) genotypes, they showed selective, global and progressive degeneration of their central noradrenergic projections. However, neuronal perikarya in the LC were found intact until late adulthood (<8 months) in Sstr2(-/-) mice. In contrast, the noradrenergic neurons in the superior cervical ganglion lacked SSTR2 and, as expected, the sympathetic innervation of the head region did not show any signs of degeneration. Our results indicate that SSTR2-mediated signaling is integral to the maintenance of central noradrenergic projections at the system level, and that early loss of somatostatin receptor 2 function may be associated with the selective vulnerability of the noradrenergic system in Alzheimer's disease.

Loss of morphine reward and dependence in mice lacking G protein-coupled receptor kinase 5.

BACKGROUND: The clinical benefits of opioid drugs are counteracted by the development of tolerance and addiction. We provide in vivo evidence for the involvement of G protein-coupled receptor kinases (GRKs) in opioid dependence in addition to their roles in agonist-selective mu-opioid receptor (MOR) phosphorylation. METHODS: In vivo MOR phosphorylation was examined by immunoprecipitation and nanoflow liquid chromatography-tandem mass spectrometry analysis. Using the hot-plate and conditioned place preference test, we investigated opioid-related antinociception and reward effects in mice lacking GRK3 or GRK5. RESULTS: Etonitazene and fentanyl stimulated the in vivo phosphorylation of multiple carboxyl-terminal phosphate acceptor sites, including threonine 370, serine 375, and threonine 379, which was predominantly mediated by GRK3. By contrast, morphine promoted a selective phosphorylation of serine 375 that was predominantly mediated by GRK5. In contrast to GRK3 knockout mice, GRK5 knockout mice exhibited reduced antinociceptive responses after morphine administration and developed morphine tolerance similar to wild-type mice but fewer signs of physical dependence. Also, morphine was ineffective in inducing conditioned place preference in GRK5 knockout mice, whereas cocaine conditioned place preference was retained. However, the reward properties of morphine were evident in knock-in mice expressing a phosphorylation-deficient S375A mutation of the MOR. CONCLUSIONS: These findings show for the first time that MOR phosphorylation is regulated by agonist-selective recruitment of distinct GRK isoforms that influence different opioid-related behaviors. Modulation of GRK5 function could serve as a new approach for preventing addiction to opioids, while maintaining the analgesic properties of opioid drugs at an effective level.

Somatostatin and its 2A receptor in dorsal root ganglia and dorsal horn of mouse and human: expression, trafficking and possible role in pain.

BACKGROUND: Somatostatin (SST) and some of its receptor subtypes have been implicated in pain signaling at the spinal level. In this study we have investigated the role of SST and its sst2A receptor (sst2A) in dorsal root ganglia (DRGs) and spinal cord. RESULTS: SST and sst2A protein and sst2 transcript were found in both mouse and human DRGs, sst2A-immunoreactive (IR) cell bodies and processes in lamina II in mouse and human spinal dorsal horn, and sst2A-IR nerve terminals in mouse skin. The receptor protein was associated with the cell membrane. Following peripheral nerve injury sst2A-like immunoreactivity (LI) was decreased, and SST-LI increased in DRGs. sst2A-LI accumulated on the proximal and, more strongly, on the distal side of a sciatic nerve ligation. Fluorescence-labeled SST administered to a hind paw was internalized and retrogradely transported, indicating that a SST-sst2A complex may represent a retrograde signal. Internalization of sst2A was seen in DRG neurons after systemic treatment with the sst2 agonist octreotide (Oct), and in dorsal horn and DRG neurons after intrathecal administration. Some DRG neurons co-expressed sst2A and the neuropeptide Y Y1 receptor on the cell membrane, and systemic Oct caused co-internalization, hypothetically a sign of receptor heterodimerization. Oct treatment attenuated the reduction of pain threshold in a neuropathic pain model, in parallel suppressing the activation of p38 MAPK in the DRGs CONCLUSIONS: The findings highlight a significant and complex role of the SST system in pain signaling. The fact that the sst2A system is found also in human DRGs and spinal cord, suggests that sst2A may represent a potential pharmacologic target for treatment of neuropathic pain.

