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Enkephalins are opioid peptides that modulate analgesia, reward, and stress. In vivo detection of enkephalins remains difficult due to transient and low endogenous concentrations and inherent sequence similarity. To begin to address this, we previously developed a system combining in vivo optogenetics with microdialysis and a highly sensitive mass spectrometry-based assay to measure opioid peptide release in freely moving rodents (Al-Hasani et al., 2018, eLife). Here, we show improved detection resolution and stabilization of enkephalin detection, which allowed us to investigate enkephalin release during acute stress. We present an analytical method for real-time, simultaneous detection of Met- and Leu-enkephalin (Met-Enk and Leu-Enk) in the mouse nucleus accumbens shell (NAcSh) after acute stress. We confirm that acute stress activates enkephalinergic neurons in the NAcSh using fiber photometry and that this leads to the release of Met- and Leu-Enk. We also demonstrate the dynamics of Met- and Leu-Enk release as well as how they correlate to one another in the ventral NAc shell, which was previously difficult due to the use of approaches that relied on mRNA transcript levels rather than posttranslational products. This approach increases spatiotemporal resolution, optimizes the detection of Met-Enk through methionine oxidation, and provides novel insight into the relationship between Met- and Leu-Enk following stress.

Measurement of neuropeptides in the brain through in vivo microdialysis sampling provides direct correlation between neuropeptide concentration and brain function. Capillary liquid chromatography-multistage mass spectrometry (CLC-MS n ) has proven to be effective at measuring endogenous neuropeptides in microdialysis samples. In the method, microliter samples are concentrated onto nanoliter volume packed beds before ionization and mass spectrometry analysis. The long times required for extensive preconcentration present a barrier to routine use because of the many samples that must be analyzed and instability of neuropeptides. In this study, we evaluated the capacity of 75 μm inner diameter (i.d.) capillary column packed with 10 μm reversed phase particles for increasing the throughput in CLC-MS n based neuropeptide measurement. Coupling a high injection flow rate for fast sample loading/desalting with a low elution flow rate to maintain detection sensitivity, this column has reduced analysis time from ∼30 min to 3.8 min for 5 μL sample, with 3 pM limit of detection (LOD) for enkephalins and 10 pM LOD for dynorphin A 1-8 in 5 μL sample. The use of isotope-labeled internal standard lowered peptide signal variation to less than 5 %. This method was validated for in vivo detection of Leu and Met enkephalin with microdialysate collected from rat globus pallidus. The improvement in speed and stability makes CLC-MS n measurement of neuropeptides in vivo more practical. Figure ᅟ

The surface chemistry of gold nanowires (AuNWs) has been systematically assessed in terms of contamination and cleaning processes. The nanomaterial’s surface quality was correlated to its performance in the matrix-free laser desorption ionization mass spectrometry (LDI-MS) analysis of low molecular weight analytes. Arrays of AuNWs were deposited on glass slides by means of the lithographically patterned nanowire electrodeposition technique. AuNWs were then characterized in terms of surface chemical composition and morphology using X-ray photoelectron spectroscopy, scanning electron microscopy and atomic force microscopy. AuNWs were subjected to a series of well-known cleaning procedures with the aim of producing the best performing surfaces for the LDI-MS detection of leucine enkephalin, chosen as a model analyte with a molar mass below 1,000 g/mol. Prolonged cyclic voltammetry in 2 M sulfuric acid and, most of all, oxygen plasma cleaning for 5 min provided the best results in terms of simpler (interference-free) and more intense mass spectrometry spectra of the reference compound. The analyte always ionized as the sodiated adduct, and leucine enkephalin limits of detection of 0.5 and 2.5 pmol were estimated for the positive and negative analysis modes, respectively. This study points out the tight correlation existing between the chemical status of the nanostructure surface and the AuNW-assisted LDI-MS performance in terms of reproducibility of spectra, intensity of analyte ions and reduction of interferences. Figure SEM (a-d) and AFM (e-f) pictures and LDI-MS spectra of leu-enk analyte (g-h) obtained with untreated (left side) and oxygen plasmatreated (right side) gold nanowire arrays supported on glass slide

A concept termed liquid-phase combinatorial synthesis (LPCS) is described. The central feature of this methodology is that it combines the advantages that classic organic synthesis in solution offers with those that solid-phase synthesis can provide, through the application of a linear homogeneous polymer. To validate this concept two libraries were prepared, one of peptide and the second of non-peptide origin. The peptide-based library was synthesized by a recursive deconvolution strategy [Erb, E., Janda, K. D. \\& Brenner, S. (1994) Proc. Natl. Acad. Sci. USA 91, 11422-11426] and several ligands were found within this library to bind a monoclonal antibody elicited against β-endorphin. The non-peptide molecules synthesized were arylsulfonamides, a class of compounds of known clinical bactericidal efficacy. The results indicate that the reaction scope of LPCS should be general, and its value to multiple, high-throughput screening assays could be of particular merit, since multimilligram quantities of each library member can readily be attained.

The performance of quadrupole ion traps using argon or air as the buffer gas was evaluated and compared to the standard helium only operation. In all cases a pure buffer gas, not mixtures of gases, was investigated. Experiments were performed on a Bruker Esquire ion trap, a Finnigan LCQ, and a Finnigan ITMS for comparison. The heavier gases were found to have some advantages, particularly in the areas of sensitivity and collision-induced dissociation efficiency; however, there is a significant resolution loss due to dissociation and/or scattering of ions. Additionally, the heavier gases were found to affect ion activation and deactivation during MS/MS, influencing the product ion intensities observed. Finally, the specific quadrupole ion trap design and the ion ejection parameters were found to be crucial in the quality of the spectra obtained in the presence of heavy gases. Operation with static pressures of heavy gases can be beneficial under certain design and operating conditions of the quadrupole ion trap.

Stored waveform inverse Fourier transform and double resonance techniques have been used in conjunction with a quadrupole ion trap to study the dissociation patterns of peptide ions. These experiments provide insight into the origin of individual product ions in an MS/MS spectrum. Results show for a series of leucine enkephalin analogues with five amino acid residues that the b 4 ion is the main product ion through which many other product ions arise. It was also observed that the percentage of the a 4 product ions that are formed directly from the protonated molecule (M+H) + depends on the nature of the fourth amino acid residue. In addition, it was determined that in the peptides studied here lower series b ions (e.g., b 3 ) arise from direct dissociation of higher series b ions (e.g., b 4 ) only about 50% of the time.

The aim of this study was the morphological and further chemical characterisation of neurons immunoreactive for leu-enkephalin (leuENK). Ten wholemounts of small and large intestinal segments from nine patients were immunohistochemically triple-stained for leuENK/neurofilament 200 (NF)/substance P (SP). Based on their simultaneous NF-reactivity and 3D reconstruction of single NF-reactive cells, 97.5% of leuENK-positive neurons displayed the appearance of stubby neurons: small somata; short, stubby dendrites and one axon. Of these leuENK-reactive stubby neurons, 91.3% did not display co-reactivity for SP whereas 8.7% were SP-co-reactive. As to their axonal projection pattern, 50.4% of the recorded leuENK stubby neurons had axons running orally whereas in 29.4% they ran anally; the directions of the remaining 20.2% could not be determined. No axons were seen to enter into secondary strands of the myenteric plexus. Somal area measurements revealed clearly smaller somata of leuENK-reactive stubby neurons (between 259+/-47 microm(2) and 487+/-113 microm(2)) than those of putative sensory type II neurons (between 700+/-217 microm(2) and 1,164+/-396 microm(2)). The ratio dendritic field area per somal area of leuENK-reactive stubby neurons was between 2.0 and 2.8 reflecting their short dendrites. Additionally, we estimated the proportion of leuENK-positive neurons in comparison to the putative whole myenteric neuron population in four leuENK/anti-Hu doublestained wholemounts. This proportion ranged between 5.9% and 8.3%. We suggest leuENK-reactive stubby neurons to be muscle motor neurons and/or ascending interneurons. Furthermore, we explain why we do not use the term \"Dogiel type I neurons\" for this population.