Deciphering µ-opioid receptor phosphorylation and dephosphorylation in HEK293 cells.

BACKGROUND AND PURPOSE: The molecular basis of agonist-selective signalling at the µ-opioid receptor is poorly understood. We have recently shown that full agonists such as [D-Ala(2)-MePhe(4)-Gly-ol]enkephalin (DAMGO) stimulate the phosphorylation of a number of carboxyl-terminal phosphate acceptor sites including threonine 370 (Thr(370)) and serine 375 (Ser(375)), and that is followed by a robust receptor internalization. In contrast, morphine promotes a selective phosphorylation of Ser(375) without causing rapid receptor internalization. EXPERIMENTAL APPROACH: Here, we identify kinases and phosphatases that mediate agonist-dependent phosphorylation and dephosphorylation of the µ-opioid receptor using a combination of phosphosite-specific antibodies and siRNA knock-down screening in HEK293 cells. KEY RESULTS: We found that DAMGO-driven phosphorylation of Thr(370) and Ser(375) was preferentially catalysed by G-protein-coupled receptor kinases (GRKs) 2 and 3, whereas morphine-driven Ser(375) phosphorylation was preferentially catalysed by GRK5. On the functional level, inhibition of GRK expression resulted in enhanced µ-opioid receptor signalling and reduced receptor internalization. Analysis of GRK5-deficient mice revealed that GRK5 selectively contributes to morphine-induced Ser(375) phosphorylation in brain tissue. We also identified protein phosphatase 1γ as a µ-opioid receptor phosphatase that catalysed Thr(370) and Ser(375) dephosphorylation at or near the plasma membrane within minutes after agonist removal, which in turn facilitates receptor recycling. CONCLUSIONS AND IMPLICATIONS: Together, the morphine-activated µ-opioid receptor is a good substrate for phosphorylation by GRK5 but a poor substrate for GRK2/3. GRK5 phosphorylates µ-opioid receptors selectively on Ser(375), which is not sufficient to drive significant receptor internalization.

Analgesic tolerance to high-efficacy agonists but not to morphine is diminished in phosphorylation-deficient S375A µ-opioid receptor knock-in mice.

Morphine is one of the most potent analgesic drugs. However, the utility of morphine in the management of chronic pain is limited by its rapid development of tolerance. Morphine exerts all of its pharmacological effects via the µ-opioid receptor. In many systems, tolerance is associated with phosphorylation and desensitization of G-protein-coupled receptors (GPCRs). In case of the µ-opioid receptor, phosphorylation occurs in an agonist-selective manner. High-efficacy agonists such as [d-Ala(2)-MePhe(4)-Gly-ol]enkephalin (DAMGO), fentanyl, or etonitazene stimulate the phosphorylation of both C-terminal threonine 370 (T370) and serine 375 (S375). In contrast, morphine promotes the phosphorylation of S375 but fails to stimulate T370 phosphorylation. Here, we have assessed the contribution of S375 phosphorylation to the development of antinociceptive tolerance to high- and low-efficacy µ agonists in vivo. We show that S375 phosphorylation of the µ-opioid receptor occurs in intact mouse brain in a dose-dependent manner after administration of morphine, fentanyl, or etonitazene. In knock-in mice expressing the phosphorylation-deficient S375A mutant of the µ-opioid receptor, morphine and fentanyl exhibited greater dose-dependent antinociceptive responses than in wild-type mice. However, acute and chronic tolerance to morphine was retained in S375A mutant mice. In contrast, antinociceptive tolerance after repeated subcutaneous application of etonitazene or repeated intracerebroventricular application of DAMGO was diminished. Thus, tolerance to µ agonists with different efficacies develops through distinct pathways. Whereas tolerance induced by DAMGO or etonitazene requires agonist-driven phosphorylation of S375, the development and maintenance of antinociceptive tolerance to morphine occurs independent of S375 phosphorylation.

Potent anti-inflammatory and antinociceptive activity of the endothelin receptor antagonist bosentan in monoarthritic mice.

INTRODUCTION: Endothelins are involved in tissue inflammation, pain, edema and cell migration. Our genome-wide microarray analysis revealed that endothelin-1 (ET-1) and endothelin-2 (ET-2) showed a marked up-regulation in dorsal root ganglia during the acute phase of arthritis. We therefore examined the effects of endothelin receptor antagonists on the development of arthritis and inflammatory pain in monoarthritic mice. METHODS: Gene expression was examined in lumbar dorsal root ganglia two days after induction of antigen-induced arthritis (AIA) using mRNA microarray analysis. Effects of drug treatment were determined by repeated assessment of joint swelling, pain-related behavior, and histopathological manifestations during AIA. RESULTS: Daily oral administration of the mixed ET(A) and ET(B) endothelin receptor antagonist bosentan significantly attenuated knee joint swelling and inflammation to an extent that was comparable to dexamethasone. In addition, bosentan reduced inflammatory mechanical hyperalgesia. Chronic bosentan administration also inhibited joint swelling and protected against inflammation and joint destruction during AIA flare-up reactions. In contrast, the ET(A)-selective antagonist ambrisentan failed to promote any detectable antiinflammatory or antinociceptive activity. CONCLUSIONS: Thus, the present study reveals a pivotal role for the endothelin system in the development of arthritis and arthritic pain. We show that endothelin receptor antagonists can effectively control inflammation, pain and joint destruction during the course of arthritis. Our findings suggest that the antiinflammatory and antinociceptive effects of bosentan are predominantly mediated via the ET(B) receptor.

Differential antiinflammatory and antinociceptive effects of the somatostatin analogs octreotide and pasireotide in a mouse model of immune-mediated arthritis.

OBJECTIVE: Clinical and preclinical evidence suggests that somatostatin exhibits potent antiinflammatory and antinociceptive properties. However, it is not known which of the 5 somatostatin receptor subtypes (SSTRs 1-5) is involved in these actions. The purpose of this study was to assess the effects of the stable somatostatin analogs octreotide and pasireotide (SOM230) in a mouse model of antigen-induced arthritis (AIA). METHODS: Studies were performed in SSTR2-deficient mice (SSTR2(-/-)) and their wild-type littermates (SSTR2(+/+)). The expression of SSTR1, SSTR2A, SSTR3, and SSTR5 in dorsal root ganglia was examined by immunohistochemistry. RESULTS: Untreated SSTR2(-/-) mice with AIA displayed joint swelling and mechanical hyperalgesia similar to that seen in SSTR2(+/+) mice. In wild-type mice, both octreotide and pasireotide significantly attenuated knee joint swelling and histopathologic manifestations of arthritis to an extent comparable to that of dexamethasone. In SSTR2(-/-) mice, the antiinflammatory effects of both octreotide and pasireotide were completely abrogated. Prolonged administration of pasireotide also inhibited joint swelling and protected against joint destruction during AIA flare reactions. In addition, both octreotide and pasireotide reduced inflammatory hyperalgesia. The antinociceptive actions of octreotide were abolished in SSTR2(-/-) mice, but those of pasireotide were retained. In dorsal root ganglia of naive wild-type mice, only SSTR1 and SSTR2A, but not SSTR3 or SSTR5, were detected in a subset of small- and medium-diameter neurons. CONCLUSION: Our findings indicate that the antinociceptive and antiinflammatory actions of octreotide and pasireotide are largely mediated via the SSTR2 receptor. In addition, we identified the SSTR1 receptor as a novel pharmacologic target for somatostatin-mediated peripheral analgesia in inflammatory pain.