Abnormalities of the enteric nervous system are thought to explain the pathophysiology of motility disorders. Our aim was to determine if particular classes of enteric neurons are affected in slow transit constipation (STC). Specimens were taken from the terminal ileum and ascending, transverse and descending colon of patients undergoing subtotal colectomy for STC. Immunohistochemistry was performed using antisera to neuron-specific enolase, tachykinin, leu-enkephalin, choline acetyltransferase, vasoactive intestinal peptide, nitric oxide synthase, tyrosine hydroxylase and neuropeptide Y. The density of nerve fibres labelled with these antibodies in each layer was compared with age-matched controls. The density of nerve fibres with tachykinin and enkephalin immunoreactivity was reduced in the colonic circular muscle of the 15 patients with STC, whereas innervation of all other layers was normal. This reduction of tachykinin-immunoreactive nerve fibres also occurred in nine of the 12 specimens of terminal ileum examined. No difference was detected in the density or distribution of nerve fibres using the other antisera. Excitatory nerve fibres are present in the circular muscle in STC but they are deficient in tachykinins and enkephalin.

Originally, intestinal motility was thought to be exclusively regulated by myenteric neurons. Some years ago, however, it was demonstrated in large mammals that submucous neurons also participate in the innervation of the circular smooth muscle layer. To date, no information is available about the submucous innervation of the longitudinal smooth muscle layer (LM). This study provides evidence that in the small intestine of large mammals, the LM is innervated not only by the myenteric plexus, but also by the inner and outer submucous plexuses (ISP and OSP). In the porcine small intestine, the involved neurons can be subdivided into the following neurochemically distinct populations: leu-enkephalin (ENK)- and/or substance P (SP)-IR neurons and nitric oxide synthase (NOS)- and/or vasoactive intestinal polypeptide (VIP)-IR neurons. In the myenteric plexus, the majority of VIP- and/or NOS-IR neurons and ENK(+)/SP(-)-IR neurons exhibit descending projections, whereas ENK(+)/SP(+)-IR neurons preferentially have ascending projections. The ENK(-)/SP(+)-IR neurons do not show a polarized pattern. In the OSP, only ENK(+)/SP(-)- and VIP(+)/NOS(-)-IR neurons display a polarized (descending) projection pattern, whereas no polarization can be noted in the ISP. Morphological analysis of the traced neurons revealed that, in general, myenteric descending LM motor neurons have larger cell bodies than ascending ones and, in addition, myenteric descending VIP- and/or NOS-IR neurons have longer projections than ENK and/or SP-IR neurons. In conclusion, the present study demonstrates the involvement of not only myenteric, but also submucous neurons in the innervation of the LM. The two major populations are descending nitrergic neurons and ascending tachykinergic motor neurons, but also other subpopulations with specific projection patterns and neurochemical features have been identified.

Immunohistochemical studies have been performed to investigate the occurrence and coexistence of two catecholamine-synthesising enzymes, tyrosine hydroxylase and dopamine-beta-hydroxylase, and several neuropeptides, including neuropeptide Y, vasoactive intestinal polypeptide, Leu5-enkephalin, somatostatin, calcitonin gene-related peptide and substance P, in nerve fibres supplying porcine accessory genital glands, the seminal vesicles, prostate (body and the disseminated part) and bulbourethral glands. Three major populations of nerve fibres supplying non-vascular elements of the glands have been distinguished (from the largest to the smallest one): (1) noradrenergic fibres, the majority of which contain Leu5-enkephalin, neuropeptide Y or, to a lesser extent, somatostatin, (2) non-noradrenergic, putative cholinergic fibres containing vasoactive intestinal polypeptide, neuropeptide Y and/or somatostatin and, (3) nonnoradrenergic, presumably sensory fibres, containing calcitonin gene-related peptide and substance P. Whilst the coexistence patterns within nerves supplying particular glands are similar, the density of innervation varies between the organs. The innervation of the seminal vesicles and prostatic body is more developed than that of the disseminated part of the prostate and bulbourethral glands. The majority of noradrenergic fibres related to blood vessels contain neuropeptide Y only, while the non-noradrenergic nerves contain mainly vasoactive intestinal polypeptide. The possible function and origin of particular nerve fibre populations are discussed.